Pectin Acetylesterase Research Articles

Transcripts of pectin-degrading enzymes and isolation of complete cDNA sequence of a pectate lyase gene induced by coffee white stem borer (Xylotrechus quadripes) in the bark tissue of Coffea canephora (robusta coffee).

Of the two commercially cultivated coffee (Coffea) species, C. arabica (arabica) is highly susceptible and C. canephora (robusta) is highly resistant to the insect pest Xylotrechus quadripes (Coleoptera: Cerambycidae), commonly known as coffee white stem borer (CWSB). We constructed a forward-subtracted cDNA library by Suppression Subtractive Hybridization (SSH) from robusta bark tissue for profiling genes induced by CWSB infestation. Among the 265 unigenes of the SSH EST library, 7 unigenes (5 contigs and 2 singletons) matching different pectin-degrading enzymes were discovered. These ESTs matched one pectate lyase, three polygalacturonases, and one pectin acetylesterase gene. Quantitative real-time PCR (qRT-PCR) revealed that CWSB infestation strongly induces the pectate lyase gene at 72h. Complete cDNA sequence of the pectate lyase gene was obtained through 3' and 5' RACE reactions. It was a 1595bp long sequence that included full CDS and both UTRs. Against C. canephora genome sequences in Coffee Genome Hub database ( http://coffee-genome.org/ ), it had 22 matches to different pectate lyase genes mapped on 9 of the 11 pseudochromosomes, the top match being Cc07_g00190 Pectate lyase. In NCBI database, it matched pectate lyase sequences of several plants. Apart from C. canephora, the closest pectate lyase matches were from Sesamum indicum and Nicotiana tabacum. The pectinolytic enzymes discovered here are thought to play a role in the production of oligogalacturonides (OGs) which act as Damage-Associated Molecular Pattern (DAMP) signals eliciting innate immunity in plants. The pectate lyase gene, induced by CWSB infestation, along with other endogenous pectinolytic enzymes and CWSB-specific elicitors, may be involved in triggering basal defense responses to protect the CWSB-damaged tissue against pathogens, as well as to contain CWSB in robusta.

Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp

The lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum. Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris. The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10).

Plant cell wall: Never too much acetate.

Plant cell walls incorporate a variety of acetylated polysaccharides. In addition to enzymes catalysing acetylation (acetyltransferases), plants could produce enzymes to remove acetyl groups (acetylesterases). Previously, pectin acetylesterases were known and now a xylan acetylesterase has been identified — and it has many surprises.

Computational identification of potential microRNAs and their targets from expressed sequence tags of marijuana (Cannabis sativa)

MicroRNAs (miRNAs) are a type of interfering RNAs which are small, ~22 nucleotides long and remain evolutionary conserved. They have been reported in a large number of plants including other organisms like viruses, insects and higher vertebrates where they have biological and metabolic activities. Their pivotal role in gene silencing has been correlated with a number of diseases; therefore miRNA prediction is an important and challenging field. In spite of being a significant medicinal plant, no specific work has been done yet on the microRNAs (miRNAs) and their potential targets in Cannabis sativa. In vitro prediction of miRNAs is a slow and laborious process as it is hard to isolate them. In our current study a well-developed, faster and powerful in silico approach, EST-based homology search is followed to identify potential miRNA of C. sativa. Here, the Expressed Sequence Tag (EST) database of Cannabis sativa was BLASTed against the previously identified plant miRNAs and following a series of filtration criteria, a total of 2 potential miRNAs were identified. Furthermore, 14 potential mRNA targets were tagged in C. sativa genome based on the complementarity between miRNAs and their targeted mRNA sequences. The predicted target genes encode mainly transcriptional regulators including R2R3-Myb transcription factor, GRAS family transcription factor, Scarecrow protein, Transcriptional regulator family protein and DNA binding protein. Some other targets have more specific functions in the developmental process of C. sativa and are associated with diverse metabolic processes for stress management as well as other metabolic functions. These include Leucine rich protein kinase family protein, Retro element pol polyprotein, Pectin acetyl Esterase, SET domain protein, HXXXD-type acyl-transferase family protein, Ankyrin repeat family protein, Hypersensitivity related protein. Overall, findings from our study will pave the way for further researches on C. sativa miRNAs and their functions.

Comparative genomics of pectinacetylesterases: Insight on function and biology

Pectin acetylation influences the gelling ability of this important plant polysaccharide for the food industry. Plant apoplastic pectinacetylesterases (PAEs) play a key role in regulating the degree of pectin acetylation and modifying their expression thus represents one way to engineer plant polysaccharides for food applications. Identifying the major active enzymes within the PAE gene family will aid in our understanding of this biological phenomena as well as provide the tools for direct trait manipulation. Using comparative genomics we propose that there is a minimal set of 4 distinct PAEs in plants. Possible functional diversification of the PAE family in the grasses is also explored with the identification of 3 groups of PAE genes specific to grasses.

Identification and functional characterization of the distinct plant pectin esterases PAE8 and PAE9 and their deletion mutants.

Main conclusion PAE8 and PAE9 have pectin acetylesterase activity and together remove one-third of the cell wall acetate associated with pectin formation in Arabidopsis leaves. In pae8 and pae9 mutants, substantial amounts of acetate accumulate in cell walls. In addition, the inflorescence stem height is decreased. Pectic polysaccharides constitute a significant part of the primary cell walls in dicotyledonous angiosperms. This diverse group of polysaccharides has been implicated in several physiological processes including cell-to-cell adhesion and pathogenesis. Several pectic polysaccharides contain acetyl-moieties directly affecting their physical properties such as gelling capacity, an important trait for the food industry. In order to gain further insight into the biological role of pectin acetylation, a reverse genetics approach was used to investigate the function of genes that are members of the Pectin AcetylEsterase gene family (PAE) in Arabidopsis. Mutations in two members of the PAE family (PAE8 and PAE9) lead to cell walls with an approximately 20 % increase in acetate content. High-molecular-weight fractions enriched in pectic rhamnogalacturonan I (RGI) extracted from the mutants had increased acetate content. In addition, the pae8 mutant displayed increased acetate content also in low-molecular-weight pectic fractions. The pae8/pae9-2 double mutant exhibited an additive effect by increasing wall acetate content by up to 37 %, suggesting that the two genes are not redundant and act on acetyl-substituents of different pectic domains. The pae8 and pae8/pae9-2 mutants exhibit reduced inflorescence growth underscoring the role of pectic acetylation in plant development. When heterologously expressed and purified, both gene products were shown to release acetate from the corresponding mutant pectic fractions in vitro. PAEs play a significant role in modulating the acetylation state of pectic polymers in the wall, highlighting the importance of apoplastic metabolism for the plant cell and plant growth.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-014-2139-6) contains supplementary material, which is available to authorized users.

Homogalacturonan-modifying enzymes: structure, expression, and roles in plants.

Understanding the changes affecting the plant cell wall is a key element in addressing its functional role in plant growth and in the response to stress. Pectins, which are the main constituents of the primary cell wall in dicot species, play a central role in the control of cellular adhesion and thereby of the rheological properties of the wall. This is likely to be a major determinant of plant growth. How the discrete changes in pectin structure are mediated is thus a key issue in our understanding of plant development and plant responses to changes in the environment. In particular, understanding the remodelling of homogalacturonan (HG), the most abundant pectic polymer, by specific enzymes is a current challenge in addressing its fundamental role. HG, a polymer that can be methylesterified or acetylated, can be modified by HGMEs (HG-modifying enzymes) which all belong to large multigenic families in all species sequenced to date. In particular, both the degrees of substitution (methylesterification and/or acetylation) and polymerization can be controlled by specific enzymes such as pectin methylesterases (PMEs), pectin acetylesterases (PAEs), polygalacturonases (PGs), or pectate lyases-like (PLLs). Major advances in the biochemical and functional characterization of these enzymes have been made over the last 10 years. This review aims to provide a comprehensive, up to date summary of the recent data concerning the structure, regulation, and function of these fascinating enzymes in plant development and in response to biotic stresses.

Phylogenetic analysis of pectin-related gene families in Physcomitrella patensand nine other plant species yields evolutionary insights into cell walls

BackgroundPectins are acidic sugar-containing polysaccharides that are universally conserved components of the primary cell walls of plants and modulate both tip and diffuse cell growth. However, many of their specific functions and the evolution of the genes responsible for producing and modifying them are incompletely understood. The moss Physcomitrella patens is emerging as a powerful model system for the study of plant cell walls. To identify deeply conserved pectin-related genes in Physcomitrella, we generated phylogenetic trees for 16 pectin-related gene families using sequences from ten plant genomes and analyzed the evolutionary relationships within these families.ResultsContrary to our initial hypothesis that a single ancestral gene was present for each pectin-related gene family in the common ancestor of land plants, five of the 16 gene families, including homogalacturonan galacturonosyltransferases, polygalacturonases, pectin methylesterases, homogalacturonan methyltransferases, and pectate lyase-like proteins, show evidence of multiple members in the early land plant that gave rise to the mosses and vascular plants. Seven of the gene families, the UDP-rhamnose synthases, UDP-glucuronic acid epimerases, homogalacturonan galacturonosyltransferase-like proteins, β-1,4-galactan β-1,4-galactosyltransferases, rhamnogalacturonan II xylosyltransferases, and pectin acetylesterases appear to have had a single member in the common ancestor of land plants. We detected no Physcomitrella members in the xylogalacturonan xylosyltransferase, rhamnogalacturonan I arabinosyltransferase, pectin methylesterase inhibitor, or polygalacturonase inhibitor protein families.ConclusionsSeveral gene families related to the production and modification of pectins in plants appear to have multiple members that are conserved as far back as the common ancestor of mosses and vascular plants. The presence of multiple members of these families even before the divergence of other important cell wall-related genes, such as cellulose synthases, suggests a more complex role than previously suspected for pectins in the evolution of land plants. The presence of relatively small pectin-related gene families in Physcomitrella as compared to Arabidopsis makes it an attractive target for analysis of the functions of pectins in cell walls. In contrast, the absence of genes in Physcomitrella for some families suggests that certain pectin modifications, such as homogalacturonan xylosylation, arose later during land plant evolution.

A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3

A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7kDa and shows optimal activity at pH 8.0 and 50°C. It is stable in the range pH 5.0–7.0 and below 50°C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, 1H NMR of polymer and LC-HILIC-MSn after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties.

Enzymatic saccharification of sugar beet pulp for the production of galacturonic acid and arabinose; a study on the impact of the formation of recalcitrant oligosaccharides

Enzymatic saccharification of sugar beet pulp was optimized on kg-scale to release the maximum amounts of monomeric galacturonic acid and arabinose with limited concomitant degradation of cellulose, using conditions that are feasible for industrial upscaling. A selected mixture of pectinases released 79% of the galacturonic acid and 82% of the arabinose as monomers from sugar beet pulp while simultaneously degrading only 17% of the cellulose. The recalcitrant structures that were obtained after hydrolysis were characterized using mass spectrometry. The most abundant structures had an average degree of polymerization of 4–5. They were identified as partially acetylated rhamnogalacturonan-oligosaccharides, mostly containing a terminal galacturonosyl residue on both reducing and non-reducing end, partially methyl esterified/acetylated homogalacturonan-oligosaccharides, mostly containing methyl and acetyl esters at contiguous galacturonosyl residues and arabinan-oligosaccharides, hypothesized to be mainly branched. It could be concluded that especially rhamnogalacturonan-galacturonohydrolase, arabinofuranosidase and pectin acetylesterase are lacking for further degradation of recalcitrant oligosaccharides.

Expression analysis of putative genes of pectin modifying enzymes in different callus lines of Cyclamen persicum and their correlation with the callus texture

Gene expression of four different pectin modifying enzymes (a putative Pectin acetylesterase (PAE) homologue, a putative Pectin esterase (PE) homologue and two different putative Pectate lyase (PL) homologues) was examined in different callus cell lines of Cyclamen persicum as well as in the corresponding explants using quantitative PCR. Transcript abundances were analysed with special regard to potential correlations with callus texture and embryogenic competence. No correlation has been detected between gene expression of the putative pectin modifying enzymes and the embryogenic competence of the different cell lines. However, expression of the putative PAE homologue correlated with the callus texture: transcript abundances of the putative PAE homologue were higher the more friable the tissue was, in callus as well as in explant tissue. Histological analyses supported the conclusion that callus texture was influenced by the explant type.

Erratum to: Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties

Erratum to: Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties

Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties

A mung bean (Vigna radiata) pectin acetyl esterase (CAA67728) was heterologously expressed in tubers of potato (Solanum tuberosum) under the control of the granule-bound starch synthase promoter or the patatin promoter in order to probe the significance of O-acetylation on cell wall and tissue properties. The recombinant tubers showed no apparent macroscopic phenotype. The enzyme was recovered from transgenic tubers using a high ionic strength buffer and the extract was active against a range of pectic substrates. Partial in vivo de-acetylation of cell wall polysaccharides occurred in the transformants, as shown by a 39% decrease in the degree of acetylation (DA) of tuber cell wall material (CWM). Treatment of CWM using a combination of endo-polygalacturonase and pectin methyl esterase extracted more pectin polymers from the transformed tissue compared to wild type. The largest effect of the pectin acetyl esterase (68% decrease in DA) was seen in the residue from this extraction, suggesting that the enzyme is preferentially active on acetylated pectin that is tightly bound to the cell wall. The effects of acetylation on tuber mechanical properties were investigated by tests of failure under compression and by determination of viscoelastic relaxation spectra. These tests suggested that de-acetylation resulted in a stiffer tuber tissue and a stronger cell wall matrix, as a result of changes to a rapidly relaxing viscoelastic component. These results are discussed in relation to the role of pectin acetylation in primary cell walls and its implications for industrial uses of potato fibres.

Acetylesterase-mediated deacetylation of pectin impairs cell elongation, pollen germination, and plant reproduction.

Pectin is a major component of the primary cell wall of higher plants. Some galacturonyl residues in the backbone of pectinaceous polysaccharides are often O-acetylated at the C-2 or C-3 position, and the resulting acetylesters change dynamically during the growth and development of plants. The processes involve both enzymatic acetylation and deacetylation. Through genomic sequence analysis, we identified a pectin acetylesterase (PAE1) from black cottonwood (Populus trichocarpa). Recombinant Pt PAE1 exhibited preferential activity in releasing the acetate moiety from sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectin in vitro. Overexpressing Pt PAE1 in tobacco (Nicotiana tabacum) decreased the level of acetyl esters of pectin but not of xylan. Deacetylation engendered differential changes in the composition and/or structure of cell wall polysaccharides that subsequently impaired the cellular elongation of floral styles and filaments, the germination of pollen grains, and the growth of pollen tubes. Consequently, plants overexpressing PAE1 exhibited severe male sterility. Furthermore, in contrast to the conventional view, PAE1-mediated deacetylation substantially lowered the digestibility of pectin. Our data suggest that pectin acetylesterase functions as an important structural regulator in planta by modulating the precise status of pectin acetylation to affect the remodeling and physiochemical properties of the cell wall's polysaccharides, thereby affecting cell extensibility.

Determination and Isolation of a Thioesterase from Passion Fruit (Passiflora edulis Sims) That Hydrolyzes Volatile Thioesters

Volatile organosulfur compounds (VOSCs) are high impact aroma chemicals characteristic of tropical fruits which are active as both free thiols and the respective thioesters. Using a simple and sensitive colorimetric enzyme assay, a thioesterase activity toward VOSCs has been identified in ripening purple passion fruit ( Passiflora edulis Sims). The assay was based on determining the release of free thiols from 2-methyl-3-furanthiol acetate using Ellman's reagent. The major thioesterase in the fruit was found to be a wall-bound protein in the mesocarp. The extracted enzyme activity was purified 150-fold and shown to be associated with a 43 kDa monomeric serine hydrolase which was selectively labeled with a fluorophosphonate suicide probe. MS-MS sequencing identified the thioesterase as a class 13 glycoside hydrolase, most similar to pectin acetylesterase, an enzyme involved in cell wall modifications in the peel of a number of fruit. Our results suggest that cell wall hydrolases in tropical fruit may have additional useful roles in biotransforming VOSCs.

Pectin acetylesterases from Aspergillus are able to deacetylate homogalacturonan as well as rhamnogalacturonan

An acetylesterase was purified from Aspergillus aculeatus on the basis of its ability to deacetylate homogalacturonan. A series of well-characterized substrates was prepared and used to investigate its specificity, including pectin with various degrees of methylation and acetylation, pectic domains with various structures, and oligomers. It was then compared to a rhamnogalacturonan acetylesterase previously isolated from the same fungus. Both enzymes were active towards various acetylated pectins, and were able to release acetyl groups from acetylated homogalacturonan, oligogalacturonates as well as rhamnogalacturonan at different extent. It was thus concluded that while the two enzymes differed in their efficiency, they are both pectin acetylesterases, able to act in the “smooth” as well as in the “hairy” regions of pectin.

Transcriptome profiling identified novel genes associated with aluminum toxicity, resistance and tolerance in Medicago truncatula

Oligonucleotide microarrays corresponding to over 16,000 genes were used to analyze changes in transcript accumulation in root tips of the Al-sensitive Medicago truncatula cultivar Jemalong genotype A17 in response to Al treatment. Out of 2,782 genes with significant changes in transcript accumulation, 324 genes were up-regulated and 267 genes were down-regulated at least twofold by Al. Up-regulated genes were enriched in transcripts involved in cell-wall modification and abiotic and biotic stress responses while down-regulated genes were enriched in transcripts involved in primary metabolism, secondary metabolism, protein synthesis and processing, and the cell cycle. Known markers of Al-induced gene expression including genes associated with oxidative stress and cell wall stiffening were differentially regulated in this study. Transcript profiling identified novel genes associated with processes involved in Al toxicity including cell wall modification, cell cycle arrest and ethylene production. Novel genes potentially associated with Al resistance and tolerance in M. truncatula including organic acid transporters, cell wall loosening enzymes, Ca(2+) homeostasis maintaining genes, and Al-binding were also identified. In addition, expression analysis of nine genes in the mature regions of the root revealed that Al-induced gene expression in these regions may play a role in Al tolerance. Finally, interfering RNA-induced silencing of two Al-induced genes, pectin acetylesterase and annexin, in A17 hairy roots slightly increased the sensitivity of A17 to Al suggesting that these genes may play a role in Al resistance.

Microarray expression analysis of normal and Fe‐hyper‐accumulating Pea (Pisum sativum L.) mutant genotypes and genetic links to Caco‐2 Fe bioavailability

Nicotianamine, plays important roles in intra and intercellular Fe movement in all higher plants but the genetic regulation is not completely understood. Three isogenic pea genotypes [Sparkle (wild type), brz and dgl] were studied. Both brz and dgl (Fe toxicity phenotypes) have been reported to over accumulate Nicotianamine. The plants were grown in full strength nutrient solutions. Pea pods at twelve stages of development and fully mature seeds were evaluated. Total RNA was isolated from mature leaves, converted to cDNA and biotin labeled. The pea targets were cross-species hybridized to nucleic acid probes of the afflymetrix Medicago genechip and/or the Arabidopsis ATH1 array. Preliminary results suggest that the highest concentration of Fe in the pods and seeds was associated with the up-regulation of methionine-adenosyl-transferese and s-adenosyl-l-homocysteinase, both precursors of nicotianamine via the methionine pathway, in dgl compared to Sparkle or brz. Furthermore, in dgl, S-adenosylmethionine synthase, S-adenosylmethionine decarboxylase, Pea root Fe transporter protein (PsIT), ferritin and pectin acetylesterase were up-regulated. Cystathionine-gamma-synthase, polygalacturonase2, and the Ps fructose-1, 6-biphosphate aldolase were down regulated in dgl. The Fe concentrations in mature dry seeds were: Sparkle, 62 mg/g; brz, 44 mg/g; dgl, 145 mg/g. The bioavailable Fe as determined using Caco-2 cells was higher in immature pods. These results suggest that up-regulation of the methionine precursors may lead to increased Fe concentration in seeds via their affects on nicotianamine biosynthesis and subsequent increased mobilization of Fe to developing seeds. Research funded by USDA-ARS.

Discovery of Pectin-degrading Enzymes and Directed Evolution of a Novel Pectate Lyase for Processing Cotton Fabric

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.

The secretome of the plant pathogenic bacterium Erwinia chrysanthemi.

Erwinia chrysanthemi causes soft-rot diseases of many plants by secreting a battery of enzymes which degrade the plant cell walls. We initiated a proteomic analysis to create a reference map of the E. chrysanthemi secretome. Extracellular proteins were isolated from E. chrysanthemi culture supernatants and resolved by two-dimensional electrophoresis. By analysis of mutants, Western blotting, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) 55 spots representing 25 unique proteins were identified. In uninduced conditions, we identified spots corresponding to the cellulase Cel5, the proteases PrtA, PrtB, and PrtC, the flagellin FliC, and some intracellular proteins whose presence probably resulted from spontaneous cell lysis. We identified another secreted protein, AvrL, homologous to an avirulence protein of Xanthomonas campestris. After culture in conditions inducing pectinase production, i.e., in the presence of galacturonate and plant extract, we identified spots corresponding to the endopectate lyases PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ, the pectin acetylesterases PaeX and PaeY, the pectin methylesterase PemA, and the polygalacturonase PehX. In the presence of other inducing compounds, we detected the rhamnogalacturonate lyase RhiE and the esterase FaeD. Analysis of mutants, altered for one type of secretion system, was performed to determine the targets of each system. The type I system Prt was necessary for the secretion of three proteases. No proteins secreted by the type III Hrp system could be detected in E. chrysanthemi supernatants. In addition to the already known substrates (eleven pectinases and one cellulase), this analysis revealed that the type II Out system mediates secretion of the esterase FaeD and of the Avr-like protein AvrL.