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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">01990</article-id><article-id pub-id-type="doi">10.7554/eLife.01990</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Plant biology</subject></subj-group></article-categories><title-group><article-title>Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-10289"><name><surname>Liu</surname><given-names>Xiaokun</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-10288"><name><surname>Grabherr</surname><given-names>Heini M</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-10290"><name><surname>Willmann</surname><given-names>Roland</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-10291"><name><surname>Kolb</surname><given-names>Dagmar</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-10292"><name><surname>Brunner</surname><given-names>Frédéric</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-10293"><name><surname>Bertsche</surname><given-names>Ute</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-10294"><name><surname>Kühner</surname><given-names>Daniel</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-14350"><name><surname>Franz-Wachtel</surname><given-names>Mirita</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-14355"><name><surname>Amin</surname><given-names>Bushra</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-14259"><name><surname>Felix</surname><given-names>Georg</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-10295"><name><surname>Ongena</surname><given-names>Marc</given-names></name><xref ref-type="aff" rid="aff5"/><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-1152"><name><surname>Nürnberger</surname><given-names>Thorsten</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con12"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-1344"><name><surname>Gust</surname><given-names>Andrea A</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con13"/><xref ref-type="fn" rid="conf2"/></contrib><aff id="aff1"><institution content-type="dept">Department of Plant Biochemistry, Center for Plant Molecular Biology</institution>, <institution>University of Tübingen</institution>, <addr-line><named-content content-type="city">Tübingen</named-content></addr-line>, <country>Germany</country></aff><aff id="aff2"><institution content-type="dept">Department of Microbial Genetics</institution>, <institution>University of Tübingen</institution>, <addr-line><named-content content-type="city">Tübingen</named-content></addr-line>, <country>Germany</country></aff><aff id="aff3"><institution content-type="dept">Proteome Center Tübingen</institution>, <institution>University of Tübingen</institution>, <addr-line><named-content content-type="city">Tübingen</named-content></addr-line>, <country>Germany</country></aff><aff id="aff4"><institution content-type="dept">Medical and Natural Sciences Research Centre</institution>, <institution>University of Tübingen</institution>, <addr-line><named-content content-type="city">Tübingen</named-content></addr-line>, <country>Germany</country></aff><aff id="aff5"><institution content-type="dept">Wallon Centre for Industrial Biology</institution>, <institution>University of Liege-Gembloux Agro-Bio Tech</institution>, <addr-line><named-content content-type="city">Gembloux</named-content></addr-line>, <country>Belgium</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Greenberg</surname><given-names>Jean T</given-names></name><role>Reviewing editor</role><aff><institution>University of Chicago</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>nuernberger@zmbp.uni-tuebingen.de</email> (TN);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>andrea.gust@zmbp.uni-tuebingen.de</email> (AAG)</corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>23</day><month>06</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01990</elocation-id><history><date date-type="received"><day>02</day><month>12</month><year>2013</year></date><date date-type="accepted"><day>20</day><month>06</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Liu et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Liu et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife01990.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01990.001</object-id><p>Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that <italic>Arabidopsis thaliana</italic>, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, <italic>LYS1</italic> mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.001">http://dx.doi.org/10.7554/eLife.01990.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01990.002</object-id><title>eLife digest</title><p>The immune response of plants and animals is triggered when cells detect small molecules that are present on the surface of the bacteria or fragments of peptidoglycans—the polymers that are a major component of the bacterial cell wall. The mechanisms by which small molecules trigger the immune response in plants have been widely studied in the model plant <italic>Arabidopsis thaliana</italic>, but less is known about the ways in which peptidoglycan fragments can initiate an immune response.</p><p>Proteins called lysozymes are known to break peptidoglycans into smaller pieces in animals. Plants do not produce lysozymes, but they do produce other enzymes such as chitinases that have similar properties. Now Liu, Grabherr, et al. have shown that a chitinase called LYS1 acts as an enzyme that catalyzes the breakdown of peptidoglycans and has a central role in triggering the immune response of Arabidopsis.</p><p>Plants that were genetically engineered to produce little or no LYS1 were highly susceptible to bacterial infection because there were no enzymes that could break the peptidoglycans into smaller fragments. However, plants that were engineered to produce very high levels of LYS1 also had a compromised immune response because the peptidoglycans were broken into fragments that were too small to be detected.</p><p>The findings of Liu, Grabherr et al. demonstrate that animals and plants employ similar strategies to break down bacterial peptidoglycans to allow them to be detected by the immune system. However, as the enzymes responsible have different structures, they are likely to have evolved separately in plants and animals.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.002">http://dx.doi.org/10.7554/eLife.01990.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>peptidoglycan</kwd><kwd>plant lysozyme</kwd><kwd>innate immunity</kwd><kwd>pattern-triggered immunity</kwd><kwd>glycan hydrolase</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Arabidopsis</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><award-id>SFB 766</award-id><principal-award-recipient><name><surname>Liu</surname><given-names>Xiaokun</given-names></name><name><surname>Grabherr</surname><given-names>Heini M</given-names></name><name><surname>Willmann</surname><given-names>Roland</given-names></name><name><surname>Kolb</surname><given-names>Dagmar</given-names></name><name><surname>Bertsche</surname><given-names>Ute</given-names></name><name><surname>Nürnberger</surname><given-names>Thorsten</given-names></name><name><surname>Gust</surname><given-names>Andrea A</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Arabidopsis lysozyme LYS1 generates soluble peptidoglycan fragments to mediate pattern-triggered immunity.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Activation of antibacterial defenses in multicellular eukaryotic organisms requires recognition of bacterial surface patterns through host-encoded pattern recognition receptors (PRRs) (<xref ref-type="bibr" rid="bib15">Chisholm et al., 2006</xref>; <xref ref-type="bibr" rid="bib39">Jones and Dangl, 2006</xref>; <xref ref-type="bibr" rid="bib35">Ishii et al., 2008</xref>; <xref ref-type="bibr" rid="bib6">Boller and Felix, 2009</xref>; <xref ref-type="bibr" rid="bib83">Vance et al., 2009</xref>; <xref ref-type="bibr" rid="bib74">Segonzac and Zipfel, 2011</xref>; <xref ref-type="bibr" rid="bib55">Monaghan and Zipfel, 2012</xref>; <xref ref-type="bibr" rid="bib11">Broz and Monack, 2013</xref>; <xref ref-type="bibr" rid="bib79">Stuart et al., 2013</xref>). Immunogenic microbial signatures are collectively referred to as pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs) (<xref ref-type="bibr" rid="bib36">Janeway and Medzhitov, 2002</xref>). Bacteria-derived PAMPs such as lipopolysaccharides (LPS) or flagellins possess immunity-stimulating activities in metazoans and plants, suggesting that the ability to sense bacterial surface structures and mount immunity is conserved across lineage borders (<xref ref-type="bibr" rid="bib60">Nürnberger et al., 2004</xref>; <xref ref-type="bibr" rid="bib6">Boller and Felix, 2009</xref>).</p><p>Likewise, peptidoglycans (PGNs) are major building blocks of the cell walls of Gram-positive and Gram-negative bacteria that have been shown to trigger host immune responses in mammalians, insects, and plants (<xref ref-type="bibr" rid="bib24">Dziarski and Gupta, 2005</xref>; <xref ref-type="bibr" rid="bib33">Gust et al., 2007</xref>; <xref ref-type="bibr" rid="bib26">Erbs et al., 2008</xref>; <xref ref-type="bibr" rid="bib47">Kurata, 2014</xref>). Structurally, PGNs are heteroglycan chains that are composed of polymeric alternating β(1,4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues (<xref ref-type="bibr" rid="bib70">Schleifer and Kandler, 1972</xref>; <xref ref-type="bibr" rid="bib32">Glauner et al., 1988</xref>). Such chains are interconnected by oligopeptide bridges which form a coordinate meshwork, thereby providing structural integrity to the bacterial envelope. Recognition of different PGN substructures in animal hosts is brought about by structurally diverse PRRs such as nucleotide-binding oligomerization domain-containing proteins (NODs), peptidoglycan recognition proteins (PGRPs/PGLYRPs), scavenger receptors, or Toll-like receptor TLR2 (<xref ref-type="bibr" rid="bib78">Strober et al., 2006</xref>; <xref ref-type="bibr" rid="bib68">Royet and Dziarski, 2007</xref>; <xref ref-type="bibr" rid="bib25">Dziarski and Gupta, 2010</xref>; <xref ref-type="bibr" rid="bib56">Müller-Anstett et al., 2010</xref>; <xref ref-type="bibr" rid="bib52">Magalhaes et al., 2011</xref>; <xref ref-type="bibr" rid="bib47">Kurata, 2014</xref>). In plants, a tripartite PGN recognition system at the plasma membrane of <italic>Arabidopsis thaliana</italic> with shared functions in PGN sensing and transmembrane signaling was recently described (<xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>). This system comprises Lysin motif (LysM) domain proteins LYM1 and LYM3 for PGN ligand binding and the transmembrane LysM receptor kinase CERK1 that is likely required for conveying the extracellular signal across the plasma membrane and for initiating intracellular signal transduction. All three proteins were shown to be indispensable for PGN sensitivity and to contribute to immunity to bacterial infection (<xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>), which is in agreement with their proposed role as a PGN sensor system. More recently, a similar PGN perception system made of LysM domain proteins LYP4 and LYP6 has been reported from rice (<xref ref-type="bibr" rid="bib50">Liu et al., 2012a</xref>).</p><p>Microbial patterns such as bacterial PGN, LPS, flagellin, or fungal chitin harbor immunogenic epitopes that are parts of supramolecular structures building microbial surfaces (<xref ref-type="bibr" rid="bib6">Boller and Felix, 2009</xref>; <xref ref-type="bibr" rid="bib45">Kumar et al., 2013</xref>; <xref ref-type="bibr" rid="bib58">Newman et al., 2013</xref>; <xref ref-type="bibr" rid="bib64">Pel and Pieterse, 2013</xref>). It is therefore assumed that recognition by host PRRs most likely requires the presence of soluble, randomly structured ligands derived from a complex matrix. X-ray structure-based insight into the binding of bacterial flagellin to the Arabidopsis receptor complex FLS2/BAK1 or of fungal chitin to the Arabidopsis receptor CERK1 supports this view (<xref ref-type="bibr" rid="bib87">Willmann and Nürnberger, 2012</xref>; <xref ref-type="bibr" rid="bib51">Liu et al., 2012b</xref>; <xref ref-type="bibr" rid="bib80">Sun et al., 2013</xref>). Moreover, the existence of fungal LysM effector proteins that scavenge soluble chitin fragments, thus preventing recognition by plant PRRs, suggests that mechanisms releasing these soluble fragments from fungal cell walls must exist (<xref ref-type="bibr" rid="bib23">de Jonge et al., 2010</xref>). Most often, however, it is an open question whether soluble ligand presentation to eukaryotic host PRRs is the result of spontaneous decomposition of the microbial extracellular matrix during infection or, alternatively, whether host-derived factors contribute to the generation of immunogenic ligands for PRR activation. For example, only monomers of bacterial flagellin induce immune responses through human TLR5 whereas filamentous flagella, in which the immunogenic flagellin structure is buried and thus is not accessible to TLR5, do not (<xref ref-type="bibr" rid="bib76">Smith et al., 2003</xref>). It was proposed that a number of circumstances cause flagellin monomer release from intact flagella. For instance, <italic>Caulobacter crescentus</italic> deliberately ejects its flagellum once it is no longer required for the bacterial life cycle (<xref ref-type="bibr" rid="bib37">Jenal and Stephens, 2002</xref>). Moreover, during infection, <italic>Pseudomonas aeruginosa</italic> produces rhamnolipids which act as surfactants and cause flagellin shedding from intact flagella, resulting in a more pronounced immune response (<xref ref-type="bibr" rid="bib31">Gerstel et al., 2009</xref>). Alternatively, host factors such as proteases or environmental conditions such as pH, temperature, or bile salts have been proposed to mediate shearing of flagella from bacterial surfaces (<xref ref-type="bibr" rid="bib66">Ramos et al., 2004</xref>). Likewise, recognition of PGN by intracellular receptors, such as mammalian NOD1 and NOD2, or by plasma membrane receptors, such as mammalian TLR2 or plant LYM1, LYM3 and CERK1 (<xref ref-type="bibr" rid="bib56">Müller-Anstett et al., 2010</xref>; <xref ref-type="bibr" rid="bib77">Sorbara and Philpott, 2011</xref>; <xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>), is facilitated by soluble ligands. Animal lysozymes have been implicated in PGN hydrolysis, bacterial lysis, and host immunity (<xref ref-type="bibr" rid="bib13">Callewaert and Michiels, 2010</xref>), probably through partial PGN degradation and generation of soluble ligands for PGN sensors (<xref ref-type="bibr" rid="bib16">Cho et al., 2005</xref>; <xref ref-type="bibr" rid="bib25">Dziarski and Gupta, 2010</xref>; <xref ref-type="bibr" rid="bib22">Davis et al., 2011</xref>).</p><p>In plants, knowledge of the mode of release of immunogenic fragments from microbial extracellular structures and their contribution to plant immunity is lacking. We here describe a plant enzyme activity (LYS1) that hydrolyzes β(1,4) linkages between N-acetylmuramic acid and N-acetylglucosamine residues in PGN and between N-acetylglucosamine residues in chitooligosaccharides, thus closely resembling metazoan lysozymes (EC 3.2.1.17). Importantly, PGN breakdown products produced by LYS1 are immunogenic in plants, and <italic>LYS1</italic> mutant genotypes were immunocompromised upon bacterial infection. Our findings suggest that plant enzymatic activities, such as LYS1, are capable of generating soluble PRR ligands that might contribute to the activation of immune responses in cells at and surrounding the site of their generation. We also infer that eukaryotic hosts more generally make concerted use of PGN hydrolytic activities and of PRRs in order to cope with bacterial infections.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Arabidopsis PGN binding proteins LYM1 and LYM3 are devoid of PGN hydrolytic activity</title><p>Soluble oligomeric PGN fragments have previously been shown to stimulate plant immune responses in Arabidopsis (<xref ref-type="bibr" rid="bib33">Gust et al., 2007</xref>; <xref ref-type="bibr" rid="bib26">Erbs et al., 2008</xref>; <xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>). As some metazoan PGRPs harbor PGN-degrading enzyme activities (<xref ref-type="bibr" rid="bib30">Gelius et al., 2003</xref>; <xref ref-type="bibr" rid="bib85">Wang et al., 2003</xref>; <xref ref-type="bibr" rid="bib4">Bischoff et al., 2006</xref>; <xref ref-type="bibr" rid="bib25">Dziarski and Gupta, 2010</xref>; <xref ref-type="bibr" rid="bib46">Kurata, 2010</xref>), we tested whether recombinant Arabidopsis PGN binding proteins LYM1 and LYM3 were able to catalyze PGN degradation. For this, we have employed a standard lysozyme assay (<xref ref-type="bibr" rid="bib62">Park et al., 2002</xref>) that is based on reduced turbidity in suspensions of Gram-positive <italic>Micrococcus luteus</italic> cell wall preparations due to PGN degradation. PGN-degrading activity of hen egg-white lysozyme served as a positive control in these assays. As shown in <xref ref-type="fig" rid="fig1">Figure 1A</xref>, lysozyme, but not recombinant LYM1 or LYM3, displayed cell wall-degrading lytic activity, suggesting that the latter are unable to release PGN fragments from bacterial cell walls. This is in agreement with a lack of sequence similarities between LYM1 or LYM3 and known metazoan PGN hydrolytic activities. We therefore conclude that LYM1 and LYM3 constitute plant PGN sensors that appear to be functionally related to non-enzymatic mammalian or Drosophila PGRPs (<xref ref-type="bibr" rid="bib16">Cho et al., 2005</xref>; <xref ref-type="bibr" rid="bib4">Bischoff et al., 2006</xref>; <xref ref-type="bibr" rid="bib25">Dziarski and Gupta, 2010</xref>; <xref ref-type="bibr" rid="bib46">Kurata, 2010</xref>).<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.003</object-id><label>Figure 1.</label><caption><title>The Arabidopsis lysozyme 1 (LYS1) gene is transcriptionally activated upon pathogen-infection.</title><p>(<bold>A</bold>) LYM1 and LYM3 do not possess peptidoglycan (PGN) hydrolytic activity. <italic>Micrococcus luteus</italic> cell wall preparations were incubated with 20 μg affinity-purified His6-tagged LYM1 or LYM3 or 0.5 μg hen egg-white lysozyme and PGN hydrolytic activity was assayed in a turbidity assay at the indicated time points. As negative control (nc), non-induced His6-tagged LYM3 bacterial lysates were used for affinity purification and eluates were subjected to turbidity assays. Means ± SD of three replicates per sample are given. Statistical significance compared with the negative control (**p<0.001, ***p<0.0001, Student’s <italic>t</italic> test) is indicated by asterisks. (<bold>B</bold>) Multiple sequence alignment of the 24 Arabidopsis chitinases using the ClustalW2 algorithm. Full length amino acid sequences were aligned and subgroups were classified according to <xref ref-type="bibr" rid="bib63">Passarinho and de Vries (2002)</xref>. Arabidopsis lysozyme 1 (LYS1, At5g24090) represents the only member of class III. (<bold>C</bold>) The expression of LYS1 in transgenic pLYS1::GUS reporter plants. Leaf halves of transgenic pLYS1::GUS or pPR1::GUS reporter plants were infiltrated with the virulent <italic>Pseudomonas syringae</italic> pv<italic>. tomato</italic> (<italic>Pto</italic>) DC3000, the type III secretion system-deficient <italic>Pto</italic> DC3000 hrcC<sup>-</sup> or the avirulent <italic>Pseudomonas syringae</italic> pv<italic>. phaseolicola</italic> (<italic>Pph</italic>) strain (10<sup>8</sup> cfu/ml) or 10 mM MgCl<sub>2</sub> as control. After 24 hr the leaves were harvested and stained for β-glucuronidase (GUS) activity. (<bold>D</bold>) Leaves of wild-type plants were treated for 3 or 24 hr with 1 µM flg22, 100 µg/ml PGN from <italic>Pto</italic> or 100 µg/ml lipopolysaccharide (LPS). Total RNA was subjected to RT-PCR using <italic>LYS1</italic> or Flagellin-responsive kinase 1 (<italic>FRK1</italic>) specific primers. <italic>EF1α</italic> transcript was used for normalization. All experiments shown in panels (<bold>A</bold>), (<bold>C</bold>) and (<bold>D</bold>) were repeated once with similar results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.003">http://dx.doi.org/10.7554/eLife.01990.003</ext-link></p></caption><graphic xlink:href="elife01990f001"/></fig></p></sec><sec id="s2-2"><title><italic>LYS1</italic> expression is activated upon bacterial infection</title><p>Lysozymes (EC 3.2.1.17) hydrolyze β(1,4) linkages between N-acetylmuramic acid and N-acetylglucosamine residues in PGNs and between N-acetylglucosamine residues in chitodextrins (<ext-link ext-link-type="uri" xlink:href="http://enzyme.expasy.org/EC/3.2.1.17">http://enzyme.expasy.org/EC/3.2.1.17</ext-link>). Plant genomes do not encode lysozyme-like proteins, but many plant species produce lysozyme-like enzyme activities such as chitinases (EC 3.2.1.14) (<xref ref-type="bibr" rid="bib2">Audy et al., 1988</xref>; <xref ref-type="bibr" rid="bib69">Sakthivel et al., 2010</xref>). Plant chitinases fall into five classes (I–V, <xref ref-type="fig" rid="fig1">Figure 1B</xref>) (<xref ref-type="bibr" rid="bib63">Passarinho and de Vries, 2002</xref>) and are grouped into structurally unrelated families 18 and 19 of glycosyl hydrolases, respectively (<xref ref-type="bibr" rid="bib34">Henrissat, 1991</xref>). Chitinases belonging to family 18 of glycosyl hydrolases are ubiquitously found in all organisms whereas chitinases of glycosyl hydrolase family 19 are found almost exclusively in plants. Class III chitinases (glycosyl hydrolase family 18) represent bifunctional plant enzymes with lysozyme-like activities. One such enzyme, hevamine from the rubber tree <italic>Hevea brasiliensis</italic> (<xref ref-type="bibr" rid="bib3">Beintema et al., 1991</xref>), has been shown to hydrolyze PGN and the structurally closely related β(1,4)-linked GlcNAc homopolymer chitin in vitro (<xref ref-type="bibr" rid="bib5">Bokma et al., 1997</xref>).</p><p>To explore host-mediated PGN degradation and its possible implication in plant immune activation, we have addressed the only class III chitinase (which we named LYS1, At5g24090) encoded by the Arabidopsis genome (<xref ref-type="bibr" rid="bib63">Passarinho and de Vries, 2002</xref>; <xref ref-type="fig" rid="fig1">Figure 1B</xref>). Bacterial infection of Arabidopsis plants stably expressing a <italic>pLYS1::GUS</italic> construct revealed that <italic>LYS1</italic> gene expression is enhanced upon infection with host non-adapted <italic>Pseudomonas</italic> <italic>syringae</italic> pv. <italic>phaseolicola</italic> (<italic>Pph</italic>) or disarmed host adapted <italic>P. syringae</italic> pv. <italic>tomato</italic> (<italic>Pto</italic>) DC3000 hrcC<sup>−</sup>. Likewise, expression of the immune response marker <italic>pathogenesis-related protein 1</italic> (<italic>PR1</italic>) was enhanced by the same treatment (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Failure to detect <italic>LYS1</italic> expression in plants infected with virulent host adapted <italic>Pto</italic> DC3000 suggests bacterial effector-mediated suppression that is reminiscent of that observed for PGN receptor proteins LYM1 and LYM3 (<xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>) as well as numerous other immunity-associated genes (<xref ref-type="bibr" rid="bib43">Kemmerling et al., 2007</xref>; <xref ref-type="bibr" rid="bib65">Postel et al., 2010</xref>). <italic>LYS1</italic> gene expression is not only triggered upon bacterial infection, but was also observed upon treatment with different MAMPs including bacterial flagellin, LPS, or PGN preparations (<xref ref-type="fig" rid="fig1">Figure 1D</xref>), similar to the immune marker gene <italic>Flagellin-responsive kinase 1</italic> (<italic>FRK1</italic>). Altogether, infection-induced <italic>LYS1</italic> transcriptional activation suggests that the LYS1 protein is implicated in immunity to bacterial infection.</p></sec><sec id="s2-3"><title>LYS1 is a plant lysozyme</title><p>To analyze the enzymatic properties of LYS1, recombinant protein production was attempted. Overexpression in <italic>Escherichia coli</italic> failed to produce active enzyme and LYS1 production in eukaryotic <italic>Pichia pastoris</italic> entirely failed to produce recombinant protein (not shown). Therefore, we resorted to generate <italic>p35S::LYS1-GFP</italic>-overexpressing (<italic>LYS1</italic><sup><italic>OE</italic></sup>) plants (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>). Notably, LYS1-GFP was glycosylated (<xref ref-type="fig" rid="fig2">Figure 2C</xref>), possibly explaining the failure to produce enzymatically active LYS1 protein in <italic>E. coli</italic>. Expression of the green fluorescent protein (GFP) fusion protein in Arabidopsis plants was accompanied by substantial proteolytic cleavage resulting in the predominant release of a protein with an approximate molecular mass of 35 kDa, most likely representing untagged LYS1 (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). Analysis of this major cleavage product by liquid chromatography-mass spectrometry (LC-MS/MS) after tryptic in-gel digestion and by peptide mass fingerprint not only confirmed the identity of LYS1 in this band but also yielded peptides spanning the whole protein sequence, except for the first 53 amino acids (data not shown), thus indicating cleavage of the LYS1-GFP fusion protein between LYS1 and GFP.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.004</object-id><label>Figure 2.</label><caption><title>Analysis of LYS1 overexpression lines.</title><p>(<bold>A</bold>) RT-qPCR analyses of transcript levels in mature leaves of two independent transgenic lines expressing <italic>p35S::LYS1-GFP</italic> (<italic>LYS1<sup>OE</sup>-1</italic>, <italic>LYS1<sup>OE</sup>-2</italic>) relative to expression levels in wild-type. <italic>EF1α</italic> transcript was used for normalization. Error bars, SD (n = 3). Statistical significance compared with wild-type (***p<0.001, Student’s <italic>t</italic> test) is indicated by asterisks. (<bold>B</bold>) Immunoblot analysis of protein extracts from leaves of two independent <italic>LYS1<sup>OE</sup></italic> lines, a LYS1 knock-down line (<italic>LYS1<sup>KD</sup>-1</italic>, see <xref ref-type="fig" rid="fig3">Figure 3</xref>) and wild-type plants. Total leaf protein was separated by SDS-PAGE and blotted onto a nitrocellulose membrane. Immunodetection was carried out using α-tobacco class III chitinase (α-Chit) or green fluorescent protein (α-GFP) (both from rabbit) and an anti-rabbit HRP-coupled secondary antibody. Ponceau S red staining of the large subunit of RuBisCO served as loading control. (<bold>C</bold>) Total protein extracts from leaves of <italic>LYS1<sup>OE</sup>-1</italic> plants were subjected to deglycosylation with a deglycosylation kit (NEB). The negative control (−) was treated as the deglycosylation sample (+) but without addition of the deglycosylation enzyme mix. Immunoblot analysis was carried out as described in (<bold>B</bold>). All experiments shown were repeated at least once.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.004">http://dx.doi.org/10.7554/eLife.01990.004</ext-link></p></caption><graphic xlink:href="elife01990f002"/></fig></p><p>Three mutant lines with T-DNA insertions in the LYS1 gene were available from the Nottingham Arabidopsis Stock Centre. However, neither the insertion in the 5' untranslated region nor the insertions in the first intron and at the end of the last exon of the coding region abolished formation of the <italic>LYS1</italic> transcript (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). As an alternative to knock-out lines, <italic>LYS1</italic> knock-down lines (<italic>LYS1</italic><sup><italic>KD</italic></sup>) were produced by artificial micro RNA technology (<xref ref-type="bibr" rid="bib72">Schwab et al., 2006</xref>; <xref ref-type="fig" rid="fig3">Figure 3</xref>). As proven by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), we obtained two genetically independent <italic>LYS1</italic><sup><italic>KD</italic></sup> lines with residual transcript levels not exceeding 10% of those detected in wild-type plants (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). In contrast, the transcription of potential off-target genes was not affected (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). Protein extracts derived from transgenic plants were tested for chitinolytic activity by employing 4-methylumbelliferyl β-D-N, N′, N″-triacetylchitotriose (4-MUCT) as substrate. Leaf protein extracts from <italic>LYS1</italic><sup><italic>OE</italic></sup> plants exhibited significant chitinase activity when compared with a <italic>Streptomyces griseus</italic> chitinase control (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). In contrast, wild-type and <italic>LYS1</italic><sup><italic>KD</italic></sup> plants exhibited only marginal chitinase activities. Likewise, using 4-MUCT in a gel electrophoretic separation-based chitinase assay produced a zymogram in which enzyme activity was solely detectable in protein extracts obtained from <italic>LYS1</italic><sup><italic>OE</italic></sup> plants, but not in those from control plants expressing secreted GFP (<italic>secGFP</italic>) (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). Thus, LYS1 indeed harbors the predicted chitinase activity. As 4-MUCT is also a typical substrate for lysozymes (<xref ref-type="bibr" rid="bib12">Brunner et al., 1998</xref>), this was the first indication that LYS1 might also harbor lysozyme activity. Next, leaf protein extracts from <italic>LYS1</italic><sup><italic>OE</italic></sup> plants were tested for their ability to solubilize complex PGN presented by intact Gram-positive <italic>M. luteus</italic> cells and to cleave preparations of complex, insoluble <italic>Bacillus subtilis</italic> PGN. Again, protein extracts from <italic>LYS1</italic><sup><italic>OE</italic></sup> plants exhibited significant PGN-degrading activity whereas wild-type and <italic>LYS1</italic><sup><italic>KD</italic></sup> plants showed basal activity levels only (<xref ref-type="fig" rid="fig4">Figure 4C,D</xref>). Likewise, PGN-solubilizing activity profiles of protoplast suspensions derived from these transgenics confirmed significant PGN-degrading activity of <italic>LYS1</italic><sup><italic>OE</italic></sup> plants (<xref ref-type="fig" rid="fig4">Figure 4E</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.005</object-id><label>Figure 3.</label><caption><title>Analysis of <italic>LYS1</italic> amiRNA lines.</title><p>(<bold>A</bold>) Predicted <italic>LYS1</italic> gene structure (exons, black bars; introns, black lines; untranslated regions, gray). The region targeted by the amiRNA construct is indicated by an arrowhead. (<bold>B</bold>) Off-target genes for the <italic>LYS1-amiRNA</italic> construct were identified using the Web microRNA Designer (WMD; <ext-link ext-link-type="uri" xlink:href="http://wmd.weigelworld.org/">http://wmd.weigelworld.org</ext-link>). The region targeted by the amiRNA is given for each gene, mismatches are indicated in red. Potential off targets either possess more than one mismatch at positions 2–12 or have mismatches at position 10 and/or 11 which will limit amiRNA function. (<bold>C</bold>) Transcript levels of the four top hits shown in (<bold>B</bold>) were determined by RT-qPCR in untreated seedlings of two independent transgenic <italic>LYS1</italic>-amiRNA knock-down lines (<italic>LYS1</italic><sup><italic>KD</italic></sup><italic>-1</italic>, <italic>LYS1</italic><sup><italic>KD</italic></sup><italic>-2</italic>) using gene-specific primers for <italic>LYS1 (At5g24090)</italic>, <italic>At4g02540</italic>, <italic>At1g05615</italic>, <italic>At5g58780</italic>, and <italic>At3g51010</italic>. <italic>EF1α</italic> transcript was used for normalization. Error bars, SD (n = 3). Statistical significance compared with the wild-type control (which was set to 1 for each primer set) is indicated by asterisks (***p<0.001, Student’s <italic>t</italic> test). The experiment was repeated once with similar results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.005">http://dx.doi.org/10.7554/eLife.01990.005</ext-link></p></caption><graphic xlink:href="elife01990f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01990.006</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Characterization of LYS1 T-DNA insertion lines.</title><p>(<bold>A</bold>) Predicted <italic>LYS1</italic> gene structure (exons, black bars; introns, black lines; untranslated regions, gray). T-DNA insertion sites are indicated by triangles. (<bold>B</bold>) The T-DNA insertion lines (each two samples) and the corresponding wild-type accessions were genotyped using the following primer combinations: LP_N853931 and RP_N853931 (WT-PCR, <italic>lys1-1</italic>), Wisc-Lba and RP_853931 (Lba-PCR, <italic>lys1-1</italic>), LP_N595362 and RP_N595362 (WT-PCR, <italic>lys1-2</italic>), Salk-Lba and RP_N595362 (Lba-PCR, <italic>lys1-2</italic>), At5g24090F1 and At5g24090R1 (WT-PCR, <italic>lys1-3</italic>), and Ds5-1 and At5g24090R1 (Lba-PCR, <italic>lys1-3</italic>). (<bold>C</bold>) The <italic>LYS1</italic> transcript analysis in mature leaves was done by semi-quantitative RT-PCR using the following primer combinations: At5g24090F and At5g24090R (<italic>lys1-1</italic> and <italic>lys1-2</italic>) and At5g24090F and At5g24090RP2 (<italic>lys1-3</italic>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.006">http://dx.doi.org/10.7554/eLife.01990.006</ext-link></p></caption><graphic xlink:href="elife01990fs001"/></fig></fig-group><fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.007</object-id><label>Figure 4.</label><caption><title>LYS1 is a glucan hydrolase.</title><p>(<bold>A-D</bold>) Protein extracts from adult wild-type or <italic>LYS</italic><sup><italic>OE</italic></sup><italic>-1</italic> and <italic>LYS</italic><sup><italic>KD</italic></sup><italic>-1</italic> homozygous lines were assayed for hydrolytic activity towards glycan substrates. Plants expressing secreted green fluorescent protein (GFP) (<italic>secGFP</italic>) served to control the effect of external GFP. (<bold>A</bold>) Leaf protein extracts from indicated transgenic plants were assayed for chitinolytic activity using the 4-methylumbelliferyl β-D-N, N′, N″-triacetylchitotriose (4-MUCT) substrate. Enzymatic activities 4 hr after treatment were calculated using <italic>Streptomyces griseus</italic> chitinase as positive control (pc). (<bold>B</bold>) Protein extracts from <italic>LYS1</italic><sup><italic>OE</italic></sup><italic>-1</italic> or <italic>secGFP</italic> plants were separated on a cetyltrimethylammonium bromide-polyacrylamide gel and hydrolytic activity was assayed by overlaying the gel with the substrate 4-MUCT. Fluorescent bands are indicative of substrate cleavage. The arrowhead indicates the position of LYS1. (<bold>C</bold> and <bold>D</bold>) <italic>Micrococcus luteus</italic> cells (<bold>C</bold>) or <italic>Bacillus subtilis</italic> peptidoglycan (PGN) (<bold>D</bold>) were subjected to hydrolysis by leaf protein extracts and PGN hydrolytic activity was calculated after 4 hr using hen egg-white lysozyme as positive control (pc). Significant differences compared with the buffer control are indicated by asterisks (*p<0.05; Student’s <italic>t</italic> test; <bold>A</bold>, <bold>C</bold>, <bold>D</bold>). (<bold>E</bold>) Protoplasts of transgenic lines were pelleted and protein extracts of the protoplast (PP) pellet or medium supernatant were subjected to the PGN hydrolysis assay as described in (<bold>C</bold>). As controls, buffer or protoplast medium (PP medium) was used. Means ± SD of two replicates per sample are given, bars with different letters are significantly different based on one-way ANOVA (p<0.05). (<bold>F</bold>) Lysis of <italic>M. luteus</italic> cells was determined in a turbidity assay with <italic>LYS1</italic><sup><italic>OE</italic></sup> leaf protein extracts as described in (<bold>C</bold>) at the indicated pH. Means ± SD of two replicates per sample are given. All experiments shown were repeated at least once.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.007">http://dx.doi.org/10.7554/eLife.01990.007</ext-link></p></caption><graphic xlink:href="elife01990f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01990.008</object-id><label>Figure 4—figure supplement 1.</label><caption><title>LYS1 is located in the plant apoplast.</title><p>(<bold>A</bold>) Apoplastic washes were prepared from leaves of wild-type Arabidopsis plants or the <italic>LYS1</italic><sup><italic>OE</italic></sup><italic>-1 and LYS1</italic><sup><italic>KD</italic></sup><italic>-1</italic> lines. Apoplastic fluids (concentrated tenfold) or total leaf protein extracts were subjected to western blot analysis using antibodies raised against green fluorescent protein (α-GFP), tobacco class III chitinase (α-chit), or the cytoplasmic mitogen-activated protein kinase 3 (MPK3). (<bold>B</bold>) The <italic>p35S::LYS1-GFP</italic> and <italic>p35S::LYS1ΔSP-GFP</italic> constructs were transiently expressed in <italic>Nicotiana benthamiana</italic> leaves using <italic>Agrobacterium tumefaciens</italic>-mediated transformation. GFP fluorescence in the leaf epidermal cells was analyzed 3 days post infection. FM4-64 was used to stain the plasma membrane. Argon/krypton laser was used for excitation of GFP at 488 nm and the 543 nm line of helium/neon laser for the excitation of FM4-64. Detection wavelengths of emitted light were 500–600 nm (GFP) and 560–615 nm (FM4-64). All experiments shown were repeated three times.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.008">http://dx.doi.org/10.7554/eLife.01990.008</ext-link></p></caption><graphic xlink:href="elife01990fs002"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01990.009</object-id><label>Figure 4—figure supplement 2.</label><caption><title>LYS1 is devoid of cellulose hydrolytic activity.</title><p>LYS1 was purified from 5-week-old <italic>LYS1</italic><sup><italic>OE</italic></sup> plants and used for cellulase activity assays. The substrate 4-methylumbelliferyl-β-D-cellobioside was incubated for 1 hr with purified LYS1, commercial reference cellulose, or buffer as control. Fluorescence was determined (ex/em = 365 nm/455 nm) after stopping the reaction with 0.2 M sodium carbonate. Means ± SD of three replicates per sample are given. Statistical significance compared with the buffer control (***p<0.001, Student’s <italic>t</italic> test) is indicated by asterisks. The experiment was repeated once with the same result.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.009">http://dx.doi.org/10.7554/eLife.01990.009</ext-link></p></caption><graphic xlink:href="elife01990fs003"/></fig></fig-group></p><p>To determine specific enzyme activities, untagged LYS1 was purified from <italic>LYS1</italic><sup><italic>OE</italic></sup> Arabidopsis lines by fast protein liquid chromatography (FPLC) and used for enzyme assays. The 4-MUCT assay yielded a Michaelis constant (K<sub>m</sub>) of 70 ± 14 µM and a V<sub>max</sub> of 378 ± 42 µM min<sup>−1</sup> mg<sup>−1</sup> for LYS1, and a K<sub>m</sub> of 53 ± 27 µM and a V<sub>max</sub> of 397 ± 145 µM min<sup>−1</sup> mg<sup>−1</sup> for commercial <italic>S. griseus</italic> chitinase. Using the turbidity assay with <italic>M. luteus</italic> cell wall preparations, a K<sub>m</sub> of 18.2 ± 2.5 mg/ml and V<sub>max</sub> of 4.4 ± 0.6 mg mg<sup>−1</sup> min<sup>−1</sup> were obtained for LYS1, and a K<sub>m</sub> of 8.4 ± 0.8 mg/ml and V<sub>max</sub> of 192 ± 120 mg mg<sup>−1</sup> min<sup>−1</sup> for commercial hen egg-white lysozyme. The K<sub>m</sub> values for LYS1 are thus comparable to the commercial enzymes.</p><p>As shown in <xref ref-type="fig" rid="fig4">Figure 4E</xref>, the majority of LYS1 activity was found in the supernatant of the protoplasts, suggesting an apoplastic localization of LYS1. To confirm this localization we prepared apoplastic washes from <italic>LYS1</italic><sup><italic>OE</italic></sup> Arabidopsis lines. Both the LYS1-GFP fusion protein as well as free LYS1 was detectable in concentrated apoplastic fluids whereas the cytoplasmic mitogen-activated protein kinase MPK3 was only present in the total leaf protein samples (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>). Moreover, transient expression in the heterologous plant system <italic>Nicotiana benthamiana</italic> of the <italic>p35S::LYS1-GFP</italic> construct resulted in labeling of the cell periphery, whereas expression of a construct lacking the <italic>LYS1</italic> signal peptide-encoding sequence yielded labeling of intracellular structures (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B</xref>). Use of the fluorescent dye FM4-64, a plasma membrane and early endosome marker (<xref ref-type="bibr" rid="bib7">Bolte et al., 2004</xref>), revealed that LYS1 signals co-localized to a large extent with the plasma membrane (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B</xref>). Thus, LYS1 likely operates in close vicinity of the plant surface. Indeed, previous identification within the Arabidopsis cell wall proteome (<xref ref-type="bibr" rid="bib48">Kwon et al., 2005</xref>) suggests that LYS1 acts in the plant apoplast. Since the plant apoplast is an acidic compartment (pH 5–6) (<xref ref-type="bibr" rid="bib71">Schulte et al., 2006</xref>), we investigated whether LYS1 is active at physiologically relevant pH conditions. For this, the <italic>M. luteus</italic> cell wall-degrading activity of an <italic>LYS1</italic><sup><italic>OE</italic></sup> leaf extract was determined at different pH values. Although active at pH values ranging from 3.2 to 7.2, a pronounced maximum of LYS1 activity was detected around pH 6 which coincided with the apoplastic pH of plant cells (<xref ref-type="fig" rid="fig4">Figure 4F</xref>).</p><p>To further confirm LYS1 glucan hydrolytic activity, an epitope-tagged <italic>LYS1</italic> fusion construct was transiently expressed in <italic>N. benthamiana</italic> (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). Similar to the Arabidopsis <italic>LYS1</italic><sup><italic>OE</italic></sup> leaf extracts, extracts from <italic>p35S::LYS1-myc</italic> expressing <italic>N. benthamiana</italic> leaves also displayed in-gel chitinolytic activity (<xref ref-type="fig" rid="fig5">Figure 5B</xref>) compared with extracts from control leaves expressing the viral silencing suppressor p19 only. Likewise, <italic>N. benthamiana</italic> protein extracts containing LYS1-myc were able to cleave preparations of complex insoluble <italic>B. subtilis</italic> PGN (<xref ref-type="fig" rid="fig5">Figure 5C</xref>).<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.010</object-id><label>Figure 5.</label><caption><title>LYS1 transiently expressed in <italic>Nicotiana benthamiana</italic> possesses hydrolytic activity.</title><p>(<bold>A</bold>) Protein extracts from <italic>N. benthamiana</italic> leaves expressing LYS1 fused to the myc-epitope tag under control of the <italic>p35S</italic> promoter were separated on an SDS-polyacrylamide gel and analyzed by western blot using antibodies raised against the myc-epitope tag. As control, plants were infiltrated with agrobacteria harboring the p19 suppressor of silencing construct (p19). Protein sizes (kDa) are indicated on the left. (<bold>B</bold>) <italic>N. benthamiana</italic> protein extracts from leaves expressing <italic>LYS1</italic><sub><italic>myc</italic></sub> or p19 were separated on a cetyltrimethylammonium bromide-polyacrylamide gel and hydrolytic activity was assayed by overlaying the gel with the substrate 4-methylumbelliferyl β-D-N, N′, N″-triacetylchitotriose. Fluorescent bands are indicative of substrate cleavage. Arrowheads indicate the positions of epitope-tagged LYS1. (<bold>C</bold>) Protein extracts from <italic>N. benthamiana</italic> leaves expressing <italic>LYS1</italic><sub><italic>myc</italic></sub> or p19 were assayed for peptidoglycan (PGN) hydrolytic activity in a turbidity assay using <italic>Bacillus subtilis</italic> PGN. Relative activities (2 hr post treatment) were calculated using hen egg-white lysozyme as standard. Statistical significance compared with the untreated control (*p<0.05, Student’s <italic>t</italic> test) is indicated by asterisks. All experiments shown were repeated at least once.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.010">http://dx.doi.org/10.7554/eLife.01990.010</ext-link></p></caption><graphic xlink:href="elife01990f005"/></fig></p><p>In sum, we provide biochemical evidence that LYS1 harbors hydrolytic activity for chitin as well as for PGN of the lysine-type (<italic>M. luteus</italic>) and diaminopimelic acid-type (<italic>B. subtilis</italic>). Importantly, LYS1 failed to exhibit activity on cellobiose as a substrate, indicating it might have no cellulose activity (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>). Thus, LYS1 resembles enzymatic activities reported for metazoan lysozymes and should be classified as lysozyme (EC 3.2.1.17) instead of chitinase (EC 3.2.1.14).</p></sec><sec id="s2-4"><title>LYS1 generates plant immunogenic PGN fragments</title><p>To analyze immunogenic activities of PGN cleavage products generated by LYS1, untagged LYS1 was purified from <italic>LYS1</italic><sup><italic>OE</italic></sup> Arabidopsis lines by FPLC and used for degradation of <italic>B. subtilis</italic> PGN. Solubilized PGN fragments found in the supernatant of LYS1-digested PGN were subsequently analyzed by high performance liquid chromatography (HPLC) (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). Only a few peaks could be detected in the supernatant of PGN incubated with a buffer control or with heat-inactivated LYS1. In contrast, PGN digests produced by native LYS1 yielded several characteristic peaks that were also detectable in the supernatants of PGN preparations treated with mutanolysin, which has been shown to cleave O-glycosidic bonds between GlcNAc and MurNAc residues in complex PGN (<xref ref-type="bibr" rid="bib89">Yokogawa et al., 1975</xref>). LYS1-generated PGN fragments were subsequently tested for their ability to trigger plant immunity-associated responses (<xref ref-type="fig" rid="fig6">Figure 6B–D</xref>). First, supernatants of PGN preparations treated with either native or heat-denatured LYS1 were used to trigger immune marker gene <italic>FRK1</italic> expression in Arabidopsis seedlings. Importantly, only supernatants from PGN digests produced by native LYS1 or mutanolysin induced <italic>FRK1</italic> expression whereas buffer controls or digests produced by heat-inactivated LYS1 did not release immunogenic soluble fragments from complex PGNs (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). Notably, activation of immune responses by LYS1-generated PGN fragments was dependent on Arabidopsis PGN receptor complex components LYM1, LYM3, and CERK1 as the respective mutant genotypes failed to respond to immunogenic PGN fragments (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). Second, we tested whether LYS1-generated PGN fragments were able to trigger an immunity-associated response, medium alkalinization, in rice cell suspensions. This plant was chosen for testing as a PGN receptor system very similar to that in Arabidopsis has recently been reported (<xref ref-type="bibr" rid="bib50">Liu et al., 2012a</xref>). As shown in <xref ref-type="fig" rid="fig6">Figure 6C</xref>, LYS1-released PGN fragments triggered medium alkalinization in cultured rice cells, suggesting that immune defense stimulation by soluble PGN fragments is not restricted to Arabidopsis only.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.011</object-id><label>Figure 6.</label><caption><title>Purified LYS1 generates immunogenic peptidoglycan (PGN) fragments.</title><p>LYS1 was purified from 5-week-old <italic>LYS1</italic><sup><italic>OE</italic></sup> plants and used for PGN digestion. (<bold>A</bold>) 500 µg <italic>Bacillus subtilis</italic> PGN were digested for 7 hr with mutanolysin (50 µg/ml), native purified LYS1 (140 µg/ml), heat-denatured purified LYS1 (140 µg/ml), or the reaction buffer alone and subjected to high performance liquid chromatography fractionation. Shown are the peak profiles of representative runs. The signal intensity is given in milliabsorbance units (mAU). (<bold>B</bold>) <italic>B. subtilis</italic> PGN was digested for 4 hr as described in (<bold>A</bold>) and Arabidopsis wild-type seedlings or the indicated mutant lines were treated for 6 hr with 25 µl/ml digest supernatant containing solubilized PGN fragments. Total seedling RNA was subjected to RT-qPCR using <italic>Flagellin responsive kinase</italic> (<italic>FRK1</italic>) specific primers. <italic>EF1α</italic> transcript was used for normalization, water treatment served as control and was set to 1. (<bold>C</bold>) Supernatants of digested PGN (25 µl/ml) were added to cultured rice cells and medium alkalinization was determined 20 min post addition. Treatment with water or MES buffer served as control. All data represent triplicate samples ± SD, bars with different letters are significantly different based on one-way ANOVA (p<0.05; <bold>B</bold> and <bold>C</bold>). (<bold>D</bold>) <italic>B. subtilis</italic> PGN was digested with native purified LYS1 for the indicated times or overnight (o/n) and digest supernatant was used to trigger medium alkalinization in rice cells as described in (<bold>C</bold>). All data represent triplicate samples ± SD, asterisks indicate significant differences compared to the buffer control (*p<0.05; **p<0.01; ***p<0.001; Student’s <italic>t</italic> test). All experiments shown were repeated at least once.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.011">http://dx.doi.org/10.7554/eLife.01990.011</ext-link></p></caption><graphic xlink:href="elife01990f006"/></fig></p><p>We further investigated the kinetics of PGN fragment release from complex PGNs. As shown in <xref ref-type="fig" rid="fig6">Figure 6D</xref>, release of immunogenic PGN fragments into solution occurred rapidly within 10 min of incubation with native LYS1. Incubation of complex PGNs with LYS1 yielded the highest immunogenic activity of the digest supernatant after 30 min, suggesting that at that time point the maximum amount of immunogenic PGN fragments was generated. However, prolonged incubation with LYS1 again resulted in a loss of activity with overnight digestion completely abolishing stimulatory activity of the PGN digest. We assume that LYS1 is capable of releasing immunogenic fragments from complex PGNs, but extensive or complete digest into PGN monomers or small PGN fragments appears to abolish the immunogenic activity of PGN fragments. This result is in accordance with our previous observations that prolonged digestion of PGN with mutanolysin diminishes its defense-inducing activity (<xref ref-type="bibr" rid="bib33">Gust et al., 2007</xref>).</p></sec><sec id="s2-5"><title>LYS1 is required for plant immunity towards bacterial infections</title><p>To examine the physiological role of LYS1 in plant immunity, <italic>LYS1</italic><sup><italic>OE</italic></sup> and <italic>LYS1</italic><sup><italic>KD</italic></sup> lines were subjected to infection with various phytopathogens. As LYS1 harbors chitinase activity (<xref ref-type="fig" rid="fig4 fig5">Figures 4A,B and 5B</xref>) and as <italic>LYS1</italic> transcripts accumulate upon fungal infection (<xref ref-type="bibr" rid="bib69a">Samac and Shah, 1991</xref>), we first analyzed the role of LYS1 in immunity towards fungal infection. Leaves of transgenic <italic>LYS1</italic><sup><italic>OE</italic></sup> or <italic>LYS1</italic><sup><italic>KD</italic></sup> lines and wild-type plants were infected with the necrotrophic fungus <italic>Botrytis cinerea</italic> and disease symptoms were monitored 2–3 days post infection. Fungal hyphal growth and necrotic leaf lesions at infection sites were detectable in all plant lines tested and hyphal outgrowth or cell death lesion sizes revealed no differences between wild-type, <italic>LYS1</italic><sup><italic>OE</italic></sup> or <italic>LYS1</italic><sup><italic>KD</italic></sup> lines (<xref ref-type="fig" rid="fig7">Figure 7</xref>). Likewise, infection with the necrotrophic fungus <italic>Alternaria brassicicola</italic> resulted in indistinguishable necrotic lesions in <italic>LYS1</italic><sup><italic>OE</italic></sup> and <italic>LYS1</italic><sup><italic>KD</italic></sup> transgenics compared to those observed in wild-type control plants (<xref ref-type="fig" rid="fig8">Figure 8</xref>). Trypan blue staining and microscopic analysis of the infection sites did not reveal major differences in fungal hyphal growth among all lines tested (<xref ref-type="fig" rid="fig8">Figure 8B,C</xref>). Although disease indices at day 11 after infection were slightly increased in <italic>LYS1</italic><sup><italic>KD</italic></sup> lines (<xref ref-type="fig" rid="fig8">Figure 8D</xref>), such subtle differences were not statistically significant. In conclusion, we failed to detect a role for LYS1 in immunity to fungal infection with <italic>B. cinerea</italic> and <italic>A. brassicicola</italic> under our experimental conditions. However, these results cannot be generalized and LYS1 might still have a role under infection regimes other than the ones used here or it might be important for defense against other fungal pathogens.<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.012</object-id><label>Figure 7.</label><caption><title><italic>LYS1</italic> lines are not impaired in resistance towards infection with <italic>Botrytis cinerea</italic>.</title><p>Five-week-old plants were infected with the necrotrophic fungus <italic>Botrytis cinerea</italic>. 5 μl spore suspension of 5 × 10<sup>5</sup> spores/ml was drop-inoculated on one half of the leaf; two leaves per plant were infected. The plants were analyzed for development of symptoms 2 and 3 days post infection (dpi). (<bold>A</bold>) Trypan blue stain showing visible symptoms after 2 dpi. (<bold>B</bold>) Microscopic analysis of the infection site and fungal hyphae 2 dpi visualized by Trypan blue stain. (<bold>C</bold>) Measurement of lesion size 3 dpi. Shown are means and standard errors (n = 16). No significant differences were observed (Student’s <italic>t</italic> test). The experiment was repeated once with the same result.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.012">http://dx.doi.org/10.7554/eLife.01990.012</ext-link></p></caption><graphic xlink:href="elife01990f007"/></fig><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.013</object-id><label>Figure 8.</label><caption><title><italic>LYS1</italic> mutation does not impinge on resistance towards <italic>Alternaria brassicicola</italic>.</title><p>Five-week-old plants were infected with the necrotrophic fungus <italic>Alternaria brassicicola</italic>. Six 5 μl droplets of a spore suspension of 5 × 10<sup>5</sup> spores/ml were inoculated on the leaf; two leaves per plant were infected. The plants were analyzed for symptom development 7, 11, and 14 days post infection (dpi). (<bold>A</bold>) Visible symptoms of four independent leaves at 14 dpi. (<bold>B</bold>) Disease symptoms 14 dpi visualized by Trypan blue stain. (<bold>C</bold>) Microscopic analysis of the infection site and fungal hyphae 14 dpi visualized by Trypan blue stain. (<bold>D</bold>) Calculation of the disease index at 7, 11, and 14 dpi. Shown are means and standard errors (n = 16). No significant differences were observed (Student’s <italic>t</italic> test). The experiment was repeated once with the same result.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.013">http://dx.doi.org/10.7554/eLife.01990.013</ext-link></p></caption><graphic xlink:href="elife01990f008"/></fig></p><p>To examine the role of LYS1 in immunity to bacterial infection, we infected wild-type plants or <italic>LYS1</italic><sup><italic>KD</italic></sup> and <italic>LYS1</italic><sup><italic>OE</italic></sup> lines with virulent <italic>Pto</italic> DC3000. Two independent <italic>LYS1</italic><sup><italic>KD</italic></sup> lines exhibited hypersusceptibility to bacterial infection (<xref ref-type="fig" rid="fig9">Figure 9A</xref>), suggesting that lack of PGN-degrading activity results in reduced plant immunity. Likewise, immunity to hypovirulent <italic>Pto</italic> DC3000 Δ<italic>AvrPto/PtoB</italic> was compromised in these lines (<xref ref-type="fig" rid="fig9">Figure 9B</xref>). Moreover, expression of the immune marker gene <italic>FRK1</italic> upon administration of complex PGNs was greatly impaired in the <italic>LYS1</italic><sup><italic>KD</italic></sup> mutants (<xref ref-type="fig" rid="fig9">Figure 9C</xref>). These findings suggest that the enzymatic activity of LYS1 on PGN contributes substantially to plant immunity against bacterial infection.<fig-group><fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.014</object-id><label>Figure 9.</label><caption><title>Manipulation of <italic>LYS1</italic> levels causes hypersusceptibility towards bacterial infection and loss of peptidoglycan (PGN)-triggered immune responses.</title><p>(<bold>A</bold> and <bold>B</bold>) Transgenic <italic>LYS1</italic> plants are hypersusceptible to bacterial infection. Growth of <italic>Pseudomonas syringae</italic> pv<italic>. tomato</italic> (<italic>Pto</italic>) DC3000 (<bold>A</bold>) or <italic>Pto</italic> DC3000 Δ<italic>AvrPto/AvrPtoB</italic> (<bold>B</bold>) was determined 2 or 4 days post infiltration of 10<sup>4</sup> colony forming units ml<sup>−1</sup> (cfu/ml). Data represent means ± SD of six replicate measurements/genotype/data point. Representative data of at least four independent experiments are shown. (<bold>C</bold>) Transgenic <italic>LYS1</italic> plants are impaired in PGN-induced immune gene expression. Leaves of wild-type plants or transgenic <italic>LYS1</italic> plants were treated for 6 hr with 100 µg <italic>Bacillus subtilis</italic> PGN and total RNA was subjected to RT-qPCR using <italic>Flagellin responsive kinase</italic> (<italic>FRK1</italic>) specific primers. <italic>EF1α</italic> transcript was used for normalization. Data represent means ± SD of triplicate samples, and shown is the result of one of three independent experiments. Statistical significance compared with wild-type (*p<0.05, Student’s <italic>t</italic> test) is indicated by asterisks.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.014">http://dx.doi.org/10.7554/eLife.01990.014</ext-link></p></caption><graphic xlink:href="elife01990f009"/></fig><fig id="fig9s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01990.015</object-id><label>Figure 9—figure supplement 1.</label><caption><title>Impact of weak <italic>LYS1</italic> overexpression.</title><p>(<bold>A</bold>) Transcript levels of <italic>LYS1</italic> and the peptidoglycan (PGN) receptors <italic>LYM1</italic>, <italic>LYM3</italic>, and <italic>CERK1</italic> in the strong <italic>LYS1</italic> overexpressor line <italic>LYS1</italic><sup><italic>OE</italic></sup>-1 compared with the weak overexpressor line <italic>LYS1</italic><sup><italic>OE</italic></sup>-3. Total RNA from untreated seedlings (top panel) or mature leaves (bottom panel) was subjected to RT-qPCR using specific primers for <italic>LYS1</italic>, <italic>LYM1</italic>, <italic>LYM3</italic>, or <italic>CERK1</italic>. <italic>EF1α</italic> transcript was used for normalization. Data represent means ± SD of triplicate samples. For mature leaves, CERK1 protein levels were also determined using an anti-CERK1 antibody (bottom panel, inset). Ponceau S red staining of the large subunit of RuBisCO served as loading control. (<bold>B</bold>) Immunoblot analysis of protein extracts from the leaves of two independent <italic>LYS1</italic><sup><italic>OE</italic></sup> lines (<italic>LYS1</italic><sup><italic>OE</italic></sup><italic>-1</italic> and <italic>LYS1</italic><sup><italic>OE</italic></sup><italic>-3</italic>) and wild-type plants. Total leaf protein was subjected to western blot analysis using α-tobacco class III chitinase (α-Chit) or green fluorescent protein (α-GFP) (both from rabbit) and an anti-rabbit HRP-coupled secondary antibody. Ponceau S red staining of the large subunit of RuBisCO served as loading control. (<bold>C</bold>) Growth of <italic>Pto</italic> DC3000 was determined 2 days post infiltration of 10<sup>4</sup> colony forming units ml<sup>−1</sup> (cfu/ml). Data represent means ± SD of six replicate measurements/genotype/data point. Statistical significance compared with wild-type (*p<0.05; **p<0.01, Student’s <italic>t</italic> test) is indicated by asterisks. All experiments shown were repeated at least once.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.015">http://dx.doi.org/10.7554/eLife.01990.015</ext-link></p></caption><graphic xlink:href="elife01990fs004"/></fig></fig-group></p><p>Unexpectedly, bacterial growth on <italic>LYS1</italic><sup><italic>OE</italic></sup> lines was also significantly enhanced compared with that observed on wild-type plants (<xref ref-type="fig" rid="fig9">Figure 9A,B</xref>). <italic>FRK1</italic> transcript accumulation upon administration of complex PGN was also strongly reduced in <italic>LYS1</italic> overexpressors (<xref ref-type="fig" rid="fig9">Figure 9C</xref>). To exclude a direct effect of LYS1 overexpression on PGN receptor abundance, we examined transcript levels of <italic>LYM1</italic>, <italic>LYM3</italic>, and <italic>CERK1</italic> but found no effect on the transcription of these receptor genes in the <italic>LYS1</italic><sup><italic>OE</italic></sup> lines (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1A</xref>). Also, CERK1 protein levels were unaltered in the <italic>LYS1</italic><sup><italic>OE</italic></sup> lines, whereas there was no CERK1 protein detectable in the <italic>cerk1-2</italic> mutant (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1A</xref>). Moreover, we included the <italic>LYS1</italic><sup><italic>OE</italic></sup>-3 line with only moderately increased <italic>LYS1</italic> transcript and protein levels in mature leaves (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1A,B</xref>). Susceptibility to <italic>Pseudomonas</italic> infection in the <italic>LYS1</italic><sup><italic>OE</italic></sup>-3 line was only slightly but not significantly increased (p=0.064, Student’s <italic>t</italic> test). These results indicate that lowering <italic>LYS1</italic> expression levels, accompanied by lower LYS1 hydrolytic activity on PGN, brings down these lines close to wild-type. Thus, massive <italic>LYS1</italic> overexpression and loss-of-function mutations are phenocopies of each other, irrespective of the fact that <italic>LYS1</italic><sup><italic>KD</italic></sup> and <italic>LYS1</italic><sup><italic>OE</italic></sup> lines show dramatic differences in LYS1 enzymatic activities (<xref ref-type="fig" rid="fig4">Figure 4</xref>).</p><p>Altogether, we propose that LYS1 contributes to plant immunity to bacterial infection by decomposition of bacterial PGNs and generation of soluble PGN-derived patterns that trigger immune activation in a LYM1-LYM3-CERK1 receptor-complex-dependent manner.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>It is generally little understood whether and how microbial patterns derived from complex extracellular assemblies, such as bacterial cell walls, are accessible to host PRRs for host immune activation in eukaryotes. This holds true for bacterial PGNs, but also for other patterns including bacterial LPS, flagellin, or fungus-derived chitin or glucan structures, all of which have been ascribed triggers of innate immunity in metazoans and plants (<xref ref-type="bibr" rid="bib6">Boller and Felix, 2009</xref>; <xref ref-type="bibr" rid="bib45">Kumar et al., 2013</xref>; <xref ref-type="bibr" rid="bib58">Newman et al., 2013</xref>; <xref ref-type="bibr" rid="bib64">Pel and Pieterse, 2013</xref>). Limited insight into the 3D structure of ligand–PRR complexes, as well as knowledge on ligand structural requirements for plant immune activation, suggests that small ligand epitopes are crucial for binding to host PRRs (<xref ref-type="bibr" rid="bib51">Liu et al., 2012b</xref>; <xref ref-type="bibr" rid="bib80">Sun et al., 2013</xref>). It is thus generally assumed that soluble fragments derived from complex microbial matrices serve as ligands for host PRRs and subsequent immune activation in both lineages.</p><p>Two possible scenarios as to how soluble PGN fragments might be generated from macromolecular assemblies of cross-linked PGNs are discussed. First, during bacterial multiplication and cell wall biogenesis, large portions of soluble PGN fragments are shed into the extracytoplasmic space from which only 50–90% are recycled (<xref ref-type="bibr" rid="bib61">Park and Uehara, 2008</xref>; <xref ref-type="bibr" rid="bib67">Reith and Mayer, 2011</xref>; <xref ref-type="bibr" rid="bib38">Johnson et al., 2013</xref>). This implies that imperfect recycling of bacterial walls might serve as a source of soluble ligands for host PRRs sensing PGN (<xref ref-type="bibr" rid="bib9">Boudreau et al., 2012</xref>; <xref ref-type="bibr" rid="bib88">Wyckoff et al., 2012</xref>). Indeed, muramylpeptides spontaneously shed by <italic>Shigella flexneri</italic> directly stimulate NOD1-dependent immune responses in mammalian immune cells, and bacterial mutants impaired in PGN recycling hyperactivate host immunity (<xref ref-type="bibr" rid="bib59">Nigro et al., 2008</xref>). Second, host lysozyme activity has been demonstrated to generate soluble PGN ligands for NOD2 receptor-mediated immune activation and clearance of <italic>Streptococcus pneumoniae</italic> colonization in mice (<xref ref-type="bibr" rid="bib13">Callewaert and Michiels, 2010</xref>; <xref ref-type="bibr" rid="bib17">Clarke and Weiser, 2011</xref>; <xref ref-type="bibr" rid="bib22">Davis et al., 2011</xref>). Importantly, <xref ref-type="bibr" rid="bib22">Davis et al. (2011)</xref> established a role for host lysozymes in PGN release from bacteria in the absence of detectable bacterial lysis. Likewise, Drosophila Gram-negative bacteria-derived binding protein 1 (GNBP1) was shown to possess PGN-hydrolyzing activity and to deliver fragmented PGN to the PGN sensor, PGRP-SA (<xref ref-type="bibr" rid="bib28">Filipe et al., 2005</xref>; <xref ref-type="bibr" rid="bib84">Wang et al., 2006</xref>). Thus, both passive and active mechanisms of PGN decomposition appear to occur simultaneously during host pathogen encounters and might not be mutually exclusive.</p><p>We here report on a lysozyme-like enzyme (LYS1) that is produced in infected Arabidopsis plants and is capable of generating soluble PGN fragments from complex bacterial PGNs. LYS1 has been demonstrated to hydrolyze β(1,4) linkages between N-acetylmuramic acid and N-acetylglucosamine residues in PGN and between N-acetylglucosamine residues in chitin oligomers, thus closely resembling metazoan lysozymes. LYS1-generated fragments trigger immunity-associated responses in a PGN receptor-dependent manner. Activation of defenses has been further shown to occur in the two plants (Arabidopsis and rice) for which PGN perception systems have been described to date (<xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>; <xref ref-type="bibr" rid="bib50">Liu et al., 2012a</xref>). Importantly, Arabidopsis plants with strongly reduced <italic>LYS1</italic> expression were impaired in immunity to bacterial infection, suggesting strongly that LYS1 function is an important element of the immune system of this plant. Notably, immunocompromised phenotypes in <italic>LYS1</italic><sup><italic>KD</italic></sup> plants were comparable to those observed in either <italic>lym1 lym3</italic> or <italic>cerk1</italic> PGN receptor mutant genotypes (<xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>). We further found that plants overexpressing LYS1 were also susceptible to bacterial infections, suggesting that defined LYS1 levels in wild-type plants are required for LYS1 immune function. The most compelling explanation for this phenotype is that PGN hyperdegradation (in <italic>LYS1</italic><sup><italic>OE</italic></sup> plants) or lack of PGN degradation (in <italic>LYS</italic><sup><italic>KD</italic></sup> mutants) are equally disadvantageous to plant immunity and that immune activation in Arabidopsis requires oligomeric PGN fragments of a particular minimum degree of polymerization (DP). This view is supported by our findings that prolonged digestion of PGN by LYS1 (<xref ref-type="fig" rid="fig6">Figure 6D</xref>) or by mutanolysin (<xref ref-type="bibr" rid="bib33">Gust et al., 2007</xref>) abolished the immunogenic activity of PGN. Likewise, immunogenic activities of fungal chitin or oomycete glucans have been reported to require defined minimum ligand sizes with a minimum DP of >5 (<xref ref-type="bibr" rid="bib14">Cheong et al., 1991</xref>; <xref ref-type="bibr" rid="bib91">Zhang et al., 2002</xref>). We therefore propose that <italic>LYS1</italic> overexpression might result in PGN fragments of insufficient size, thereby mimicking the physiological status in <italic>LYS1</italic><sup><italic>KD</italic></sup> mutants lacking major PGN hydrolytic activities.</p><p>Plants produce various carbohydrate-degrading hydrolytic enzyme activities, some of which have been implicated in plant immunity to microbial infection, such as glucanases and chitinases (<xref ref-type="bibr" rid="bib82">van Loon et al., 2006</xref>). While it is often not entirely clear how these enzymes contribute to plant immunity, it is widely assumed that this is due to microcidal activities of these proteins. In our study we have shown that Arabidopsis LYS1 cleaves O-glycosidic bonds formed between GlcNAc (indicative of chitinolytic activity) as well as those formed between GlcNAc and MurNAc (indicative of peptidoglycanolytic activity). However, we have been unable to demonstrate any deleterious effect of LYS1 overexpression on fungal infections, suggesting that <italic>B. cinerea</italic> and <italic>A. brassicicola</italic> at least are not affected by LYS1 function. Likewise, we have been unable to demonstrate direct bactericidal activity of LYS1 to <italic>P. syringae</italic> (not shown), suggesting that the positive role of LYS1 in plant immunity to bacterial infection is not due to its direct inhibitory effect on bacterial fitness. This view is further supported by the fact that <italic>LYS1</italic><sup><italic>OE</italic></sup> plants with strongly enhanced PGN hydrolytic activity do not exhibit enhanced immunity to <italic>Pseudomonas</italic> infections but become hypersusceptible to infection (<xref ref-type="fig" rid="fig9">Figure 9</xref>). We cannot rule out at this point LYS1-mediated bacterial lysis, which would likely also result in the release of immunogenic PGN fragments. We would like to emphasize, however, that our findings are in agreement with a predominant role of LYS1 in the generation of PGN fragments that subsequently can trigger plant immunity via PRRs. Hence, plant LYS1 functionally resembles recently described mammalian lysozymes that were shown to generate soluble PGN fragments for PGN receptor NOD2, thereby mediating immunity to <italic>S. pneumoniae</italic> infection in mice (<xref ref-type="bibr" rid="bib22">Davis et al., 2011</xref>).</p><p><italic>LYS1</italic> gene expression is strongly enhanced upon PAMP administration or bacterial infection while expression levels in naive plants are low. It is conceivable that the low constitutive LYS1 levels are sufficient to generate soluble PGN fragments from bulk PGN-containing bacterial walls which are then perceived via the LYM1-LYM3-CERK1 receptor complex. It is possible that the pathogen-inducible later increase in LYS1 activity could have further roles for generating diffusible signals that might serve innate immune activation, not only in cells that are directly in contact with invading microbes but also in cell layers adjacent to infection sites.</p><p>A role for plant glycosyl hydrolases in immunogenic PAMP generation and immune activation has been proposed previously (<xref ref-type="bibr" rid="bib54">Mithöfer et al., 2000</xref>; <xref ref-type="bibr" rid="bib29">Fliegmann et al., 2004</xref>). An extracellular soluble bipartite soybean glucan binding protein (GBP) was shown to harbor 1,3-β-glucanase activity and binding activity for glucan fragments of DP >6 derived from intact glucans. Complex glucans constitute major constituents of various <italic>Phytophthora</italic> species, many of which are plant pathogens (<xref ref-type="bibr" rid="bib44">Kroon et al., 2011</xref>). It was therefore suggested that, during infection, GBP endoglucanase activity produces soluble <italic>Phytophthora</italic>-derived oligoglucoside fragments as ligands for the high-affinity binding site within this protein (<xref ref-type="bibr" rid="bib29">Fliegmann et al., 2004</xref>). While this study supported the concept of plant hydrolases tailor-making ligands for plant PRRs, causal evidence for the involvement of the endoglucanase activity in plant immunity was not provided.</p><p>Eukaryotic PGN recognition proteins (PGRP, PGLYRP) are conserved from insects to mammals, bind PGN, and function in antibacterial immunity (<xref ref-type="bibr" rid="bib16">Cho et al., 2005</xref>; <xref ref-type="bibr" rid="bib4">Bischoff et al., 2006</xref>; <xref ref-type="bibr" rid="bib25">Dziarski and Gupta, 2010</xref>; <xref ref-type="bibr" rid="bib46">Kurata, 2010</xref>, <xref ref-type="bibr" rid="bib47">2014</xref>). Some PGRP family members are non-enzymatic PRRs (NOD1, NOD2) while others possess PGN-degrading activities (<xref ref-type="bibr" rid="bib30">Gelius et al., 2003</xref>; <xref ref-type="bibr" rid="bib85">Wang et al., 2003</xref>; <xref ref-type="bibr" rid="bib4">Bischoff et al., 2006</xref>; <xref ref-type="bibr" rid="bib25">Dziarski and Gupta, 2010</xref>; <xref ref-type="bibr" rid="bib46">Kurata, 2010</xref>). PGN hydrolytic enzyme activities such as lysozymes have been ascribed functions in direct bacterial killing (<xref ref-type="bibr" rid="bib16">Cho et al., 2005</xref>) and in generating soluble PGN fragments as ligands for PRRs (<xref ref-type="bibr" rid="bib84">Wang et al., 2006</xref>; <xref ref-type="bibr" rid="bib22">Davis et al., 2011</xref>). LYS1 constitutes the first plant lysozyme-type activity for which a role in host immunity has been established. LYS1 is capable of generating immunogenic fragments from complex PGNs, which themselves serve as ligands for the LYM1-LYM3-CERK1-PGN recognition complex in Arabidopsis. It is noteworthy that LYM1 and LYM3 are PGN recognition proteins that lack apparent intrinsic PGN-degrading activity. We conclude that metazoans and plants employ hydrolytic activities for the decomposition of bacterial PGNs during host immune activation. In addition to the established role of PGNs in pattern-triggered immune activation, host-mediated degradation of bacterial PGNs constitutes another conserved feature of innate immunity in both lineages. However, as the molecular components involved differ structurally among phyla, both facets of PGN-mediated immunity might have evolved convergently.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Plant growth conditions and infections</title><p><italic>A. thaliana</italic> Columbia-0 wild-type and <italic>N. benthamiana</italic> plants were grown on soil as previously described (<xref ref-type="bibr" rid="bib10">Brock et al., 2010</xref>). T-DNA insertion lines for <italic>LYS1</italic> (<italic>lys1-1</italic>, WiscDsLox387C11; <italic>lys1-</italic>2, SALK_095362; <italic>lys1-3</italic>, CSHL_ET14179) were obtained from the Nottingham Arabidopsis Stock Centre. The transgenic <italic>pPR1::GUS</italic> and <italic>secGFP</italic> lines and the <italic>lym1 lym3</italic> and <italic>cerk1-2</italic> mutants have been described previously (<xref ref-type="bibr" rid="bib75">Shapiro and Zhang, 2001</xref>; <xref ref-type="bibr" rid="bib81">Teh and Moore, 2007</xref>; <xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>). Rice (<italic>Oryza sativa</italic>) suspension cell cultures were grown in MS-medium (4.41 g/l MS salt, 6% [wt/vol] sucrose, 50 mg/l MES, 2 mg/l 2,4-D) at 150 rpm and sub-cultured every week. Bacterial strains <italic>P. syringae</italic> pv. <italic>tomato</italic> DC3000 or <italic>Pto</italic> DC3000 <italic>ΔAvrPto/AvrPto</italic>, <italic>A. brassicicola</italic> isolate MUCL 20297, and <italic>B. cinerea</italic> isolate BO5-10 were grown and used for infection assays on Arabidopsis leaves of 4–5-week-old plants as described previously (<xref ref-type="bibr" rid="bib49">Lin and Martin, 2005</xref>; <xref ref-type="bibr" rid="bib43">Kemmerling et al., 2007</xref>). To visualize plant cell death and fungal growth on a cellular level, infected plants were stained with Trypan blue in lactophenol and ethanol as described elsewhere (<xref ref-type="bibr" rid="bib43">Kemmerling et al., 2007</xref>).</p></sec><sec id="s4-2"><title>Materials</title><p>Flg22 peptide has been described previously (<xref ref-type="bibr" rid="bib27">Felix et al., 1999</xref>). The purification of <italic>P. syringae</italic> pv. <italic>tomato</italic> PGN was performed as described previously (<xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>). <italic>M. luteus</italic> cell wall preparations and <italic>B. subtilis</italic> PGN were purchased from Invivogen (San Diego, California, United States), Cecolabs (Tübingen, Germany), and Sigma-Aldrich (Hamburg, Germany). PGNs and LPS (from <italic>P. aeruginosa</italic>, Sigma-Aldrich) were dissolved in water at a concentration of 10 mg/ml and stored at −20°C. Mutanolysin was purchased from Sigma-Aldrich.</p></sec><sec id="s4-3"><title>Constructs and transgenic lines</title><p>Recombinant His6-LYM1 and His6-LYM3 were expressed in <italic>E. coli</italic> and purified as previously described (<xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>). As negative control, a protein purification using non-induced cultures harboring the His6-LYM3 construct was performed.</p><p>For the <italic>p35S::LYS1</italic> fusion constructs, a 903 bp fragment of the <italic>LYS1</italic> coding sequence without STOP codon was cloned using the primers At5g24090gatF and At5g24090gatR (<xref ref-type="table" rid="tbl1">Table 1</xref>). In a second PCR, the recombination sites of the inserts were completed using the Gateway adaptor primers attB1 and attB2 (Invitrogen, Darmstadt, Germany). The resulting fragments were then subcloned into pDONR201 (Invitrogen) by using the BP clonase reaction according to the manufacturer’s protocol (Invitrogen) and inserted into the binary expression vectors pK7FWG2.0 (C-terminal GFP-tag) (<xref ref-type="bibr" rid="bib42">Karimi et al., 2002</xref>, <xref ref-type="bibr" rid="bib41">2005</xref>) or pGWB17 (C-terminal myc-tag) (<xref ref-type="bibr" rid="bib57">Nakagawa et al., 2007</xref>) by using the LR clonase reaction following the manufacturer’s protocol (Invitrogen). For the <italic>pLYS1::GUS</italic> reporter construct, a 1948 bp fragment of the <italic>LYS1</italic> promoter sequence was amplified from Arabidopsis Col-0 genomic DNA using the primers At5g24090gatF2 and At5g24090gatR2 (<xref ref-type="table" rid="tbl1">Table 1</xref>), extended in a second PCR with Gateway adaptor primers attB1 and attB2 and subcloned into pDONR207 (Invitrogen) before being inserted into the binary expression vector pBGWFS7 (<xref ref-type="bibr" rid="bib42">Karimi et al., 2002</xref>, <xref ref-type="bibr" rid="bib41">2005</xref>).<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01990.016</object-id><label>Table 1.</label><caption><p>Primers used in this study</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01990.016">http://dx.doi.org/10.7554/eLife.01990.016</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th><italic>AGI</italic></th><th>Primer name</th><th>Sequence 5′ → 3′</th></tr></thead><tbody><tr><td rowspan="23"><italic>At5g24090 (LYS1)</italic></td><td>At5g24090F1</td><td>CCAGAGGTGGCATAGCCATC</td></tr><tr><td>At5g24090R1</td><td>CATCTGGTGGGATATAGCCAC</td></tr><tr><td>At5g24090F</td><td>ATGACCAACATGACTCTTCG</td></tr><tr><td>At5g24090R</td><td>TCACACACTAGCCAATATAG</td></tr><tr><td>At5g24090RP2</td><td>TGATGCCACGAGACTGAC</td></tr><tr><td>LP_N853931</td><td>TGACGAACCATGATAAATGGG</td></tr><tr><td>RP_N853931</td><td>CATAACCTCACACTGTGCTCG</td></tr><tr><td>LP_N595362</td><td>TAGTGCATGCATGTTAAACCG</td></tr><tr><td>RP_N595362</td><td>AGCTCCTCAATGTCCATTTCC</td></tr><tr><td>Salk-Lba</td><td>TGGTTCACGTAGTGGGCCATCG</td></tr><tr><td>Ds5-1</td><td>GAAACGGTCGGGAAACTAGCTCTAC</td></tr><tr><td>Wisc-Lba (p745)</td><td>AACGTCCGCAATGTGTTATTAAGTTGTC</td></tr><tr><td>At5g24090Fq</td><td>CACTTGCACCCATTTTGGC</td></tr><tr><td>At5g24090Rq</td><td>CCTCGACCCAATCGAGTA</td></tr><tr><td>At5g24090miR-s</td><td>GATTTGACGTAAGCATACCGCCCTCTCTCTTTTGTATTCC</td></tr><tr><td>At5g24090miR-a</td><td>GAGGGCGGTATGCTTACGTCAAATCAAAGAGAATCAATGA</td></tr><tr><td>At5g24090miR*s</td><td>GAGGACGGTATGCTTTCGTCAATTCACAGGTCGTGATATG</td></tr><tr><td>At5g24090miR*s</td><td>GAATTGACGAAAGCATACCGTCCTCTACATATATATTCCT</td></tr><tr><td>At5g24090gatF</td><td>AAAAAGCAGGCTACATGACCAACATGACTCTTCG</td></tr><tr><td>At5g24090gatR</td><td>AGAAAGCTGGGTACACACTAGCCAATATAGATG</td></tr><tr><td>At5g24090gatR-STOP</td><td>AGAAAGCTGGGTATCACACACTAGCCAATATAG</td></tr><tr><td>At5g24090gatF2</td><td>AAAAAGCAGGCTATGCCGTAGGCGAGTGTTTC</td></tr><tr><td>At5g24090gatR2</td><td>AGAAAGCTGGGTGTTTTTGGTTAAAGATGTTTG</td></tr><tr><td rowspan="2"><italic>At1g07920/30/40(EF1α)</italic></td><td>Ef1α-100-f</td><td>GAGGCAGACTGTTGCAGTCG</td></tr><tr><td>Ef1α-100-r</td><td>TCACTTCGCACCCTTCTTGA</td></tr><tr><td rowspan="2"><italic>At2g19190 (FRK1)</italic></td><td>FRK1-F</td><td>AAGAGTTTCGAGCAGAGGTTGAC</td></tr><tr><td>FRK1-R</td><td>CCAACAAGAGAAGTCAGGTTCGTG</td></tr><tr><td rowspan="2"><italic>At4g02540</italic></td><td>At4g02540-qf1</td><td>GTACCACGCCTATCTATT</td></tr><tr><td>At4g02540-qr1</td><td>CTCATAGAAGAAACCAGCA</td></tr><tr><td rowspan="2"><italic>At1g05615</italic></td><td>At1g05615-qf1</td><td>GGATTCCTATCTCTACCT</td></tr><tr><td>At1g05615-qr1</td><td>TTCTTTACCCTCATCAACC</td></tr><tr><td rowspan="2"><italic>At5g58780</italic></td><td>At5g58780-qf1</td><td>CTCTCTTCTCTTTTATCTCTCC</td></tr><tr><td>At5g58780-qr1</td><td>CTCCTCCACTCCTACCACA</td></tr><tr><td rowspan="2"><italic>At3g51010</italic></td><td>At3g51010-qf1</td><td>GCGTCGTGCTTTTATACTG</td></tr><tr><td>At3g51010-qr1</td><td>TTCTTCCTCTTCGCCTCT</td></tr><tr><td rowspan="2"><italic>At1g21880 (LYM1)</italic></td><td>Lym1-100-f</td><td>TACAACGGTATAGCCAACGGCACT</td></tr><tr><td>Lym1-100-r</td><td>GTGGAGCTAGAAGCGGCGCA</td></tr><tr><td rowspan="2"><italic>At1g77630 (LYM3)</italic></td><td>Lym3-100-f</td><td>ACTTCGCAGCAGAGTAGCTC</td></tr><tr><td>Lym3-100-r</td><td>AGCGGTGCTAATTGTTGCGG</td></tr><tr><td rowspan="2"><italic>At3g21630 (CERK1)</italic></td><td>CERK1-100-f</td><td>GGGCAAGGTGGTTTTGGGGCT</td></tr><tr><td>CERK1-100-r</td><td>CCGCCAAGAACTGTTTCGATGCC</td></tr><tr><td/><td>attB1</td><td>GGGGACAACTTTGTACAAAAAAGCAGGCT</td></tr><tr><td/><td>attB2</td><td>GGGGACCACTTTGTAC AAGAAAGCTGGGT</td></tr></tbody></table></table-wrap></p><p>For the generation of <italic>pLYS1::GUS</italic> and <italic>p35S::LYS1-GFP</italic> overexpression lines (<italic>LYS1</italic><sup><italic>OE</italic></sup>), wild-type Col-0 plants were transformed. Stable transgenic lines were generated using standard <italic>Agrobacterium tumefaciens</italic>-mediated gene transfer by the floral dip procedure (<xref ref-type="bibr" rid="bib18">Clough and Bent, 1998</xref>). Expression of GFP fusion proteins was confirmed by immunoblot analysis using an anti-GFP antibody (Acris Antibodies GmbH) and anti-tobacco class III chitinase antibody (kindly provided by Michel Legrand, IBMP Strasbourg, France). The histochemical detection of β-glucuronidase (GUS) enzyme activity in whole leaves of <italic>pLYS1::GUS</italic> or <italic>pPR-1::GUS</italic> transgenic Arabidopsis (<xref ref-type="bibr" rid="bib75">Shapiro and Zhang, 2001</xref>) was determined as described earlier (<xref ref-type="bibr" rid="bib33">Gust et al., 2007</xref>).</p><p>Artificial microRNA-mediated gene silencing was used to specifically knock down <italic>LYS1</italic> in the Col-0 background as mutant lines carrying T-DNA insertions in the <italic>LYS1</italic> gene were unavailable. The Web microRNA Designer (WMD; <ext-link ext-link-type="uri" xlink:href="http://wmd.weigelworld.org/">http://wmd.weigelworld.org</ext-link>) was used to select the primers At5g24090miR-s, At5g24090miR-a, At5g24090miR*s, and At5g24090miR*s (<xref ref-type="table" rid="tbl1">Table 1</xref>) for the generation of an artificial 21mer microRNA (<xref ref-type="bibr" rid="bib73">Schwab et al., 2005</xref>). The <italic>LYS1</italic>-specific amiRNA was then introduced into the vector miR319a pBSK (pRS300) by directed mutagenesis. Knock-down of the <italic>LYS1</italic> transcript level in stably transformed Col-0 plants (<italic>LYS1</italic> knock-down line, <italic>LYS1</italic><sup><italic>KD</italic></sup>) was determined by RT-qPCR using primers At5g24090Fq and At5g24090Rq listed in <xref ref-type="table" rid="tbl1">Table 1</xref>. Off-target genes were identified using the Web microRNA Designer and transcript levels of the four top hits were determined by RT-qPCR using primers listed in <xref ref-type="table" rid="tbl1">Table 1</xref>.</p></sec><sec id="s4-4"><title>Transient protein expression</title><p><italic>A. tumefaciens</italic>-mediated transient transformation of <italic>N. benthamiana</italic> was performed as described previously (<xref ref-type="bibr" rid="bib10">Brock et al., 2010</xref>). The leaves were examined for expression of tagged fusion proteins 3–4 days post infection. Expression of fusion proteins was confirmed by immunoblot analysis using anti-myc antibodies (Sigma-Aldrich) and localization studies of GFP fusion proteins were carried out using a confocal laser-scanning microscope, as described elsewhere (<xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>).</p></sec><sec id="s4-5"><title>LYS1 purification from <italic>LYS1</italic><sup><italic>OE</italic></sup> plants</title><p>From 5-week-old <italic>LYS1</italic><sup><italic>OE</italic></sup> Arabidopsis plants, 100 g leaf tissue was frozen in liquid nitrogen and ground to fine powder. After addition of buffer A (20 mM sodium acetate, pH 5.2, 0.01% [vol/vol] β-mercaptoethanol), the extract was incubated on ice overnight. After filtration through four layers of cheesecloth, the homogenate was centrifuged at 10,000× g for 30 min. The supernatant was loaded on a cation exchange column (SP Sepharose, GE Healthcare, München, Germany) equilibrated with buffer A. The column was washed with buffer A and proteins were eluted with a 0 to 1 M NaCl gradient in buffer A. The elution fractions were monitored for LYS1 activity with the 4-MUCT assay and protein purification was further confirmed by SDS-PAGE. 4-MUCT-active fractions were pooled and exchanged to buffer A using Vivaspin 3 kDa columns (GE Healthcare). Protein concentration was determined using the Bradford assay.</p><p>For LC-MS analysis, the Coomassie Blue-stained band of the major cleavage product of the purified LYS1-GFP sample was cut and in-gel digested with trypsin, as described elsewhere (<xref ref-type="bibr" rid="bib8">Borchert et al., 2010</xref>). LC-MS analyses of the peptides were done on an EasyLC nano-HPLC (Proxeon Biosystems) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific) as described elsewhere (<xref ref-type="bibr" rid="bib19">Conzelmann et al., 2013</xref>). MS data were processed using the software suite MaxQuant, version 1.2.2.9 (<xref ref-type="bibr" rid="bib20">Cox and Mann, 2008</xref>) and searched using Andromeda search engine (<xref ref-type="bibr" rid="bib21">Cox et al., 2011</xref>) against a target-decoy <italic>A. thaliana</italic> database containing 33,351 forward protein sequences, the sequence of the LYS1-GFP fusion protein, and 248 frequently observed protein contaminants. MS data were processed twice, once considering only fully tryptic peptides and once considering only semi-tryptic peptides. In each case, two missed cleavage sites were allowed, carbamidomethylation of cysteine was set as the fixed modification, and N-terminal acetylation and methionine oxidation were set as variable modifications. Mass tolerance was set to 6 parts per million (ppm) at the precursor ion and 20 ppm at the fragment ion level. Identified peptide spectrum matches (PSM) were statistically scored by MaxQuant software by calculation of posterior error probabilities (PEP) (<xref ref-type="bibr" rid="bib40">Käll et al., 2008</xref>) for each PSM. All PSMs having a PEP below 0.01 were considered as valid.</p><p>For matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), protein digestion was performed as described elsewhere (<xref ref-type="bibr" rid="bib53">Maurer et al., 2013</xref>; <xref ref-type="bibr" rid="bib1">Amin et al., 2014</xref>). Briefly, the Coomassie Blue-stained band of the major cleavage product of the FPLC-purified LYS1-GFP sample was cut from the gel and destained with 30% (vol/vol) acetonitrile in 50 mM ammonium bicarbonate buffer. Disulfide bonds were reduced with 10 mM dithiothreitol (DTT), 50 mM iodoacetamide was used to alkylate the cysteines followed by overnight protein digestion with mass spectrometry grade trypsin (Promega, Manheim, Germany) at 37°C. The digests were acidified by the addition of trifluoric acid (TFA) to a final concentration of 0.5%. Extracted peptides were desalted and mixed with an equal volume of 2,5-dihydroxybenzoic acid for Reflex-IV MALDI-TOF-MS (Bruker Daltonics, Bremen, Germany) measurements. Each spectrum was processed internally for trypsin autolysis before database search. The identity of protein was annotated using the SwissProt database (542782 sequences; 193019802 residues). To achieve the best possible results, the search parameters were as follows: one miscleavage was set for trypsin specificity and carbamidomethyl modification of cysteine and oxidation of methionine were selected as fixed and optional modifications, respectively. At a mass tolerance of 5 ppm, only protein scores greater than 70 (p<0.05) were assigned significant with an expected value of 10<sup>−7</sup>.</p></sec><sec id="s4-6"><title>Protein extraction and enzymatic assays</title><p>Apoplastic washes were obtained from mature leaves of 4-week-old Arabidopsis plants by vacuum-infiltrating complete rosettes with 20 mM sodium acetate, pH 5.2. Afterwards, leaf tissue was dipped dry on paper towels, placed in 50 ml Falcon tubes and spun at 1000× g for 5 min at 4°C. Collected fluids were concentrated tenfold using Vivaspin 500 columns with a 3 kDa cut-off (GE Healthcare).</p><p>Isolation of mesophyll protoplasts from leaves of 4–5-week-old Arabidopsis plants was performed according to a protocol described previously (<xref ref-type="bibr" rid="bib90">Yoo et al., 2007</xref>). Isolated protoplasts were resuspended in W5 solution (2 mM MES, pH 5.7, 154 mM sodium chloride, 125 mM calcium chloride, 5 mM potassium chloride) and incubated overnight at room temperature in the dark (2 × 10<sup>5</sup> protoplasts in 1 ml W5 solution). Subsequently, protoplasts were removed by centrifugation (20 s, 800 rpm, 4°C) and secreted proteins in the medium were concentrated using Vivaspin 2 columns with a 10 kDa cut-off (GE Healthcare).</p><p>Total protein extracts from the harvested protoplast pellet of 4–5-week-old leaves of <italic>A. thaliana</italic> or <italic>N. benthamiana</italic> were prepared using 20 mM sodium acetate, pH 5.2, supplemented with 15 mM β-mercaptoethanol and proteinase inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Approximately 40–60 µg total protein of the leaf extracts or 15 µg of the protoplast samples were added to the enzyme assays. For all in-tube enzyme assays described in the supplemental information, the reaction mix was incubated with shaking at 37°C in 20 mM sodium acetate, pH 5.2.</p><p>The 4-MUCT chitinase assay was performed as described (<xref ref-type="bibr" rid="bib12">Brunner et al., 1998</xref>). Briefly, the hydrolytic activity towards 4-MUCT (Sigma-Aldrich) was measured for 30 min and compared with that of 2 µg <italic>S. griseus</italic> chitinase (Sigma-Aldrich). After enzyme incubation in 250 µl final volume of 0.05% (wt/vol) 4-MUCT, 20 µl of the reaction mixture were removed and added to 980 µl 0.2 M sodium carbonate solution. Free 4-MU (Sigma-Aldrich) was used for the generation of a standard curve. The intensity of the fluorescence was monitored with an MWG Sirius HT fluorescence microplate reader. For the zymogram, discontinuous cetyltrimethylammonium bromide (CTAB) polyacrylamide gel electrophoresis was performed using a 12% separating gel (43 mM potassium hydroxide [KOH], 280 mM acetic acid, pH 4.0, 12% [vol/vol] acrylamide bisacrylamide 37.5:1, 8% [vol/vol] glycerol, 1.3% ammonium persulphate and 0.16% N, N, N, N-tetramethylethylene diamine [TEMED]) overlaid by a 4% stacking gel (64 mM KOH, 94 mM acetic acid, pH 5.1, 4% acrylamide, 1.25% ammonium persulphate and 0.125% TEMED). Prior to loading, the gel was pre-run using anode buffer (40 mM beta-alanine, 70 mM acetic acid, 0.1% CTAB, pH 4.0) and cathode buffer (50 mM KOH, 56 mM acetic acid, pH 5.7, 0.1% CTAB) for 1 hr at 250 Volts. Crude protein extracts were mixed with an equal volume of loading buffer (5 M urea, 25 mM potassium acetate, pH 6.8, methylene blue) and separated for 2 hr at 150 Volts and 4°C. After electrophoresis the CTAB gel was washed with 20 mM sodium acetate, then sprayed with 0.00625% (wt/vol) 4-MUCT in 20 mM sodium acetate, pH 5.2, and incubated at 37°C for 30 min. Fluorescent bands were documented under UV light using the Infinity-3026WL/26MX gel imaging system (PeqLab, Erlangen, Germany).</p><p>The turbidity assay was done as described previously (<xref ref-type="bibr" rid="bib62">Park et al., 2002</xref>). Lytic activity towards <italic>M. luteus</italic> cell wall preparations or <italic>B. subtilis</italic> peptidoglycan (Invivogen, Cecolabs) was measured for 4 hr and compared with that of 1 µg hen egg-white lysozyme (Sigma-Aldrich). 1 ml 0.02% (wt/vol) <italic>M. luteus</italic> cells or PGN suspension were incubated together with the enzyme and the decrease in absorbance at 570 nm of the suspension was measured with a spectrophotometer over time.</p><p>The 4-MUC cellulase assay was performed using 4-methylumbelliferyl-β-D-cellobioside (4-MUC; Sigma-Aldrich) as substrate. 1 mM 4-MUC was incubated in 20 mM sodium acetate (pH 5.2) at 37°C for 1 hr in a 96 well plate with either 40 µg purified LYS1 or cellulase (Duchefa, Haarlem, The Netherlands) in a total volume of 100 µl. The reaction was stopped with 0.2 M sodium carbonate and the intensity of the fluorescence was monitored with an MWG Sirius HT fluorescence microplate reader using excitation and emission wavelengths of 365 nm and 455 nm, respectively.</p></sec><sec id="s4-7"><title>HPLC analysis</title><p>500 µg/ml <italic>B. subtilis</italic> PGN was incubated with 140 µg LYS1 purified from <italic>LYS1</italic><sup><italic>OE</italic></sup> plants or controls in 20 mM sodium acetate, pH 5.2, at 37°C with shaking for 7 hr. After stopping the reaction by heating at 100°C for 10 min, the reaction was centrifuged and the supernatant analyzed by HPLC. The analyses were done by Cecolabs on an Agilent 1200 system with a Prontosil C18-RP column (Bischoff Chromatography, Leonberg, Germany). The mobile phase was (A) 100 mM sodium phosphate, 5% (vol/vol) methanol and (B) 100 mM sodium phosphate, 30% (vol/vol) methanol.</p></sec><sec id="s4-8"><title>Immune responses</title><p>RNA isolation, semi-quantitative RT-PCR and RT-qPCR analysis were performed as described previously (<xref ref-type="bibr" rid="bib43">Kemmerling et al., 2007</xref>; <xref ref-type="bibr" rid="bib86">Willmann et al., 2011</xref>). For RT-qPCR, all quantifications were made in duplicate on RNA samples obtained from three independent experiments, each performed with a pool of 3–5 seedlings or two leaves. <italic>EF1α</italic> transcripts served normalization; corresponding water controls were set to 1. The sequences of the primers used for PCR amplifications are given in <xref ref-type="table" rid="tbl1">Table 1</xref>. The histochemical detection of β-glucuronidase (GUS) enzyme activity in whole leaves of <italic>pLYS1::GUS</italic> or <italic>pPR-1::GUS</italic> transgenic Arabidopsis (<xref ref-type="bibr" rid="bib75">Shapiro and Zhang, 2001</xref>) was determined as described earlier (<xref ref-type="bibr" rid="bib33">Gust et al., 2007</xref>). For the measurement of extracellular pH, 300 µl of cultured rice cells were transferred to 48 well plates and equilibrated at 150 rpm for 30 min. After addition of elicitors, the pH in the cell culture was monitored with an InLab Micro electrode (Mettler Toledo, Gießen, Germany).</p><p>For assays with LYS1-digested PGN, 100 µg/ml <italic>B. subtilis</italic> PGN was incubated with 40 µg LYS1 purified from <italic>LYS1</italic><sup><italic>OE</italic></sup> plants or controls in 2.5 mM MES, pH 5.2, at 37°C with shaking for 4 hr. After stopping the reaction by heating at 100°C for 10 min, the reaction was centrifuged and the supernatant used for triggering immune responses.</p></sec><sec id="s4-9"><title>Statistical methods</title><p>Statistical significance between two groups has been checked using the Student’s <italic>t</italic> test. Asterisks represent significant differences (*p<0.05; **p<0.01; ***p<0.001). One-way analysis of variance (ANOVA) was performed for multiple comparisons combined with Duncan’s multiple range test indicating significant differences with different letters (p<0.05).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Andreas Kulik and Friedrich Götz for bacterial fermentation and Gary Stacey and Michel Legrand for providing the anti-CERK1 and anti-class III chitinase antibody, respectively.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>TN: Reviewing editor, <italic>eLife</italic>.</p></fn><fn fn-type="conflict" id="conf2"><p>The other authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>XL, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con2"><p>HMG, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>RW, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con4"><p>DK, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>UB, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>DK, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>MF-W, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con8"><p>BA, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con9"><p>FB, Conception and design, Drafting or revising the article</p></fn><fn fn-type="con" id="con10"><p>GF, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con11"><p>MO, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con12"><p>TN, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con13"><p>AAG, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Amin</surname><given-names>B</given-names></name><name><surname>Maurer</surname><given-names>A</given-names></name><name><surname>Voelter</surname><given-names>W</given-names></name><name><surname>Melms</surname><given-names>A</given-names></name><name><surname>Kalbacher</surname><given-names>H</given-names></name></person-group><year>2014</year><article-title>New poteintial serum biomarkers in multiple sclerosis identified by 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T</given-names></name><role>Reviewing editor</role><aff><institution>University of Chicago</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Jean Greenberg (Reviewing editor).</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The authors are interested in whether plants help generate soluble MAMPs from pathogens that can then be detected by plant receptors to induced defenses. They focus on LYS1, which they show can generate PGN fragments; the LYS1 gene is induced by various MAMPs and “Non-adapted” or non-pathogenic <italic>P. syringae</italic>. Recombinant LYS1 is not active, so they overexpress LYS1-GFP in plants and use this as a source for enzyme to characterize. They also characterize the overexpression plants and knockdowns. The biochemical effects are for the most part convincing but might be more so if they were presented as specific activities with some kinetic analyses that were compared with the known enzymes they use. A major finding is that the plants with OE and KD seem to have the same phenotypes with respect to hypersusceptibility to <italic>P. syringae</italic> and lack of MAMP response (at least FRK1 expression) with PGN treatment. Several very important issues came up in the review process that need to be addressed:</p><p>1) Claims of specificity: Since LYS1 has chitinolytic activity, there is the potential that this activity could be playing a role in anti-fungal defense. Although the authors tested this, they can’t exclude that with certain fungal isolate/host combinations LYS1 might have a role in fungal defense. Therefore, we advise toning down the claim of specificity for responses to bacteria.</p><p>2) Organization and analysis methods: Figure panels should be organized in the order they are discussed. Add something about how many times each experiment was replicated. Statistical analysis needs to be improved. In complex experiments where multiple comparisons can/should be made, use ANOVA and multiple comparison post hoc test. It’s especially difficult to understand the comparisons made in 3E and <xref ref-type="fig" rid="fig5">Figure 5</xref>.These data should all be analysed together and grouped into significance groups.</p><p>3) Concerning the KD and overexpression plants: the specificity of the artificial miRNA needs to be established. The authors need to show that only the LYS1 gene is being silenced and not other genes that may have some sequence similarity. Because the results of <xref ref-type="fig" rid="fig8">Figure 8</xref> are confusing, it would be good to use a knockout plant such as a SALK mutant (e.g. SALK_095362). <xref ref-type="fig" rid="fig2">Figure 2E</xref> is the only example where the KD seems to affect the relative PGN hydrolysis activity. Therefore, it’s especially important to do the correct statistical analysis. The effect of the KD would be more convincing if we had conditions where the level of protein in WT can be detected (or maybe one of the bands in D in the Col lane is right?). Did the authors try using apoplast washes to enrich for the activity? How about inducing conditions (e.g. MAMP treatment?) Since LYS expression is normally PAMP-induced, it is important to show that a PAMP-induced increase in PGN-degrading activity is decreased in extracts from <italic>LYS1-KD</italic> lines, when compared to extracts from WT plants.</p><p>The overexpression lines with 300 fold higher than wild type seems way too high (<xref ref-type="fig" rid="fig2">Figure 2B</xref>) and could be causing unforeseen effects. At a minimum the authors should check that the consequence of OE is not causing downregulation of the receptors that perceive the PGNs. If this were the case, it would change the interpretation of the experiments. Some lines where the overexpression is more modest would be helpful for determining if increased resistance can be achieved. In any case with <xref ref-type="fig" rid="fig8">Figure 8.C</xref>, the authors should check the kinetics to see if there is a time point at which the response of OE is faster/higher than WT.</p><p>4) Localization: The experiments performed in protoplasts (<xref ref-type="fig" rid="fig3">Figure 3E</xref>) is not sufficient to claim that LYS1 localizes to the apoplast, as it is highly likely that the protoplast medium also contains damaged protoplasts. The authors should transiently express LYS1-GFP in <italic>N. benthamiana</italic>, and show by immunoblot analysis that LYS1-GFP is enriched in apoplastic fluids after PAMP treatment. This experiment should be very straight-forward in this system.</p><p>5) Consequence of down and up-regulation for susceptibility to infection: The results of <xref ref-type="fig" rid="fig8">Figure 8A and 8B</xref> are very puzzling. They show that over-expression lines have the same phenotype, with regard to bacterial virulence, as the silenced lines. This is not due to co-suppression since the authors measured significant levels of both mRNA and protein levels in these over-expression lines. In the Discussion, the authors postulate that higher levels of LYS1 results in a more extensive hydrolysis of the PGN resulting in the loss of the correct sized fragment for induction of innate immunity. This seems a reasonable hypothesis but one that requires experimental validation. This requires the use of a quantitative assay for the immune response that can be followed over time. There are many such relatively easy assays that could be used, such as ROS production or gene expression. The authors should test whether the <italic>LYS1</italic> OE and KD lines differ in their temporal response to both PGN and bacterial inoculation using such assays. If their hypothesis is correct, then one might predict that the OE lines would show some response very early but this would decline rapidly as the elicitor is completely hydrolyzed. One might also predict, for example, that adding higher levels of PGN elicitor might elicit a response on the OE lines (i.e., one might be able to saturate the enzyme and slow hydrolysis to the point where elicitation can be measured).</p><p>6) Association of PGN-degrading activity with LYS1. The authors must demonstrate that the PGN-degrading activity detectable upon over-expression of <italic>LYS1</italic> is indeed due to LYS1 itself, and not to another enzyme whose expression or activity is increased by <italic>LYS1</italic> over-expression. For this, the authors should transiently express a catalytically-inactive variant of LYS1 (based on the knowledge on similar enzymes in other kingdoms) and compare the PGN-degrading activity of the corresponding extract when compared to an extract from leaves over-expressing WT LYS1.</p><p>7) What is LYS1 generating? In <xref ref-type="fig" rid="fig5">Figure 5A</xref>, the authors say that the mutants don't respond to the fragment generated, but the data shows there is some response. In fact, it looks like the basal FRK1 level with buffer is much lower in the mutants than in the Col-O. Therefore, are the fold inductions are retained and just the basal responses are altered? As mentioned above, all the data should be analysed together for significance groups. LYM1, LYM3 and CERK1 genes are essential for PGN signal transduction, so the mutants should have almost no response to soluble PGN. So it’s not clear whether this LYS1-generated peptidoglycan (PGN) fragments is soluble PGN or not, if <italic>cerk1</italic> mutants are still responding to it.</p><p>Other issues to address:</p><p><xref ref-type="fig" rid="fig1">Figure 1</xref> text says suspensions of Micrococcus luteus cell but legend and methods say it’s really cell wall prep/extract. It’s not clear what the negative control is. This should be documented in the Methods.</p><p><xref ref-type="fig" rid="fig1">Figure 1D</xref> 100 µg PGN but 100 µg/ml LPS. Need consistent description (per ml )</p><p><xref ref-type="fig" rid="fig2">Figure 2D</xref>: how can the authors be sure that the fast-migrating band corresponds to the full-length LYS1 protein, or actually, event to LYS1. Did the authors attempt to confirm the identity of this band by mass spectrometry?</p><p>In <xref ref-type="fig" rid="fig3">Figure 3</xref>, the relative hydrolytic activity of LYS1 was calculated using chitinase as a standard. It would be better if the authors could directly measure LYS1 kinetic parameters (e.g., Kd). This would be an important measurement since it would get directly to the issue of physiological relevance. These are standard techniques, which should be within the authors' ability given their access to purified LYS1 protein.</p><p>In <xref ref-type="fig" rid="fig2">Figure 2C</xref>, there is another band which located between 35Kd and 40 Kd shown in LYS1OE samples, please clarify what it is. If it is possible, please do this experiment again; the picture looks very bad. What are the possible effects of proteolysis on the protein, which seems essential for its purification? To be thorough, the authors should identify the cleavage site so that they have a better idea of how much of the LYS1 protein remains. For example, could proteolysis be eliminating a key regulatory portion of the protein?</p><p>[Editors' note: further clarifications were requested prior to acceptance, as described below.]</p><p>The authors did a good job trying to respond to the comments to improve the manuscript. They added analysis of an additional O/E line that does not seem to cause statistically significant increase in <italic>P. syringae</italic> growth after 2 days; this line shows only moderate overexpression of <italic>LYS1</italic>. However, there are a few issues with part of the analysis that need to be addressed before final acceptance.</p><p>1) The authors need to harmonize the description of the statistical analysis with what is presented in the figures. The authors say they added multiple comparison tests, but the way they describe these is confusing. For example in <xref ref-type="fig" rid="fig4">Figure 4E</xref>, if the multiple comparison test was done, then all values in the panel should be labeled by grouping into like categories (i.e. A, B etc). Similarly in 6B (or 6C), significance at P<0.001 are given, but we cant tell whether LYS1 is different from mutanolysin or whether all the treatments in the mutants are giving the same or different values. If all the data was analyzed together, you should be able to label each group (all values that are similar get a different letter).</p><p>2) For the P. syringae infection experiments, the authors say in the discussion that overexpression or down-regulation of LYS1 causes the same magnitude of defect. Actually, they don't do any multiple comparison test of the data in <xref ref-type="fig" rid="fig9">Fig 9</xref> and it looks like the O/E are even more susceptible than the down-regulated plants. The authors should use multiple comparison test here to show whether the effects are the same or not. A couple of other things about the description of this experiment. (1) the title of the <xref ref-type="fig" rid="fig9">Figure 9</xref> should be changed, since there is no “mutation of LYS1”. (2) This experiment does not measure the growth rate since only one endpoint is taken. Therefore no conclusion about the rate can be given.</p><p>3) The authors tested the transcript level of receptors that would detect PGN fragments liberated by LYS1 in the <italic>LYS1</italic> overexpression plants; this is important for excluding the possibility that high overexpression of <italic>LYS1</italic> has unintended consequences on the receptor levels. A more informative experiment would be to check protein levels, as this is the most relevant assay (transcript levels don't always reflect protein levels). There is an available antibody for CERK1- did the authors try using this antibody?</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01990.018</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) Claims of specificity: Since Lys1 has chitinolytic activity, there is the potential that this activity could be playing a role in anti-fungal defense. Although the authors tested this, they can’t exclude that with certain fungal isolate/host combinations LYS1 might have a role in fungal defense. Therefore, we advise toning down the claim of specificity for responses to bacteria</italic>.</p><p>As suggested by the Reviewers we toned down the claim of specificity for responses to bacteria.</p><p><italic>2) Organization and analysis methods: Figure panels should be organized in the order they are discussed. Add something about how many times each experiment was replicated. Statistical analysis needs to be improved. In complex experiments where multiple comparisons can/should be made, use ANOVA and multiple comparison post hoc test. It’s especially difficult to understand the comparisons made in 3E and</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref><italic>.These data should all be analysed together and grouped into significance groups</italic>.</p><p>All figure panels, particularly in <xref ref-type="fig" rid="fig2">Figure 2</xref>, are now discussed in the text as they are appearing in the figure(s). Each figure legend now also includes a statement about how many repetitions were performed for the experiments.</p><p>As suggested by the Referees, we now analyzed complex data shown in <xref ref-type="fig" rid="fig4 fig6">Figure 4E, 6B and 6C</xref> (corresponds to old <xref ref-type="fig" rid="fig3 fig5">Figures 3E, 5B and 5C</xref>) using one way analysis of variance (ANOVA) followed by Turkey’s multiple comparison post hoc test. This is now stated in the respective figure legends and in the Methods section. Notably, the ANOVA analysis validated significant differences obtained initially with the Student’s t-test with the only exception in <xref ref-type="fig" rid="fig4">Figure 4E</xref>, now showing no significant differences between supernatant samples from wild type and <italic>LYS1</italic><sup><italic>KD</italic></sup> lines (also see Reply 3.3.). Thus, in this case we deleted the corresponding statement from the main text.</p><p><italic>3.1) Concerning the KD and overexpression plants: the specificity of the artificial miRNA needs to be established. The authors need to show that only the LYS1 gene is being silenced and not other genes that may have some sequence similarity</italic>.</p><p>To test specificity of the target sequence used for the artificial miRNA construct, we performed qPCR analyses of potential off-target genes. BLAST searches indicated that no other chitinase gene was a potential off-target. As stated in the Methods section, off-target genes were identified using the Web microRNA Designer (new <xref ref-type="fig" rid="fig3">Figure 3B</xref>) and transcript levels of the four top hits were determined by qRT-PCR (new <xref ref-type="fig" rid="fig3">Figure 3C</xref>). In contrast to <italic>LYS1</italic> transcript levels in the <italic>LYS1</italic><sup><italic>KD</italic></sup> lines, the transcription of potential off-target genes was not affected.</p><p><italic>3.2) Because the results of</italic> <xref ref-type="fig" rid="fig8"><italic>Figure 8</italic></xref> <italic>are confusing, it would be good to use a knockout plant such as a SALK mutant (e.g. SALK_095362)</italic>.</p><p>As suggested by the Referees, we initially obtained three independent T-DNA insertion lines, SALK_095362 (<italic>lys1-2</italic>) as suggested by the Referees, and additionally WiscDsLox387C11 (<italic>lys1-1</italic>) and CSHL_ET14179 (<italic>lys1-3</italic>). For all lines we could identify homozygous progeny, however, <italic>LYS1</italic> transcript levels appeared to be like wild type. These results are now presented in <xref ref-type="fig" rid="fig3s1">Figure 3–figure supplement 1</xref>. Consequently, we generated amiRNA lines and only used these KD-lines in our experiments.</p><p><italic>3.3)</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2E</italic></xref> <italic>is the only example where the KD seems to affect the relative PGN hydrolysis activity. Therefore, it’s especially important to do the correct statistical analysis</italic>.</p><p>As requested by the Referees, we now applied ANOVA and multiple comparison post hoc test to our data obtained in the protoplast system (now <xref ref-type="fig" rid="fig4">Figure 4E</xref>). However, differences between wild type supernatant samples and <italic>LYS1</italic><sup><italic>KD</italic></sup> supernatant samples did not proof to be significantly different in the one-way ANOVA analysis followed by a Turkey multiple comparison post hoc test. Thus, as also stated in Reply 2 we deleted this statement from the main text.</p><p><italic>3.4) The effect of the KD would be more convincing if we had conditions where the level of protein in WT can be detected (or maybe one of the bands in D in the Col lane is right?). Did the authors try using apoplast washes to enrich for the activity? How about inducing conditions (e.g. MAMP treatment?) Since LYS expression is normally PAMP-induced, it is important to show that a PAMP-induced increase in PGN-degrading activity is decreased in extracts from</italic> LYS1-KD <italic>lines, when compared to extracts from WT plants</italic>.</p><p>Concerning the detection of native LYS1 in the wild type plants we repeated the western blot formerly shown in <xref ref-type="fig" rid="fig2">Figure 2D</xref> (now new <xref ref-type="fig" rid="fig2">Figure 2B</xref>) but now additionally included protein extracts from one <italic>LYS1</italic><sup><italic>KD</italic></sup>-line. However, we could not detect any differences in the background band pattern between wild type and the <italic>LYS1</italic><sup><italic>KD</italic></sup>-line. Also, in apoplastic fluids we could not detect any protein band in the wild type samples that was absent in the <italic>LYS1</italic><sup><italic>KD</italic></sup>-line (data not shown). Thus wild type LYS1 protein levels are most likely too low to be detected.</p><p>As suggested by the Referees, we infected wild type, <italic>LYS1</italic><sup><italic>OE</italic></sup> and <italic>LYS1</italic><sup><italic>KD</italic></sup> lines with <italic>Pseudomonas syringae</italic> pv <italic>phaseolicola</italic> (which induces <italic>LYS1</italic> gene expression, <xref ref-type="fig" rid="fig1">Figure 1C</xref>) and used protein extracts from infected versus uninfected leaves for enzyme assays. However, slight but not significant increases in hydrolytic activities against 4-MUCT could be observed in both the wild type and the <italic>LYS1</italic><sup><italic>KD</italic></sup> samples and even in the <italic>LYS1</italic><sup><italic>OE</italic></sup> line. As chitinases belong to the family of pathogenesis-related proteins which are generally induced upon pathogen attack it can be assumed that also other chitinases/hydrolytic activities are induced upon <italic>Pph</italic> infection, overlaying the effect of <italic>LYS1</italic> downregulation.</p><p><italic>3.5) The overexpression lines with 300 fold higher than wild type seems way too high (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2B</italic></xref><italic>) and could be causing unforeseen effects. At a minimum the authors should check that the consequence of OE is not causing downregulation of the receptors that perceive the PGNs. If this were the case, it would change the interpretation of the experiments</italic>.</p><p>We acknowledge that <italic>LYS1</italic> overexpression might indeed have side effects. To exclude a direct effect of LYS1-overexpression on PGN receptor expression, we examined transcript levels of <italic>LYM1</italic>, <italic>LYM3</italic> and <italic>CERK1</italic>. However, transcription of these receptor genes was not affected in the <italic>LYS1</italic><sup><italic>OE</italic></sup> lines and the results are now presented in <xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>.</p><p><italic>3.6) Some lines where the overexpression is more modest would be helpful for determining if increased resistance can be achieved</italic>.</p><p>We now include a new line (<italic>LYS1</italic><sup><italic>OE</italic></sup><italic>-3</italic>) in the <italic>Pseudomonas</italic> infection assays (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>). In accordance with ten-fold less increased <italic>LYS1</italic> transcript levels in mature leaves of the <italic>LYS1</italic><sup><italic>OE</italic></sup>-3 line compared to the <italic>LYS1</italic><sup><italic>OE</italic></sup>-1 line, also leaf protein levels of LYS1-GFP and free LYS1 are less abundant in the <italic>LYS</italic><sup><italic>OE</italic></sup>-3 line. Moreover, susceptibility to <italic>Pseudomonas</italic> infection was only slightly but not significantly (P = 0,064, Student’s t-test) increased. It should, however, be noted that differences in bacterial growth smaller than half log are difficult to statistically validate. These results however confirm that lowering <italic>LYS1</italic> expression levels, accompanied by lower LYS1 hydrolytic activity on PGN, brings down these lines close to wild-type.</p><p><italic>3.7) In any case with</italic> <xref ref-type="fig" rid="fig8"><italic>Figure 8.C</italic></xref><italic>, the authors should check the kinetics to see if there is a time point at which the response of OE is faster/higher than WT</italic>.</p><p>As suggested by the Referees we compared the kinetics of early defense responses in wild type, <italic>LYS1</italic><sup><italic>OE</italic></sup> and <italic>LYS1</italic><sup><italic>KD</italic></sup> lines. We conducted ion leakage experiments after <italic>Pto</italic> DC3000 infection and determined <italic>FRK1</italic> gene expression after treatment with either 100 or 500 µg/ml PGN (see <xref ref-type="fig" rid="fig10">author response image 1</xref> below). For ion leakage we measured every hour starting 1 hour post infection up to 6 hours and then every 12 hours up to 36 hours, but in three independent experiments we could not observe any differences between the responses in the different lines at these early time points. For the determination of <italic>FRK1</italic> transcript levels we took samples after 30 min, 1 hour, 3 hours and 6 hours. Here, at early time points such as 30 min to 1 hour, <italic>FRK1</italic> gene expression is not really induced by PGN, thus error bars are very big. At time points 3 and 6 hours, however, we observed <italic>FRK1</italic> induction in WT samples but not in <italic>LYS1</italic><sup><italic>KD</italic></sup> lines, confirming our results shown in <xref ref-type="fig" rid="fig9">Figure 9C</xref>. Likewise, <italic>LYS1</italic><sup><italic>OE</italic></sup> samples treated for 6 hours with 100 µg/ml PGN mimicked the lack of <italic>FRK1</italic> gene expression also observed in the <italic>LYS1</italic><sup><italic>KD</italic></sup> mutant (see also <xref ref-type="fig" rid="fig9">Figure 9C</xref>). Notably, treatment of <italic>LYS1</italic><sup><italic>OE</italic></sup> seedlings with 500 µg/ml PGN resulted in <italic>FRK1</italic> gene induction, however, at no time point did we observe a stronger response in <italic>LYS1</italic><sup><italic>OE</italic></sup> lines than in the wild type. As information for the Referees, these data are included below:<fig id="fig10" position="float"><label>Author response image 1.</label><caption><title>Author response image 1: Early defense responses are not enhanced in <italic>LYS1</italic><sup><italic>OE</italic></sup>-lines.</title><p>(A) Ion leakage measured in leaves of WT, <italic>LYS1</italic><sup><italic>OE</italic></sup> or <italic>LYS1</italic><sup><italic>KD</italic></sup> lines at indicated time points after infiltration of <italic>Pto</italic> DC3000. (B) Determination of <italic>FRK1</italic> transcript levels in seedlings of wild type plants or transgenic <italic>LYS1</italic> plants treated for 30 min, or 1, 3, or 6 hours with 100 or 500 µg/ml <italic>B. subtilis</italic> PGN. Total RNA was subjected to RT-qPCR using <italic>FRK1</italic> specific primers, <italic>EF1α</italic> transcript was used for normalization. Data represent means ± S.D. of triplicate samples.</p></caption><graphic xlink:href="elife01990f010"/></fig></p><p>We would like to emphasize that in the <italic>LYS1</italic><sup><italic>OE</italic></sup> lines, an enhanced level of LYS1 enzyme activity is present all the time, thus also right at the beginning of both the <italic>Pto</italic> infection and the PGN treatment. Hence, we consider it being very challenging to find a time point at which the <italic>LYS1</italic><sup><italic>OE</italic></sup> line might show increased immune responses compared to wild type plants.</p><p>Moreover, we would like to draw the Reviewers’ attention again to <xref ref-type="fig" rid="fig6">Figure 6D</xref> (old <xref ref-type="fig" rid="fig5">figure 5D</xref>). Here, both the PGN preparation treated with buffer only AND the PGN preparation treated overnight with LYS1 lack immunogenic activity in the respective supernatant. We conclude from this experiment that a lack of PGN hydrolysis in buffer controls in <xref ref-type="fig" rid="fig6">Figure 6D</xref> (old <xref ref-type="fig" rid="fig5">Figure 5D</xref>) accompanied with no induction of immune responses is reminiscent of the situation in <italic>LYS1</italic><sup><italic>KD</italic></sup> lines, whereas a complete PGN digestion overnight (new <xref ref-type="fig" rid="fig6">Figure 6D</xref>), again accompanied with no induction of immune responses, is reminiscent of the situation in <italic>LYS1</italic><sup><italic>OE</italic></sup> lines.</p><p><italic>4) Localization: The experiments performed in protoplasts (</italic><xref ref-type="fig" rid="fig3"><italic>Figure 3E</italic></xref><italic>) is not sufficient to claim that LYS1 localizes to the apoplast, as it is highly likely that the protoplast medium also contains damaged protoplasts. The authors should transiently express LYS1-GFP in</italic> N. benthamiana<italic>, and show by immunoblot analysis that LYS1-GFP is enriched in apoplastic fluids after PAMP treatment. This experiment should be very straight-forward in this system</italic>.</p><p>As suggested, we transiently expressed a <italic>p35S::LYS1-GFP</italic> construct in <italic>Nicotiana benthamiana</italic> which resulted in labelling of the cell periphery, whereas expression of a construct lacking the <italic>LYS1</italic> signal peptide-encoding sequence yielded labelling of intracellular structures (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B</xref>). Use of the fluorescent dye FM4-64, a plasma membrane and early endosome marker (<xref ref-type="bibr" rid="bib7">Bolte et al., 2004</xref>), revealed that LYS1 signals co-localized to a large extent with the plasma membrane (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B</xref>). Thus, LYS1 likely operates in close vicinity of the plant surface.</p><p>To confirm a LYS1-localization in the apoplast, we prepared apoplastic washes from <italic>LYS1</italic><sup><italic>OE</italic></sup> Arabidopsis lines. Both the LYS1-GFP fusion protein as well as free LYS1 was detectable in concentrated apoplastic fluids whereas the cytoplasmic protein MPK3 was only present in the total leaf protein samples (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>). As expression of the constructs we used was driven by the 35S-promoter we did not see any further enrichment after PAMP treatment (data not shown).</p><p>Together with the previous identification of LYS1 within the <italic>Arabidopsis</italic> cell wall proteome (<xref ref-type="bibr" rid="bib48">Kwon et al., 2005</xref>, as already stated in the previous manuscript) our data suggest that LYS1 acts in the plant apoplast.</p><p><italic>5) Consequence of down and up-regulation for susceptibility to infection: The results of</italic> <xref ref-type="fig" rid="fig8"><italic>Figure 8A and 8B</italic></xref> <italic>are very puzzling. They show that over-expression lines have the same phenotype, with regard to bacterial virulence, as the silenced lines. This is not due to co-suppression since the authors measured significant levels of both mRNA and protein levels in these over-expression lines. In the Discussion, the authors postulate that higher levels of LYS1 results in a more extensive hydrolysis of the PGN resulting in the loss of the correct sized fragment for induction of innate immunity. This seems a reasonable hypothesis but one that requires experimental validation. This requires the use of a quantitative assay for the immune response that can be followed over time. There are many such relatively easy assays that could be used, such as ROS production or gene expression. The authors should test whether the</italic> LYS1 <italic>OE and KD lines differ in their temporal response to both PGN and bacterial inoculation using such assays. If their hypothesis is correct, then one might predict that the OE lines would show some response very early but this would decline rapidly as the elicitor is completely hydrolyzed. One might also predict, for example, that adding higher levels of PGN elicitor might elicit a response on the OE lines (i.e., one might be able to saturate the enzyme and slow hydrolysis to the point where elicitation can be measured)</italic>.</p><p>We agree that the results of the increased susceptibility of the <italic>LYS1</italic><sup><italic>OE</italic></sup> lines to <italic>Pseudomonas</italic> infection presented in <xref ref-type="fig" rid="fig9">Figure 9</xref> (old <xref ref-type="fig" rid="fig8">Figure 8</xref>) are rather unexpected. However, there are other prominent examples in the literature where overexpression of a protein mimics the effect of genetic inactivation. For instance, strong overexpression of BAK1, a co-receptor of leucine-rich-repeat receptor kinases, triggers inappropriate plant cell death (Belkhadir et al., PNAS 2012, 109,297-302) as is also observed in <italic>bak1</italic> mutants (Kemmerling et al., Curr. Biol.17, 1116–1122). Hence, it can be assumed that wild-type levels of proteins are optimized during evolution and it is therefore not surprising that either too little or too much activity (at least in some cases) might have a deleterious effect.</p><p>However, as suggested by the Referees and as explained in more detail in Reply 3.7., we measured ion leakage in leaves infected with <italic>Pto</italic> DC3000, but could not observe any differences in wild type plants compared to <italic>LYS1</italic><sup><italic>KD</italic></sup> or <italic>LYS1</italic><sup><italic>OE</italic></sup> lines. In all lines, ion leakage was increased starting at 2 to 3 hours post bacterial infiltration. Moreover, we analysed <italic>FRK1</italic> transcript levels at early time points, also using PGN concentrations up to 500 µg/ml, but again we could not observe an enhanced <italic>FRK1</italic> gene expression in the <italic>LYS1</italic><sup><italic>OE</italic></sup> lines at any time point. Notably, in our hands PGN does not induce an oxidative burst and was thus not used as an assay here.</p><p>As only <italic>LYS1</italic><sup><italic>OE</italic></sup> lines with a massively increased LYS1 protein level (lines 1 and 2) show an increased susceptibility to bacterial infection we believe it to be quite challenging to find the right time point for detecting differences in immune responses in <italic>LYS1</italic><sup><italic>OE</italic></sup> lines compared to wild type or <italic>LYS1</italic><sup><italic>KD</italic></sup> lines as the enhanced LYS1 enzyme activity is present at the beginning of all experiments. We might have simply missed the short time frame in which such differences might occur, if they do occur at all.</p><p>For further information, please refer to Reply 3.7.</p><p><italic>6) Association of PGN-degrading activity with LYS1. The authors must demonstrate that the PGN-degrading activity detectable upon over-expression of</italic> LYS1 <italic>is indeed due to LYS1 itself, and not to another enzyme whose expression or activity is increased by</italic> LYS1 <italic>over-expression. For this, the authors should transiently express a catalytically-inactive variant of LYS1 (based on the knowledge on similar enzymes in other kingdoms) and compare the PGN-degrading activity of the corresponding extract when compared to an extract from leaves over-expressing WT LYS1</italic>.</p><p>As suggested, we generated Ala-replacement mutants for the two amino acid residues Asn154 and Glu156, positions which were shown to be crucial for enzymatic activity of rubber tree hevamine (<xref ref-type="bibr" rid="bib5">Bokma et al., 1997</xref>). However, protein extracts from <italic>N. benthamiana</italic> leaves transiently expressing the LYS1<sup>N</sup>154<sup>A/E156A</sup>-myc or LYS1<sup>N</sup>154<sup>A/E156A</sup>-HA variants yielded the same enzyme activity towards 4-MUCT as the corresponding wild type extracts, hence these mutations did not render LYS1 catalytically inactive.</p><p>For hevamine a mutation of the D<sup>125</sup>E<sup>127</sup> motif to A<sup>125</sup>A<sup>127</sup> rendered the enzyme completely inactive. However, an amino acid alignment of hevamine and LYS1 indicated that LYS1 contains a N<sup>154</sup>E<sup>156</sup> motif instead of the DE motif. Additional Aspartate residues are spread around this motif and any of those might be required for enzymatic activity. As we do not have any information from crystal structures for LYS1 it will be challenging to determine which Aspartate residue will be the most important one for catalytic activity.</p><p>Thus, our experimental data indeed cannot exclude the theoretical risk of an up-regulation of the activity of an enzyme other than LYS1 by LYS1 over-expression, but are there any precedents/examples in the literature showing such scenarios?</p><p><italic>7) What is LYS1 generating? In</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5A</italic></xref><italic>, the authors say that the mutants don't respond to the fragment generated, but the data shows there is some response. In fact, it looks like the basal FRK1 level with buffer is much lower in the mutants than in the Col-O. Therefore, are the fold inductions are retained and just the basal responses are altered? As mentioned above, all the data should be analysed together for significance groups. LYM1, LYM3 and CERK1 genes are essential for PGN signal transduction, so the mutants should have almost no response to soluble PGN. So it’s not clear whether this LYS1-generated peptidoglycan (PGN) fragments is soluble PGN or not, if</italic> cerk1 <italic>mutants are still responding to it</italic>.</p><p>As requested, we now analyzed all data shown in new <xref ref-type="fig" rid="fig6">Figure 6B and 6C</xref> with ANOVA and the multiple comparison post hoc test. Following this analysis, only the wild type samples treated with supernatants from the digest with native LYS1 and mutanolysin are significantly different (p < 0.001) from all other samples. Additionally, the mutanolysin-digest was also significantly less active (p < 0.05) in the <italic>lym1 lym3</italic> double mutant. For a better understanding, <xref ref-type="fig" rid="fig6">Figure 6B</xref> now also includes the water control samples, which were set to 1 for each mutant.</p><p>However, we would like to point out that none of the PGN receptor mutants <italic>lym1</italic>, <italic>lym3</italic> and <italic>cerk1</italic> are completely devoid of any responses to PGN treatment, which we detected in our comprehensive micro-array studies (Willmann et al., PNAS 2011). This is most likely due to contaminations which are present in PGN preparations, and might not be removed by LYS1 digest and subsequent HPLC purification. Thus a null-response cannot be expected but, as demonstrated in <xref ref-type="fig" rid="fig9">Figure 9B</xref> (formerly <xref ref-type="fig" rid="fig8">Figure 8B</xref>), a massive reduction in PGN responses in the mutants is evident.</p><p><italic>Other issues to address:</italic></p><p><xref ref-type="fig" rid="fig1"><italic>Figure 1</italic></xref> <italic>text says suspensions of Micrococcus luteus cell but legend and methods say it’s really cell wall prep/extract. It’s not clear what the negative control is. This should be documented in the Methods</italic>.</p><p>We apologize for any confusion and now clearly state in the main text that <italic>Micrococcus luteus</italic> cell wall preparations were used.</p><p>To clarify what the negative control exactly is, the legend to <xref ref-type="fig" rid="fig1">Figure 1A</xref> now reads as follows:</p><p>“As negative control (nc) non-induced His6-tagged LYM3 bacterial lysates were used for affinity purification and eluates were subjected to turbidity assays.”</p><p>Also, we added this information in the Methods section:</p><p>“As negative control, a protein purification using non-induced cultures harbouring the His6-LYM3 construct was performed.”</p><p><xref ref-type="fig" rid="fig1"><italic>Figure 1D</italic></xref> <italic>100 µg PGN but 100 µg/ml LPS. Need consistent description (per ml )</italic></p><p>Changed as requested.</p><p><xref ref-type="fig" rid="fig2"><italic>Figure 2D</italic></xref><italic>: how can the authors be sure that the fast-migrating band corresponds to the full-length LYS1 protein, or actually, event to LYS1. Did the authors attempt to confirm the identity of this band by mass spectrometry?</italic></p><p>The fast-migrating band visible on the western blot in <xref ref-type="fig" rid="fig2">Figure 2D</xref> (now <xref ref-type="fig" rid="fig2">Figure 2B</xref> with the western blot replaced as requested in Inquiry 12) is only visible in the <italic>LYS1</italic><sup><italic>OE</italic></sup> lines, thus representing most likely LYS1. We agree with the referees that from the western blot analysis it cannot be judged if this fast-migrating band indeed represents the full-length LYS1 protein. Hence we purified LYS1 from the <italic>LYS1</italic><sup><italic>OE</italic></sup> line and analysed the purified LYS1 protein with MS analysis. For further information please also refer to Reply 12.</p><p><italic>In</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref><italic>, the relative hydrolytic activity of LYS1 was calculated using chitinase as a standard. It would be better if the authors could directly measure LYS1 kinetic parameters (e.g., Kd). This would be an important measurement since it would get directly to the issue of physiological relevance. These are standard techniques, which should be within the authors' ability given their access to purified LYS1 protein</italic>.</p><p>We agree with the Referees and now indicate specific enzyme activities as calculated in comparison to the commercially available chitinase and lysozyme, for which activities are now also shown (new <xref ref-type="fig" rid="fig4">Figure 4</xref>).</p><p>We also determined K<sub>m</sub> values for purified LYS1.</p><p><italic>In</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2C</italic></xref><italic>, there is another band which located between 35Kd and 40 Kd shown in LYS1OE samples, please clarify what it is. If it is possible, please do this experiment again; the picture looks very bad. What are the possible effects of proteolysis on the protein, which seems essential for its purification? To be thorough, the authors should identify the cleavage site so that they have a better idea of how much of the LYS1 protein remains. For example, could proteolysis be eliminating a key regulatory portion of the protein?</italic></p><p>We apologize for this low quality western blot and repeated this experiment (now <xref ref-type="fig" rid="fig2">Figure 2B</xref>). Additional bands in the <italic>LYS1</italic><sup><italic>OE</italic></sup> line were most of the time hardly visible but most likely represent degradation products derived from the LYS1-GFP fusion protein.</p><p>As the analysis of the total mass of the cleavage product by MALDI was not possibly due to the large size of the protein with approximately 35 kDa, we analysed the major cleavage product by LC-MS/MS after tryptic in-gel digestion and by peptide mass fingerprint, analyses which were conducted in two independent laboratories (Boris Macek, Proteome Center Tübingen, and Hubert Kalbacher, Medical and Natural Sciences Research Centre, both University of Tübingen). As stated in Reply 10, this not only confirmed the identity of LYS1 in this band but also yielded peptides spanning almost the whole protein sequence. We lacked the first 53 amino acids at the N-terminus (with the signal peptide comprising 22 amino acids); however, as these analysis rarely yield a 100 % peptide coverage and as the far N- terminus might not ionize easily, we are confident that the cleavage of the LYS1-GFP fusion protein occurs between LYS1 and GFP yielding an untagged, full length LYS1 protein.</p><p>[Editors' note: further clarifications were requested prior to acceptance, as described below.]</p><p><italic>The authors did a good job trying to respond to the comments to improve the manuscript. They added analysis of an additional O/E line that does not seem to cause statistically significant increase in</italic> P. syringae <italic>growth after 2 days; this line shows only moderate overexpression of</italic> LYS1<italic>. However, there are a few issues with part of the analysis that need to be addressed before final acceptance</italic>.</p><p><italic>1) The authors need to harmonize the description of the statistical analysis with what is presented in the figures. The authors say they added multiple comparison tests, but the way they describe these is confusing. For example in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4E</italic></xref><italic>, if the multiple comparison test was done, then all values in the panel should be labeled by grouping into like categories (i.e. A, B etc). Similarly in 6B (or 6C), significance at P<0.001 are given, but we cant tell whether LYS1 is different from mutanolysin or whether all the treatments in the mutants are giving the same or different values. If all the data was analyzed together, you should be able to label each group (all values that are similar get a different letter)</italic>.</p><p>As requested, the data in <xref ref-type="fig" rid="fig4 fig6">Figure 4E, 6B and 6C</xref> are now presented as significance groups labeled with letters rather than asterisks.</p><p><italic>2) For the P. syringae infection experiments, the authors say in the discussion that overexpression or down-regulation of LYS1 causes the same magnitude of defect. Actually, they don't do any multiple comparison test of the data in</italic> <xref ref-type="fig" rid="fig9"><italic>Fig 9</italic></xref> <italic>and it looks like the O/E are even more susceptible than the down-regulated plants. The authors should use multiple comparison test here to show whether the effects are the same or not. A couple of other things about the description of this experiment. (1) the title of the</italic> <xref ref-type="fig" rid="fig9"><italic>Figure 9</italic></xref> <italic>should be changed, since there is no “mutation of LYS1”. (2) This experiment does not measure the growth rate since only one endpoint is taken. Therefore no conclusion about the rate can be given</italic>.</p><p>We agree that the statement “We further found that plants overexpressing LYS1 were as susceptible as knock down mutants to bacterial infections, suggesting that defined LYS1 levels in wild-type plants are required for LYS1 immune function” in the Discussion was incorrect and misleading. We have therefore changed the statement into:</p><p>“We further found that plants overexpressing LYS1 were also susceptible to bacterial infections, suggesting that defined LYS1 levels in wild-type plants are required for LYS1 immune function.”</p><p>It was never intended to claim that <italic>LYS1</italic><sup><italic>OE</italic></sup> lines were AS susceptible as the <italic>LYS1</italic><sup><italic>KD</italic></sup> lines. The Referee is very much entitled to say that to state this would require more in-depth statistical analysis. However, we would like to state that by Student’s t-test, <italic>LYS1</italic><sup><italic>OE</italic></sup> lines as well as <italic>LYS1</italic><sup><italic>KD</italic></sup> lines were both more susceptible to bacterial infection than WT plants in a statistically significant manner. Student’s t-test is the most commonly used statistical analysis in current literature. We therefore prefer to represent our data using this statistical method.</p><p>As suggested, we changed the title of <xref ref-type="fig" rid="fig9">Figure 9</xref> which now reads as follows: “Manipulation of LYS1 levels causes hyper-susceptibility towards bacterial infection and loss of PGN-triggered immune responses.”</p><p>We agree that for bacterial infections, no growth rates can be given. However, the figure legends state “bacterial growth” (and not growth rates), which can be determined by counting colony forming units at different times after inoculation.</p><p><italic>3) The authors tested the transcript level of receptors that would detect PGN fragments liberated by LYS1 in the</italic> LYS1 <italic>overexpression plants; this is important for excluding the possibility that high overexpression of</italic> LYS1 <italic>has unintended consequences on the receptor levels. A more informative experiment would be to check protein levels, as this is the most relevant assay (transcript levels don't always reflect protein levels). There is an available antibody for CERK1- did the authors try using this antibody?</italic></p><p>We agree with the Referees that protein levels are more informative than transcript levels. We have thus conducted a Western blot analysis with total protein from mature leaves using an anti-CERK1 antibody. New <xref ref-type="fig" rid="fig9s1">Figure 9 – supplement 1A</xref> shows that there is no difference in CERK1 protein levels in WT and LYS1<sup>OE</sup> lines.</p></body></sub-article></article> |