Cell Wall Degrading Enzyme Genes Research Articles

Insights into the Ecological Diversification of the Hymenochaetales based on Comparative Genomics and Phylogenomics With an Emphasis on Coltricia.

To elucidate the genomic traits of ecological diversification in the Hymenochaetales, we sequenced 15 new genomes, with attention to ectomycorrhizal (EcM) Coltricia species. Together with published data, 32 genomes, including 31 Hymenochaetales and one outgroup, were comparatively analyzed in total. Compared with those of parasitic and saprophytic members, EcM species have significantly reduced number of plant cell wall degrading enzyme genes, and expanded transposable elements, genome sizes, small secreted proteins, and secreted proteases. EcM species still retain some of secreted carbohydrate-active enzymes (CAZymes) and have lost the key secreted CAZymes to degrade lignin and cellulose, while possess a strong capacity to degrade a microbial cell wall containing chitin and peptidoglycan. There were no significant differences in secreted CAZymes between fungi growing on gymnosperms and angiosperms, suggesting that the secreted CAZymes in the Hymenochaetales evolved before differentiation of host trees into gymnosperms and angiosperms. Nevertheless, parasitic and saprophytic species of the Hymenochaetales are very similar in many genome features, which reflect their close phylogenetic relationships both being white rot fungi. Phylogenomic and molecular clock analyses showed that the EcM genus Coltricia formed a clade located at the base of the Hymenochaetaceae and divergence time later than saprophytic species. And Coltricia remains one to two genes of AA2 family. These indicate that the ancestors of Coltricia appear to have originated from saprophytic ancestor with the ability to cause a white rot. This study provides new genomic data for EcM species and insights into the ecological diversification within the Hymenochaetales based on comparative genomics and phylogenomics analyses.

Electrostatic Atomized Water Particles Induce Disease Resistance in Muskmelon (Cucumis melo L.) against Postharvest Fruit Rot Caused by Fusarium incarnatum.

The postharvest quality of muskmelon can be affected by fruit rot caused by the fungus Fusarium incarnatum, resulting in loss of quality. The utilization of electrostatic atomized water particles (EAWPs) in agriculture applications has been shown to induce disease resistance in plants. Therefore, in this study, we determined the effect of electrostatic atomized water particles (EAWPs) on the disease resistance of muskmelon fruits against postharvest fruit rot caused by F. incarnatum. EAWPs were applied to muskmelon fruits for 0, 30, 60, and 90 min. EAWP-treated muskmelon fruits were inoculated with F. incarnatum, and disease progress was measured. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of the chitinase (CmCHI) and β-1,3-glucanase (CmGLU) genes of Cucumis melo (muskmelon) was performed for EAWP-treated and -untreated muskmelon fruits. The activities of cell-wall-degrading enzymes (CWDEs), chitinase, and β-1,3-glucanase were also assayed in EAWP-treated and -untreated muskmelon fruits. The results showed that disease progress was limited by EAWP treatment for 30 min prior to pathogen inoculation. Muskmelon fruits treated with EAWPs for 30 min showed an upregulation of CWDE genes, CmCHI and CmGLU, as observed by qRT-PCR, leading to high chitinase and β-1,3-glucanase activities, as observed through enzyme assays. The results of SEM microscopy revealed that the effect of the crude enzymes of EAWP-treated muskmelon caused morphological changes in F. incarnatum mycelia. Furthermore, treatment with EAWPs preserved postharvest quality in muskmelon, including with regard to texture stiffness and total chlorophyll contents, compared to untreated muskmelon. These results demonstrate that the pretreatment of muskmelon with EAWPs suppresses the development of F. incarnatum in the early stage of infection by regulating gene expression of CWDEs and elevating the activities of CWDEs, while also maintaining postharvest muskmelon quality.

Effects of calcium on cell wall metabolism enzymes and expression of related genes associated with peel creasing in Citrus fruits.

Fruit peel creasing is a serious pre-harvest physiological disorder in citrus, influencing fruit quality, storage, and yield. Four- and eight-year-old 'Hongjiang' oranges grafted onto Canton lemon rootstocks were treated with calcium and calcium inhibitors, respectively, to study the effects of different treatments on fruit creasing rate, mechanical properties of the peel, cell wall metabolism enzyme activities, and the expression of related genes. Foliar application of 0.5% calcium nitrate significantly reduced the fruit creasing rate, while treatment with EGTA and LaCl3, inhibitors of calcium uptake, increased the fruit creasing rate; But the effect of calcium nitrate treatment on changing the mechanical properties of pericarp and inhibiting the activity of hydrolase (PG, Cx and PE) was not very significant. Furthermore, it was observed that the expression levels of genes (PG, Cx, and PE) encoding cell wall-degrading enzymes were significantly lower in the normal fruit peel than in the creased fruit peel. Meanwhile, the expression levels of PG, Cx, and PE were higher in the peel of shaded fruit than in the peel of exposed fruit. During the high incidence period of fruit creasing, calcium nitrate treatment down-regulated the expression of PG, Cx, and PE, while EGTA treatment up-regulated the expression of these genes. In conclusion, foliar spraying of calcium nitrate at the fruit rapid enlargement stage can increase the Ca content in the peel of 'Hongjiang' orange and significantly suppress the expression of cell wall degrading enzymes genes (PG, PE and Cx) in 'Hongjiang' orange peel during the high occurrence period of fruit creasing, resulting in reducing the occurrence of fruit creasing and cracking.

Genomic and Functional Dissections of Dickeya zeae Shed Light on the Role of Type III Secretion System and Cell Wall-Degrading Enzymes to Host Range and Virulence.

ABSTRACTDickeya zeae is a worldwide destructive pathogen that causes soft rot diseases on various hosts such as rice, maize, banana, and potato. The strain JZL7 we recently isolated from clivia represents the first monocot-specific D. zeae and also has reduced pathogenicity compared to that of other D. zeae strains (e.g., EC1 and MS2). To elucidate the molecular mechanisms underlying its more restricted host range and weakened pathogenicity, we sequenced the complete genome of JZL7 and performed comparative genomic and functional analyses of JZL7 and other D. zeae strains. We found that, while having the largest genome among D. zeae strains, JZL7 lost almost the entire type III secretion system (T3SS), which is a key component of the virulence suite of many bacterial pathogens. Importantly, the deletion of T3SS in MS2 substantially diminished the expression of most type III secreted effectors (T3SEs) and MS2’s pathogenicity on both dicots and monocots. Moreover, although JZL7 and MS2 share almost the same repertoire of cell wall-degrading enzymes (CWDEs), we found broad reduction in the production of CWDEs and expression levels of CWDE genes in JZL7. The lower expression of CWDEs, pectin lyases in particular, would probably make it difficult for JZL7 to break down the cell wall of dicots, which is rich in pectin. Together, our results suggest that the loss of T3SS and reduced CWDE activity together might have contributed to the host specificity and virulence of JZL7. Our findings also shed light on the pathogenic mechanism of Dickeya and other soft rot Pectobacteriaceae species in general.IMPORTANCE Dickeya zeae is an important, aggressive bacterial phytopathogen that can cause severe diseases in many crops and ornamental plants, thus leading to substantial economic losses. Strains from different sources showed significant diversity in their natural hosts, suggesting complicated evolution history and pathogenic mechanisms. However, molecular mechanisms that cause the differences in the host range of D. zeae strains remain poorly understood. This study carried out genomic and functional dissections of JZL7, a D. zeae strain with restricted host range, and revealed type III secretion system (T3SS) and cell wall-degrading enzymes (CWDEs) as two major factors contributing to the host range and virulence of D. zeae, which will provide a valuable reference for the exploration of pathogenic mechanisms in other bacteria and present new insights for the control of bacterial soft rot diseases on crops.

Pangenomic Analysis of Dickeya dianthicola Strains Related to the Outbreak of Blackleg and Soft Rot of Potato in the United States

Dickeya dianthicola has caused an outbreak of blackleg and soft rot of potato in the eastern half of the United States since 2015. To investigate genetic diversity of the pathogen, a comparative analysis was conducted on genomes of D. dianthicola strains. Whole genomes of 16 strains from the United States outbreak were assembled and compared with 16 previously sequenced genomes of D. dianthicola isolated from potato or carnation. Among the 32 strains, eight distinct clades were distinguished based on phylogenomic analysis. The outbreak strains were grouped into three clades, with the majority of the strains in clade I. Clade I strains were unique and homogeneous, suggesting a recent incursion of this strain into potato production from alternative hosts or environmental sources. The pangenome of the 32 strains contained 6,693 genes, 3,377 of which were core genes. By screening primary protein subunits associated with virulence from all U.S. strains, we found that many virulence-related gene clusters, such as plant cell wall degrading enzyme genes, flagellar and chemotaxis related genes, two-component regulatory genes, and type I/II/III secretion system genes, were highly conserved but that type IV and type VI secretion system genes varied. The clade I strains encoded two clusters of type IV secretion systems, whereas the clade II and III strains encoded only one cluster. Clade I and II strains encoded one more VgrG/PAAR spike protein than did clade III. Thus, we predicted that the presence of additional virulence-related genes may have enabled the unique clade I strain to become predominant in the U.S. outbreak.

Dual species dynamic transcripts reveal the interaction mechanisms between Chrysanthemum morifolium and Alternaria alternata

BackgroundChrysanthemum (Chrysanthemum morifolium) black spot disease caused by Alternaria alternata is one of the plant’s most destructive diseases. Dual RNA-seq was performed to simultaneously assess their transcriptomes to analyze the potential interaction mechanism between the two species, i.e., host and pathogen.ResultsC. morifolium and A. alternata were subjected to dual RNA-seq at 1, 12, and 24 h after inoculation, and differential expression genes (DEGs) in both species were identified. This analysis confirmed 153,532 DEGs in chrysanthemum and 14,932 DEGs in A. alternata, which were involved in plant-fungal interactions and phytohormone signaling. Fungal DEGs such as toxin synthesis related enzyme and cell wall degrading enzyme genes played important roles during chrysanthemum infection. Moreover, a series of key genes highly correlated with the early, middle, or late infection stage were identified, together with the regulatory network of key genes annotated in the Plant Resistance Genes database (PRGdb) or Pathogen-Host Interactions database (PHI-base). Highly correlated genes were identified at the late infection stage, expanding our understanding of the interplay between C. morifolium and A. alternata. Additionally, six DEGs each from chrysanthemum and A. alternata were selected for quantitative real-time PCR (qRT-PCR) assays to validate the RNA-seq output.ConclusionsCollectively, data obtained in this study enriches the resources available for research into the interactions that exist between chrysanthemum and A. alternata, thereby providing a theoretical basis for the development of new chrysanthemum cultivars with resistance to pathogen.

Phloretin, an Apple Phytoalexin, Affects the Virulence and Fitness of Pectobacterium brasiliense by Interfering With Quorum-Sensing

The effects of phloretin a phytoalexin from apple, was tested on Pectobacterium brasiliense (Pb1692), an emerging soft-rot pathogen of potato. Exposure of Pb1692 to 0.2 mM phloretin a concentration that does not affect growth, or to 0.4 mM a 50% growth inhibiting concentration (50% MIC), reduced motility, biofilm formation, secretion of plant cell wall-degrading enzymes, production of acyl–homoserine lactone (AHL) signaling molecules and infection, phenotypes that are associated with bacterial population density-dependent system known as quorum sensing (QS). To analyze the effect of growth inhibition on QS, the activity of ciprofloxacin, an antibiotic that impairs cell division, was compared to that of phloretin at 50% MIC. Unlike phloretin, the antibiotic hardly affected the tested phenotypes. The use of DH5α, a QS-negative Escherichia coli strain, transformed with an AHL synthase (ExpI) from Pb1692, allowed to validate direct inhibition of AHL production by phloretin, as demonstrated by two biosensor strains, Chromobacterium violaceaum (CV026) and E. coli (pSB401). Expression analysis of virulence-related genes revealed downregulation of QS-regulated genes (expI, expR, luxS, rsmB), plant cell wall degrading enzymes genes (pel, peh and prt) and motility genes (motA, fim, fliA, flhC and flhD) following exposure to both phloretin concentrations. The results support the inhibition of ExpI activity by phloretin. Docking simulations were used to predict the molecular associations between phloretin and the active site of ExpI, to suggest a likely mode of action for the compound’s inhibition of virulence.

Characterization of sucrose non-fermenting-1 (SNF1) homologue gene in Fusarium udum WSP-V2 and its regulation by the biocontrol agent Pseudomonas fluorescens OKC.

Sucrose non-fermenting 1 (SNF1) is a protein kinase and plays an important role in the energy homeostasis of glucose repressible gene transcription. It derepresses glucose repressed genes and associated with pathogenesis and production of cell wall degrading enzymes in fungal species. In the present study, we identified and characterized SNF1 homologue FuSNF1 in the F. udum strain WSP-V2. Transcript analysis of FuSNF1 along with the MAP kinases and some cell wall degrading enzyme (CWDE) genes of F. udum during interaction with pigeonpea revealed that most MAP kinases and CWDE genes was positively correlated with the FuSNF1 gene. Interestingly, transcript accumulation of all these genes was lowered when pigeonpea seeds were bioprimed with a PGPR strain Pseudomonas fluorescens OKC. Transcript accumulation of FuSNF1 was observed from the day of inoculation and reached maximum level on day 7 in OKC non-bioprimed plants. However, transcript accumulation was low (1.5 fold) in F. udum inoculated with pigeonpea plants bioprimed with OKC. Transcript accumulation patterns of the F. udum MAP Kinases genes and CWDE genes also showed a similar trend and their transcript accumulation was lowered in the OKC bioprimed treatment. The results thus indicate a prime role of FuSNF1 in regulating pathogenicity and virulence of F. udum. The results further emphasize the importance of application of effective PGPR strains in regulating virulence of F. udum. In silico analysis of the SNF1 reference proteins from different fungal species showed that their homologue FuSNF1 is likely to be thermostable and acidic in nature.

Molecular Events Occurring During Softening of Strawberry Fruit.

Changes in fruit texture taking place during ripening, described as softening, are mainly due to alterations in structure and/or composition of the cell wall. Several non-covalent interactions between the three carbohydrate polymers of the cell wall, cellulose, pectins and hemicellulose, and many structural proteins and ions, enable a complex structure. During softening, the disassembly of the cell wall structure takes place, mediated by a complete set of cell wall degrading enzymes or proteins. Softening is a coordinated event that requires the orchestrated participation of a wide variety of proteins. Plant hormones and a set of transcription factors are the organizers of this multi-protein effort. Strawberry is a non climacteric fruit that softens intensively during the last stages of development. The Chilean strawberry fruit (Fragaria chiloensis), the maternal relative of the commercial strawberry (F. × ananassa), softens even faster than commercial strawberry. Softening of the Chilean strawberry fruit has been studied at different levels: changes in cell wall polymers, activity of cell wall degrading enzymes and transcriptional changes of their genes, providing a general view of the complex process. The search for the ‘orchestra director’ that could coordinate softening events in strawberry fruit has been focussed on hormones like ABA and auxins, and more precisely the relation ABA/AUX. These hormones regulate the expression of many cell wall degrading enzyme genes, and this massive transcriptional change that takes place involves the participation of key transcriptional factors (TF). This review provides an update of the present knowledge regarding the softening of strawberry fruit. Nevertheless, the entire softening process is still under active research especially for the great influence of texture on fruit quality and its high impact on fruit shelf life, and therefore it is expected that new and promising information will illuminate the field in the near future.

A two-component regulatory system VfmIH modulates multiple virulence traits in Dickeya zeae.

Bacterial pathogen Dickeya zeae strain EC1 produces antibiotics-like phytotoxins called zeamines, which are major virulence determinants encoded by the zms gene cluster. In this study, we identified a zeamine-deficient mutant with a Tn5 insertion in a gene designated as vfmI encoding a two-component system (TCS) sensor histidine kinase (HK), which is accompanied by vfmH encoding a response regulator (RR) at the same genetic locus. Domain analysis shows this TCS is analogous to the VfmIH of D. dadantii, with typical characteristics of sensor HK and RR, respectively, and sharing the same operon. Deletion of either vfmI or vfmH resulted in decreased production of zeamines and cell wall degrading enzymes (CWDEs), and alleviated virulence on rice seeds and potato tubers. In D. dadantii 3937, VfmH was shown to bind to the promoters of vfmA and vfmE, while in D. zeae EC1, VfmH could bind to the promoters of vfmA, vfmE and vfmF. RNA-seq analysis of strain EC1 and its vfmH mutant also showed that the TCS positively regulated a range of virulence genes, including zms, T1SS, T2SS, T3SS, T6SS, flagellar and CWDE genes.

Two members of the velvet family, VmVeA and VmVelB, affect conidiation, virulence and pectinase expression in Valsa mali.

Velvet protein family members are important fungal-specific regulators which are involved in conidial development, secondary metabolism and virulence. To gain a broader insight into the physiological functions of the velvet protein family of Valsa mali, which causes a highly destructive canker disease on apple, we conducted a functional analysis of two velvet protein family members (VmVeA and VmVelB) via a gene replacement strategy. Deletion mutants of VmVeA and VmVelB showed increased melanin production, conidiation and sensitivity to abiotic stresses, but exhibited reduced virulence on detached apple leaves and twigs. Further studies demonstrated that the regulation of conidiation by VmVeA and VmVelB was positively correlated with the melanin synthesis transcription factor VmCmr1. More importantly, transcript levels of pectinase genes were shown to be decreased in deletion mutants compared with those of the wild-type during infection. However, the expression of other cell wall-degrading enzyme genes, including cellulase, hemi-cellulase and ligninase genes, was not affected in the deletion mutants. Furthermore, the determination of pectinase activity and immunogold labelling of pectin demonstrated that the capacity for pectin degradation was attenuated as a result of deletions of VmVeA and VmVelB. Finally, the interaction of VmVeA with VmVelB was identified through co-immunoprecipitation assays. VmVeA and VmVelB play critical roles in conidiation and virulence, probably via the regulation of the melanin synthesis transcription factor VmCmr1 and their effect on pectinase gene expression in V.mali, respectively.

A mitogen-activated protein kinase gene (VmPmk1) regulates virulence and cell wall degrading enzyme expression in Valsa mali

Mitogen-activated protein kinases (MAPKs) play critical roles in the regulation of different developmental processes and hydrolytic enzyme production in many fungal plant pathogens. In this study, an FUS3/KSS1-related MAPK gene, VmPmk1, was identified and characterized in Valsa mali, which causes a highly destructive canker disease on apple. VmPmk1 deletion mutant showed a significant reduction in growth rate in vitro, and could not produce pycnidium, indicating that the MAPK gene is important for growth and asexual development. Also, VmPmk1 played a significant role in response to oxidative stress and in the maintenance of cell wall integrity. More importantly, when deletion mutant was inoculated onto detached apple leaves and twigs, an obvious decrease in lesion size was observed. Furthermore, expression of many cell wall degrading enzyme (CWDE) genes declined in the VmPmk1 deletion mutant during infection. VmPmk1 deletion mutant also showed a significant reduction in activities of CWDEs in both induced media and infection process. Finally, the determination of immunogold labeling of pectin demonstrated that the capacity of degradation pectin was attenuated due to the deletion of VmPmk1. These results indicated that VmPmk1 plays important roles in growth, asexual development, response to oxidative stress, and maintenance of cell wall integrity. More importantly, VmPmk1 is involved in pathogenicity of V. mali mainly by regulating CWDE genes expression.

The genome of the emerging barley pathogen Ramularia collo-cygni.

BackgroundRamularia collo-cygni is a newly important, foliar fungal pathogen of barley that causes the disease Ramularia leaf spot. The fungus exhibits a prolonged endophytic growth stage before switching life habit to become an aggressive, necrotrophic pathogen that causes significant losses to green leaf area and hence grain yield and quality.ResultsThe R. collo-cygni genome was sequenced using a combination of Illumina and Roche 454 technologies. The draft assembly of 30.3 Mb contained 11,617 predicted gene models. Our phylogenomic analysis confirmed the classification of this ascomycete fungus within the family Mycosphaerellaceae, order Capnodiales of the class Dothideomycetes. A predicted secretome comprising 1053 proteins included redox-related enzymes and carbohydrate-modifying enzymes and proteases. The relative paucity of plant cell wall degrading enzyme genes may be associated with the stealth pathogenesis characteristic of plant pathogens from the Mycosphaerellaceae. A large number of genes associated with secondary metabolite production, including homologs of toxin biosynthesis genes found in other Dothideomycete plant pathogens, were identified.ConclusionsThe genome sequence of R. collo-cygni provides a framework for understanding the genetic basis of pathogenesis in this important emerging pathogen. The reduced complement of carbohydrate-degrading enzyme genes is likely to reflect a strategy to avoid detection by host defences during its prolonged asymptomatic growth. Of particular interest will be the analysis of R. collo-cygni gene expression during interactions with the host barley, to understand what triggers this fungus to switch from being a benign endophyte to an aggressive necrotroph.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2928-3) contains supplementary material, which is available to authorized users.

Transgenic Plant-Produced Hydrolytic Enzymes and the Potential of Insect Gut-Derived Hydrolases for Biofuels.

Various perennial C4 grass species have tremendous potential for use as lignocellulosic biofuel feedstocks. Currently available grasses require costly pre-treatment and exogenous hydrolytic enzyme application to break down complex cell wall polymers into sugars that can then be fermented into ethanol. It has long been hypothesized that engineered feedstock production of cell wall degrading (CWD) enzymes would be an efficient production platform for of exogenous hydrolytic enzymes. Most research has focused on plant overexpression of CWD enzyme-coding genes from free-living bacteria and fungi that naturally break down plant cell walls. Recently, it has been found that insect digestive tracts harbor novel sources of lignocellulolytic biocatalysts that might be exploited for biofuel production. These CWD enzyme genes can be located in the insect genomes or in symbiotic microbes. When CWD genes are transformed into plants, negative pleiotropic effects are possible such as unintended cell wall digestion. The use of codon optimization along with organelle and tissue specific targeting improves CWD enzyme yields. The literature teaches several important lessons on strategic deployment of CWD genes in transgenic plants, which is the focus of this review.

RNAi-induced silencing of an effector confers transcriptional oscillation in another group of effectors in the root-knot nematode, Meloidogyne incognita

The sophisticated parasitic tactic of sedentary endoparasitic nematodes seems to involve the simultaneous alteration of the expression of multitude of its effector genes in order to hijack the plant metabolic and developmental pathway. In concordance with this hypothesis, we have targeted some candidate effector genes of Meloidogyne incognita to understand the possible interaction among those effectors for successful infection of the host plant. In vitro RNAi strategy was used to knock down M. incognita-specific pioneer effector genes, such as msp-18, msp-20, msp-24, msp-33 and msp-16 (known to interact with plant transcription factor), to investigate their possible effect on the expression of key cell wall-degrading enzymes (CWDE) and vice versa. Supported by the phenotypic data, intriguingly our study revealed that induced suppression of these pioneer genes cause transcriptional alteration of CWDE genes in M. incognita. This remarkable finding may provide some useful links for future research on nematode effector interaction.

Comparative expression of genes controlling cell wall-degrading enzymes in Verticillium dahliae isolates from olive, potato and sunflower

Verticillium dahliae is a pathogen causing vascular wilts in a broad range of plant species. The production of cell wall degrading enzymes (CWDE) is one of the pathogenicity factors contributing to this disease. The expression of eight CWDE genes was estimated in 15 V. dahliae isolates with different hosts of origin and aggressiveness levels. The tested isolates had similar levels of gene expression after12 h, 24 h, 48 h, and 72 h in the presence of host plant root extracts. Nevertheless, correlation tests revealed a clear association between the pathogenicity and gene expression of pectinase, cutinase, 1,4-β glucosidase, and SNF protein kinase. In contrast, xylanase and endoglucanase G1 correlated negatively with pathogenicity. Over time, V. dahliae produced CWDEs in a sequence, in which pectinases were produced earlier than xylanase and endoglucanase G1. Correlation of gene expression results with the respective hosts of origin of the tested V. dahliae isolates indicated that the latter may be producing their CWDEs according to the cell wall composition of the hosts. Phylogenetic analysis of cDNA-AFLP profiles using V. dahliae mRNA revealed a cohesive clade formed by the isolates from olive and potato, whereas those from sunflower formed a genetically isolated clade. EST sequencing data revealed the exclusive expression of acetylxylan esterase and ornithine decarboxylase in the case of sunflower isolates.

Histone H3K4 methyltransferase globally regulates substrate-dependent activation of cell-wall-degrading enzymes in Magnaporthe oryzae

Here we addressed the involvement of histone methylation at H3 lysine 4 (H3K4me) in substrate-dependent activation of cell-wall-degrading enzymes (CWDEs) in Magnaporthe oryzae. Transcriptional activation of 36 putative CWDEs including cellobiohydrolases, endoglucanases, β-glucosidases, and endoxylanases was assessed by qRT-PCR. Among the CWDE genes, 16 were upregulated during infection and also in media containing their respective substrates with an exception. Interestingly, the substrate-dependent activation of most CWDE genes was severely impaired in a deletion mutant of MoSET1, histone H3K4 methyltransferase in M. oryzae, suggesting that MoSET1 directly or indirectly regulates a wide range of CWDE genes.

The role of transcriptional activator Mg-XlnR in regulation of xylanases, cellulases and virulence of the rice blast fungus Magnaporthe oryzae

Transcriptional activator, XlnR, has been known to play an important role in regulation of cell wall degrading enzymes (CWDEs) in some pathogenic fungi. Here, we used RNA silencing to simultaneously knock-down the expression of two XlnR like genes (Mg-XlnR) in the genome of rice blast fungus Magnaporthe oryzae. Using Northern analysis, various levels of silencing of Mg-XlnR genes were observed in knock-down mutants compared with those in the wildtype. Moreover, the reduction of MG-XLNR mRNA observed in knock-down mutants resulted in the degradation of CWDEs mRNA and their extracellular enzymes such as xylanases and cellulases. The decrease of blast lesions and their expansion on infected leaves observed in silenced mutants indicated an important role of Mg-XlnR in regulation of CWDEs genes, particularly xylanases and virulence of the rice blast fungus.

Mycoparasitism studies of Trichoderma harzianum against Sclerotinia sclerotiorum: evaluation of antagonism and expression of cell wall-degrading enzymes genes.

Trichoderma spp. are known for their biocontrol activity against several plant pathogens. A specific isolate of Trichoderma harzianum, 303/02, has the potential to inhibit the growth of Sclerotinia sclerotiorum, an important agent involved in several crop diseases. In this study, the interaction between T. harzianum 303/02 and mycelia, sclerotia and apothecia of S. sclerotiorum was studied by scanning electron microscopy. RT-qPCR was used to examine the expression of 11 genes potentially involved in biocontrol. T. harzianum 303/02 parasitizes S. sclerotiorum by forming branches that coil around the hyphae. The fungus multiplied abundantly at the sclerotia and apothecia surface, forming a dense mycelium that penetrated the inner surface of these structures. The levels of gene expression varied according to the type of structure with which T. harzianum was interacting. The data also showed the presence of synergistic action between the cell-wall degrading enzymes.

Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi.

Approximately a tenth of all described fungal species can cause diseases in plants. A common feature of this process is the necessity to pass through the plant cell wall, an important barrier against pathogen attack. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, i.e., cellulose, hemicellulose, and pectin. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell wall-degrading enzyme (CWDE) families that are specifically present or enhanced in plant-pathogenic fungi. In this review, we discuss differences between the CWDE arsenal of plant-pathogenic and non-plant-pathogenic fungi, highlight the importance of individual enzyme families for pathogenesis, illustrate the secretory pathway that transports CWDEs out of the fungal cell, and report the transcriptional regulation of expression of CWDE genes in both saprophytic and phytopathogenic fungi.