Horizontally Transferred DNA in the Genome of the Fungus Pyricularia oryzae is Associated With Repressive Histone Modifications.

Horizontal gene transfer (HGT) is a means of exchanging genetic material asexually. The process by which horizontally transferred genes are domesticated by the host genome is of great interest but is not well understood. In this study, we determined the telomere-to-telomere genome sequence of the wheat-infecting Pyricularia oryzae strain Br48. SNP analysis indicated that the Br48 strain is a hybrid of wheat- and Brachiaria-infecting strains by a sexual or parasexual cross. Comparative genomic analysis identified several megabase-scale "insertions" in the Br48 genome, some of which were possibly gained by HGT-related events from related species, such as P. pennisetigena or P. grisea. Notably, the mega-insertions often contained genes whose phylogeny is not congruent with the species phylogeny. Moreover, some of the genes have a close homolog even in distantly related organisms, such as basidiomycetes or prokaryotes, implying the involvement of multiple HGT events. Interestingly, the levels of the silent epigenetic marks H3K9me3 and H3K27me3 in a genomic region tended to be negatively correlated with the phylogenetic concordance of genes in the same region, suggesting that horizontally transferred DNA is preferentially targeted for epigenetic silencing. Indeed, the putative HGT-derived genes were activated when MoKmt6, the gene responsible for H3K27me3 modification, was deleted. Notably, these genes also tended to be up-regulated during infection, suggesting that they are now under host control and have contributed to establishing a fungal niche. In conclusion, this study suggests that epigenetic modifications have played an important role in the domestication of HGT-derived genes in the P. oryzae genome.

Novel insights into host specificity of Pyricularia oryzae and Pyricularia grisea in the infection of gramineous plant roots.

Pyricularia oryzae and Pyricularia grisea are pathogens that cause blast disease in various monocots. It has been reported that P. oryzae infects the leaves and roots of rice via different mechanisms. However, it is unclear to what extent the tissue types affect the host specificities of P. oryzae and P. grisea. Here, we evaluated the tissue-specific infection strategies of P. oryzae and P. grisea in various gramineous plants. Generally, mycelial plug inoculation caused root browning but the degree of browning did not simply follow the disease index on leaves. Interestingly, the Triticum and Digitaria pathotypes caused strong root growth inhibition in rice, wheat, and barley. Moreover, the Digitaria pathotype inhibited root branching only in rice. Culture filtrate reproduced these inhibitory effects on root, suggesting that some secreted molecules are responsible for the inhibitions. Observation of root sections revealed that most of the infection hyphae penetrated intercellular spaces and further extended into root cells, regardless of pathotype and host plant. The infection hyphae of Digitaria and Triticum pathotypes tended to localize in the outer layer of rice roots, but not in those of wheat and barley roots. The infection hyphae of the Oryza pathotype were distributed in both the intercellular and intracellular spaces of rice root cells. Pathogenesis-related genes and reactive oxygen species accumulation were induced after root inoculation with all combinations. These results suggest that resistance reactions were induced in the roots of gramineous plants against the infection with Pyricularia isolates but failed to prevent fungal invasion.

Copy number-dependent DNA methylation of the Pyricularia oryzae MAGGY retrotransposon is triggered by DNA damage.

Transposable elements are common targets for transcriptional and post-transcriptional gene silencing in eukaryotic genomes. However, the molecular mechanisms responsible for sensing such repeated sequences in the genome remain largely unknown. Here, we show that machinery of homologous recombination (HR) and RNA silencing play cooperative roles in copy number-dependent de novo DNA methylation of the retrotransposon MAGGY in the fungus Pyricularia oryzae. Genetic and physical interaction studies revealed that RecA domain-containing proteins, including P. oryzae homologs of Rad51, Rad55, and Rad57, together with an uncharacterized protein, Ddnm1, form complex(es) and mediate either the overall level or the copy number-dependence of de novo MAGGY DNA methylation, likely in conjunction with DNA repair. Interestingly, P. oryzae mutants of specific RNA silencing components (MoDCL1 and MoAGO2) were impaired in copy number-dependence of MAGGY methylation. Co-immunoprecipitation of MoAGO2 and HR components suggested a physical interaction between the HR and RNA silencing machinery in the process.

Evolution of an Eleusine-Specific Subgroup of Pyricularia oryzae Through a Gain of an Avirulence Gene.

Eleusine isolates (members of the Eleusine pathotype) of Pyricularia oryzae are divided into two subgroups, EC-I and EC-II, differentiated by molecular markers. A multilocus phylogenetic analysis revealed that these subgroups are very close to Eragrostis isolates. EC-II and Eragrostis isolates were exclusively virulent on finger millet and weeping lovegrass, respectively, while EC-I isolates were virulent on both. The avirulence of EC-II on weeping lovegrass was conditioned by an avirulence gene, PWL1. All EC-II isolates shared a peculiar structure (P structure) that was considered to be produced by an insertion (or translocation) of a DNA fragment carrying PWL1. On the other hand, all EC-I and Eragrostis isolates were noncarriers of PWL1 and shared a gene structure that should have predated the insertion of the PWL1-containing fragment. These results, together with phylogenetic analyses using whole-genome sequences, suggest that the Eleusine-specific subgroup (EC-II) evolved through a loss of pathogenicity on weeping lovegrass caused by a gain of PWL1.

Three ourmia-like viruses and their associated RNAs in Pyricularia oryzae.

Three ourmia-like viruses, designated Pyricularia oryzae ourmia-like virus (PoOLV) 1 to 3, were identified in a wheat-infecting isolate of P. oryzae. The sizes of the full-length PoOLV1-3 genomes were determined to be 2,528, 1,671, and 2,557 nt. Interestingly, we also found two abundant single-stranded RNAs sharing their 5' terminal 25 and 255 nt with PoOLV1 RNA and PoOLV3 RNA, respectively. The PoOLV1- and PoOLV3-associated RNAs (ARNA1 and ARNA3) were 639 and 514 nt in length, and possessed one and two small ORFs, respectively. In the field isolates of P. oryzae, PoOLVs and ARNAs were detectable at varying levels, and the levels of PoOLV1 and ARNA1 as well as those of PoOLV3 and ARNA3, were tightly correlated. In addition, gene silencing of PoOLV1 and PoOLV3 resulted in a reduction of ARNA1 and ARNA3, respectively. There results indicated that replication of ARNA1 and ARNA3 was associated with PoOLV1 and PoOLV3, respectively.

Blast Fungal Genomes Show Frequent Chromosomal Changes, Gene Gains and Losses, and Effector Gene Turnover.

Pyricularia is a fungal genus comprising several pathogenic species causing the blast disease in monocots. Pyricularia oryzae, the best-known species, infects rice, wheat, finger millet, and other crops. As past comparative and population genomics studies mainly focused on isolates of P. oryzae, the genomes of the other Pyricularia species have not been well explored. In this study, we obtained a chromosomal-level genome assembly of the finger millet isolate P. oryzae MZ5-1-6 and also highly contiguous assemblies of Pyricularia sp. LS, P. grisea, and P. pennisetigena. The differences in the genomic content of repetitive DNA sequences could largely explain the variation in genome size among these new genomes. Moreover, we found extensive gene gains and losses and structural changes among Pyricularia genomes, including a large interchromosomal translocation. We searched for homologs of known blast effectors across fungal taxa and found that most avirulence effectors are specific to Pyricularia, whereas many other effectors share homologs with distant fungal taxa. In particular, we discovered a novel effector family with metalloprotease activity, distinct from the well-known AVR-Pita family. We predicted 751 gene families containing putative effectors in 7 Pyricularia genomes and found that 60 of them showed differential expression in the P. oryzae MZ5-1-6 transcriptomes obtained under experimental conditions mimicking the pathogen infection process. In summary, this study increased our understanding of the structural, functional, and evolutionary genomics of the blast pathogen and identified new potential effector genes, providing useful data for developing crops with durable resistance.

A fungal Argonaute interferes with RNA interference.

Small RNA (sRNA)-mediated gene silencing phenomena, exemplified by RNA interference (RNAi), require a unique class of proteins called Argonautes (AGOs). An AGO protein typically forms a protein-sRNA complex that contributes to gene silencing using the loaded sRNA as a specificity determinant. Here, we show that MoAGO2, one of the three AGO genes in the fungus Pyricularia oryzae (Magnaporthe oryzae) interferes with RNAi. Gene knockout (KO) studies revealed that MoAGO1 and MoAGO3 additively or redundantly played roles in hairpin RNA- and retrotransposon (MAGGY)-triggered RNAi while, surprisingly, the KO mutants of MoAGO2 (Δmoago2) showed elevated levels of gene silencing. Consistently, transcript levels of MAGGY and mycoviruses were drastically reduced in Δmoago2, supporting the idea that MoAGO2 impeded RNAi against the parasitic elements. Deep sequencing analysis revealed that repeat- and mycovirus-derived small interfering RNAs were mainly associated with MoAGO2 and MoAGO3, and their populations were very similar based on their size distribution patterns and positional base preference. Site-directed mutagenesis studies indicated that sRNA binding but not slicer activity of MoAGO2 was essential for the ability to diminish the efficacy of RNAi. Overall, these results suggest a possible interplay between distinct sRNA-mediated gene regulation pathways through a competition for sRNA.

Ralfuranones contribute to mushroom-type biofilm formation by Ralstonia solanacearum strain OE1-1.

After invasion into intercellular spaces of tomato plants, the soil-borne, plant-pathogenic Ralstonia solanacearum strain OE1-1 forms mushroom-shaped biofilms (mushroom-type biofilms, mBFs) on tomato cells, leading to its virulence. The strain OE1-1 produces aryl-furanone secondary metabolites, ralfuranones (A, B, J, K and L), dependent on the quorum sensing (QS) system, with methyl 3-hydroxymyristate (3-OH MAME) synthesized by PhcB as a QS signal. Ralfuranones are associated with the feedback loop of the QS system. A ralfuranone productivity-deficient mutant (ΔralA) exhibited significantly reduced growth in intercellular spaces compared with strain OE1-1, losing its virulence. To analyse the function of ralfuranones in mBF formation by OE1-1 cells, we observed cell aggregates of R. solanacearum strains statically incubated in tomato apoplast fluids on filters under a scanning electron microscope. The ΔralA strain formed significantly fewer microcolonies and mBFs than strain OE1-1. Supplementation of ralfuranones A, B, J and K, but not L, significantly enhanced the development of mBF formation by ΔralA. Furthermore, a phcB- and ralA-deleted mutant (ΔphcB/ralA) exhibited less formation of mBFs than OE1-1, although a QS-deficient, phcB-deleted mutant formed mBFs similar to OE1-1. Supplementation with 3-OH MAME significantly reduced the formation of mBFs by ΔphcB/ralA. The application of each ralfuranone significantly increased the formation of mBFs by ΔphcB/ralA supplied with 3-OH MAME. Together, our findings indicate that ralfuranones are implicated not only in the development of mBFs by strain OE1-1, but also in the suppression of QS-mediated negative regulation of mBF formation.

The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces.

The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants remains unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to biofilm formation by OE1-1, which is required for OE1-1 virulence.

MoSET1 (Histone H3K4 Methyltransferase in Magnaporthe oryzae) Regulates Global Gene Expression during Infection-Related Morphogenesis.

Here we report the genetic analyses of histone lysine methyltransferase (KMT) genes in the phytopathogenic fungus Magnaporthe oryzae. Eight putative M. oryzae KMT genes were targeted for gene disruption by homologous recombination. Phenotypic assays revealed that the eight KMTs were involved in various infection processes at varying degrees. Moset1 disruptants (Δmoset1) impaired in histone H3 lysine 4 methylation (H3K4me) showed the most severe defects in infection-related morphogenesis, including conidiation and appressorium formation. Consequently, Δmoset1 lost pathogenicity on wheat host plants, thus indicating that H3K4me is an important epigenetic mark for infection-related gene expression in M. oryzae. Interestingly, appressorium formation was greatly restored in the Δmoset1 mutants by exogenous addition of cAMP or of the cutin monomer, 16-hydroxypalmitic acid. The Δmoset1 mutants were still infectious on the super-susceptible barley cultivar Nigrate. These results suggested that MoSET1 plays roles in various aspects of infection, including signal perception and overcoming host-specific resistance. However, since Δmoset1 was also impaired in vegetative growth, the impact of MoSET1 on gene regulation was not infection specific. ChIP-seq analysis of H3K4 di- and tri-methylation (H3K4me2/me3) and MoSET1 protein during infection-related morphogenesis, together with RNA-seq analysis of the Δmoset1 mutant, led to the following conclusions: 1) Approximately 5% of M. oryzae genes showed significant changes in H3K4-me2 or -me3 abundance during infection-related morphogenesis. 2) In general, H3K4-me2 and -me3 abundance was positively associated with active transcription. 3) Lack of MoSET1 methyltransferase, however, resulted in up-regulation of a significant portion of the M. oryzae genes in the vegetative mycelia (1,491 genes), and during infection-related morphogenesis (1,385 genes), indicating that MoSET1 has a role in gene repression either directly or more likely indirectly. 4) Among the 4,077 differentially expressed genes (DEGs) between mycelia and germination tubes, 1,201 and 882 genes were up- and down-regulated, respectively, in a Moset1-dependent manner. 5) The Moset1-dependent DEGs were enriched in several gene categories such as signal transduction, transport, RNA processing, and translation.

Substrate-induced transcriptional activation of the MoCel7C cellulase gene is associated with methylation of histone H3 at lysine 4 in the rice blast fungus Magnaporthe oryzae.

The mechanisms involved in substrate-dependent regulation of a Magnaporthe oryzae gene encoding a cellulase which we designate MoCel7C (MGG_14954) were investigated. The levels of MoCel7C transcript were dramatically increased more than 1,000-fold, 16 to 24 h after transfer to a medium containing 2% carboxymethylcellulose (CMC), while levels were very low or undetectable in conventional rich medium. Green fluorescent protein reporter assays showed that the MoCel7C promoter was activated by cello-oligosaccharides larger than a pentamer. CMC-induced activation of the MoCel7C promoter was suppressed by glucose and cellobiose. Chromatin immunoprecipitation assays revealed that histone H3 methylation on lysine 4 (H3K4) at the MoCel7C locus was associated with activation of the gene by CMC. Consistently, CMC-induced MoCel7C gene activation was drastically diminished in a knockout (KO) mutant of the MoSET1 gene, which encodes a histone lysine methyltransferase that catalyzes H3K4 methylation in M. oryzae. Interestingly, however, MoCel7C transcript levels under noninducing conditions were significantly increased in the MoSET1 KO mutant, suggesting that MoSET1 directly or indirectly plays a role in both activation and suppression of the MoCel7C gene in response to environmental signals. In addition, gene expression and silencing vectors using the MoCel7C promoter were constructed.

Is the fungus Magnaporthe losing DNA methylation?

The long terminal repeat retrotransposon, Magnaporthe gypsy-like element (MAGGY), has been shown to be targeted for cytosine methylation in a subset of Magnaporthe oryzae field isolates. Analysis of the F1 progeny from a genetic cross between methylation-proficient (Br48) and methylation-deficient (GFSI1-7-2) isolates revealed that methylation of the MAGGY element was governed by a single dominant gene. Positional cloning followed by gene disruption and complementation experiments revealed that the responsible gene was the DNA methyltransferase, MoDMT1, an ortholog of Neurospora crassa Dim-2. A survey of MAGGY methylation in 60 Magnaporthe field isolates revealed that 42 isolates from rice, common millet, wheat, finger millet, and buffelgrass were methylation proficient while 18 isolates from foxtail millet, green bristlegrass, Japanese panicgrass, torpedo grass, Guinea grass, and crabgrass were methylation deficient. Phenotypic analyses showed that MoDMT1 plays no major role in development and pathogenicity of the fungus. Quantitative polymerase chain reaction analysis showed that the average copy number of genomic MAGGY elements was not significantly different between methylation-deficient and -proficient field isolates even though the levels of MAGGY transcript were generally higher in the former group. MoDMT1 gene sequences in the methylation-deficient isolates suggested that at least three independent mutations were responsible for the loss of MoDMT1 function. Overall, our data suggest that MoDMT1 is not essential for the natural life cycle of the fungus and raise the possibility that the genus Magnaporthe may be losing the mechanism of DNA methylation on the evolutionary time scale.

Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific, toxin-producing fungus Alternaria alternata†Japanese pear pathotype.

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant-pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.

Molecular cloning of the apoptosis-related calcium-binding protein AsALG-2 in Avena sativa.

Victorin, the host-selective toxin produced by the fungus Cochliobolus victoriae, induces programmed cell death (PCD) in victorin-sensitive oat lines with characteristic features of animal apoptosis, such as mitochondrial permeability transition, chromatin condensation, nuclear DNA laddering and rRNA/mRNA degradation. In this study, we characterized a calcium-binding protein, namely AsALG-2, which might have a role in the victorin-induced PCD. AsALG-2 is homologous to the Apoptosis-Linked Gene ALG-2 identified in mammalian cells. Northern blot analysis revealed that the accumulation of AsALG-2 transcripts increased during victorin-induced PCD, but not during necrotic cell death. Salicylic acid, chitosan and chitin strongly activated the expression of general defence response genes, such as PR-10; however, neither induced cell death nor the accumulation of AsALG-2 mRNA. Pharmacological studies indicated that victorin-induced DNA laddering and AsALG-2 expression were regulated through similar pathways. The calcium channel blocker, nifedipine, moderately inhibited the accumulation of AsALG-2 mRNA during cell death. Trifluoperazine (calmodulin antagonist) and K252a (serine-threonine kinase inhibitor) reduced the victorin-induced phytoalexin accumulation, but did not prevent the victorin-induced DNA laddering or accumulation of AsALG-2 mRNA. Taken together, our investigations suggest that there is a calcium-mediated signalling pathway in animal and plant PCD in common.

Cellulases belonging to glycoside hydrolase families 6 and 7 contribute to the virulence of Magnaporthe oryzae.

Upon infection, phytopathogenic fungi secrete an array of hydrolytic enzymes that can degrade components of the host epidermis, including waxes, the cuticle, and cell walls. Cellulases, which can hydrolyze crystalline cellulose in the plant cell wall, are among these hydrolytic enzymes. Here, we provide RNAi-based evidence to show that cellulases belonging to glycosyl hydrolase (GH) families 6 and 7 contribute to the penetration of the host epidermis and further invasion by the phytopathogenic fungus Magnaporthe oryzae. The GH6 and GH7 cellulases likely include all members of the cellobiohydrolase family and some endoglucanases in M. oryzae. Quantitative reverse-transcriptase polymerase chain reaction analysis indicated that more than half of the cellulases were highly induced during infection. We constructed knock-down (KD) mutants of these cellulases using the building blocks method we reported previously. The transcript levels of the target genes and cellulase activity were considerably reduced in the KD mutants. The KD mutants resulted in fewer lesions, less penetration, and infection of fewer cells compared with the parent strain. Cytological analyses showed that a high rate of papilla formation blocked invasion of the KD mutants into host cells. These results suggest that the GH6 and GH7 cellulases play roles in the virulence of M. oryzae.

Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species.

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.

The Trickster in the genome: contribution and control of transposable elements.

In mythology, the Trickster is an archetype who typically behaves selfishly and delights in playing tricks and breaking ordinary rules. In many myths and folktales, however, the Trickster also brings new knowledge and, ultimately, has positive effects on the community. Transposable elements (TEs) might have played such a role in the story of genome evolution. TEs can cause nonroutine genetic events like insertional mutations and ectopic recombination that provide a fundamental source of genetic variation, but they can also be a potential threat to genome integrity. Thus, the activity of TEs is usually controlled by an array of sophisticated mechanisms for genome defense. Recent findings indicate that TEs are important components of eukaryotic genomes, often to a much larger extent than ever anticipated. In this review, I focus on the contributions of TEs to various aspects of genome evolution. In addition, why TEs are specific targets for the genome defense mechanisms is discussed.

Simultaneous silencing of endo-ß-1,4 xylanase genes reveals their roles in the virulence of Magnaporthe oryzae.

Due to functional redundancy, it is often difficult to genetically analyse the biological function of fungal cell wall-degrading enzymes that belong to a gene family. To overcome this difficulty, we used RNAi to knock-down (KD) multiple xylanase genes to elucidate their roles in the pathogenicity of the blast fungus, Magnaporthe oryzae. To obtain the maximum average efficiency of gene silencing for the xylanase genes, we used the 'building blocks method', in which a 40 bp sequence was chosen from an endoxylanase gene, and 10 such sequences from 10 endoxylanases were combined to make an artificial RNAi trigger by synthetic DNA. Quantitative RT-PCR analysis revealed that the transcript levels of all the expressed xylanase genes were significantly reduced in KD mutants with the artificial RNAi trigger. Even though the KD mutants did not completely lose their pathogenicity to host plants, the number of lesions, rate of penetration and extent of infected cells were all reduced in KD mutant-infected leaves. The degree of pathogenicity reduction was associated with the silencing levels of xylanase mRNA and enzymatic activity in the KD mutants. Cytological analysis indicated that xylanases play significant roles in both vertical penetration and horizontal expansion of M. oryzae in infected plants.