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Diversity of beetle genes encoding novel plant cell wall degrading enzymes.

ABSTRACT: Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterostomes was inferred to be able to digest, or even synthesize, cellulose using endogenous genes, all model insects whose complete genomes have been sequenced lack genes encoding such enzymes. To establish if the apparent "disappearance" of PCWDEs from insects is simply a sampling problem, we used 454 mediated pyrosequencing to scan the gut transcriptomes of beetles that feed on a variety of plant derived diets. By sequencing the transcriptome of five beetles, and surveying publicly available ESTs, we describe 167 new beetle PCWDEs belonging to eight different enzyme families. This survey proves that these enzymes are not only present in non-model insects but that the multigene families that encode them are apparently undergoing complex birth-death dynamics. This reinforces the observation that insects themselves, and not just their microbial symbionts, are a rich source of PCWDEs. Further it emphasises that the apparent absence of genes encoding PCWDEs from model organisms is indeed simply a sampling artefact. Given the huge diversity of beetles alive today, and the diversity of their lifestyles and diets, we predict that beetle guts will emerge as an important new source of enzymes for use in biotechnology.

SUBMITTER: Pauchet Y

PROVIDER: S-EPMC3003705 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Diversity of beetle genes encoding novel plant cell wall degrading enzymes.

Pauchet Yannick Y Wilkinson Paul P Chauhan Ritika R Ffrench-Constant Richard H RH

PloS one 20101217 12

Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterostome ...[more]

PMID: 21179425

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Project description:BACKGROUND: A critical aspect of plant infection by the majority of pathogens is penetration of the plant cell wall. This process requires the production and secretion of a broad spectrum of pathogen enzymes that target and degrade the many complex polysaccharides in the plant cell wall. As a necessary framework for a study of the expression of cell wall degrading enzymes (CWDEs) produced by the broad host range phytopathogen, Phytophthora parasitica, we have conducted an in-depth bioinformatics analysis of the entire complement of genes encoding CWDEs in this pathogen's genome. RESULTS: Our bioinformatic analysis indicates that 431 (2%) of the 20,825 predicted proteins encoded by the P. parasitica genome, are carbohydrate-active enzymes (CAZymes) involved in the degradation of cell wall polysaccharides. Of the 431 proteins, 337 contain classical N-terminal secretion signals and 67 are predicted to be targeted to the non-classical secretion pathway. Identification of CAZyme catalytic activity based on primary protein sequence is difficult, nevertheless, detailed comparisons with previously characterized enzymes has allowed us to determine likely enzyme activities and targeted substrates for many of the P. parasitica CWDEs. Some proteins (12%) contain more than one CAZyme module but, in most cases, multiple modules are from the same CAZyme family. Only 12 P. parasitica CWDEs contain both catalytically-active (glycosyl hydrolase) and non-catalytic (carbohydrate binding) modules, a situation that contrasts with that in fungal phytopathogens. Other striking differences between the complements of CWDEs in P. parasitica and fungal phytopathogens are seen in the CAZyme families that target cellulose, pectins or ?-1,3-glucans (e.g. callose). About 25% of P. parasitica CAZymes are solely directed towards pectin degradation, with the majority coming from pectin lyase or carbohydrate esterase families. Fungal phytopathogens typically contain less than half the numbers of these CAZymes. The P. parasitica genome, like that of other Oomycetes, is rich in CAZymes that target ?-1,3-glucans. CONCLUSIONS: This detailed analysis of the full complement of P. parasitica cell wall degrading enzymes provides a framework for an in-depth study of patterns of expression of these pathogen genes during plant infection and the induction or repression of expression by selected substrates.

Project description:BackgroundThe primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs). The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae.ResultsUsing a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH) families: GH11 (xylanases), GH28 (polygalacturonases or pectinases), and GH45 (β-1,4-glucanases or cellulases). Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs) families as well as polygalacturonase-inhibiting proteins (PGIPs) were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome analyses could be partially explained by differences in transcriptional level.ConclusionsCombining proteome and transcriptome sequencing analyses proved to be a powerful tool for the discovery of active PCWDEs in a non-model species. Our data represent the starting point of an in-depth functional and evolutionary characterization of PCWDE gene families in phytophagous beetles and their contribution to the adaptation of these highly successful herbivores to their host plants.

Project description:BACKGROUND: The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides. RESULTS: Large-scale screening identified a range of hydrolytic activities among 348 unique isolates representing 156 species of plant pathogenic and non-pathogenic fungi. Hierarchical clustering was used to identify groups of species with similar hydrolytic profiles. Among moderately and highly active species, plant pathogenic species were found to be more active than non-pathogens on six of eight substrates tested, with no significant difference seen on the other two substrates. Among the pathogenic fungi, greater hydrolysis was seen when they were tested on biomass and hemicellulose derived from their host plants (commelinoid monocot or dicot). Although T. reesei has a hydrolytic profile that is highly active on cellulose and pretreated biomass, it was less active than some natural isolates of fungi when tested on xylans and untreated biomass. CONCLUSIONS: Several highly active isolates of plant pathogenic fungi were identified, particularly when tested on xylans and untreated biomass. There were statistically significant preferences for biomass type reflecting the monocot or dicot host preference of the pathogen tested. These highly active fungi are promising targets for identification and characterization of novel cell wall degrading enzymes for industrial applications.

Project description:BackgroundThe fungivorus nematode, Aphelenchus avenae is widespread in soil and is found in association with decaying plant material. This nematode is also found in association with plants but its ability to cause plant disease remains largely undetermined. The taxonomic position and intermediate lifestyle of A. avenae make it an important model for studying the evolution of plant parasitism within the Nematoda. In addition, the exceptional capacity of this nematode to survive desiccation makes it an important system for study of anhydrobiosis. Expressed sequence tag (EST) analysis may therefore be useful in providing an initial insight into the poorly understood genetic background of A. avenae.ResultsWe present the generation, analysis and annotation of over 5,000 ESTs from a mixed-stage A. avenae cDNA library. Clustering of 5,076 high-quality ESTs resulted in a set of 2,700 non-redundant sequences comprising 695 contigs and 2,005 singletons. Comparative analyses indicated that 1,567 (58.0%) of the cluster sequences had homologues in Caenorhabditis elegans, 1,750 (64.8%) in other nematodes, 1,321(48.9%) in organisms other than nematodes, and 862 (31.9%) had no significant match to any sequence in current protein or nucleotide databases. In addition, 1,100 (40.7%) of the sequences were functionally classified using Gene Ontology (GO) hierarchy. Similarity searches of the cluster sequences identified a set of genes with significant homology to genes encoding enzymes that degrade plant or fungal cell walls. The full length sequences of two genes encoding glycosyl hydrolase family 5 (GHF5) cellulases and two pectate lyase genes encoding polysaccharide lyase family 3 (PL3) proteins were identified and characterized.ConclusionWe have described at least 2,214 putative genes from A. avenae and identified a set of genes encoding a range of cell-wall-degrading enzymes. This EST dataset represents a starting point for studies in a number of different fundamental and applied areas. The presence of genes encoding a battery of cell-wall-degrading enzymes in A. avenae and their similarities with genes from other plant parasitic nematodes suggest that this nematode can act not only as a fungal feeder but also a plant parasite. Further studies on genes encoding cell-wall-degrading enzymes in A. avenae will accelerate our understanding of the complex evolutionary histories of plant parasitism and the use of genes obtained by horizontal gene transfer from prokaryotes.

Project description:BACKGROUND:Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. METHODOLOGY/PRINCIPAL FINDINGS:The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. CONCLUSIONS/SIGNIFICANCE:The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.

Project description:RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response.

Dataset Information

Diversity of beetle genes encoding novel plant cell wall degrading enzymes.

Publications

Diversity of beetle genes encoding novel plant cell wall degrading enzymes.

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