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.

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.

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.

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.

The ACE1 secondary metabolite gene cluster is a pathogenicity factor of wheat blast fungus.

Wheat blast caused by Pyricularia oryzae pathotype Triticum is now becoming a very serious threat to global food security. Here, we report an essential pathogenicity factor of the wheat blast fungus that is recognized and may be targeted by a rice resistance gene. Map-based cloning of Pwt2 showed that its functional allele is the ACE1 secondary metabolite gene cluster of the wheat blast fungus required for its efficient penetration of wheat cell walls. ACE1 is required for the strong aggressiveness of Triticum, Eleusine, and Lolium pathotypes on their respective hosts, but not for that of Oryza and Setaria pathotypes on rice and foxtail millet, respectively. All ACE1 alleles found in wheat blast population are recognized by a rice resistance gene, Pi33, when introduced into rice blast isolates. ACE1 mutations for evading the recognition by Pi33 do not affect the aggressiveness of the rice blast fungus on rice but inevitably impair the aggressiveness of the wheat blast fungus on wheat. These results suggest that a blast resistance gene already defeated in rice may be revived as a durable resistance gene in wheat by targeting an Achilles heel of the wheat blast fungus.

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.

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.

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.

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.

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.

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 the Eleusine subgroup of Pyricularia oryzae inferred from rearrangement at the Pwl1 locus.

Eleusine isolates (members of the Eleusine subgroup) of Pyricularia oryzae are divided into two groups, Ec-I and Ec-II, differentiated by molecular markers. A multilocus phylogenetic analysis and DNA fingerprinting suggested that Ec-I isolates are very close to Eragrostis isolates rather than Ec-II isolates. Infection assays revealed that Ec-II and Eragrostis isolates were exclusively virulent on finger millet and weeping lovegrass, respectively, whereas Ec-I isolates were virulent on both. The avirulence or virulence on weeping lovegrass perfectly corresponded to the presence or absence of an avirulence gene, PWL1; all Ec-II isolates carried an identical, functional PWL1, whereas none of Ec-I isolates or Eragrostis isolates carried it. A comparison of PWL1 flanking regions revealed that Ec-II isolates had a peculiar structure produced by an insertion (or translocation) of a DNA fragment carrying PWL1. Based on these results, a model was constructed which illustrated possible pathways to the establishment of the Eleusine subgroup.

Methods to study PAMP-triggered immunity using tomato and Nicotiana benthamiana.

Understanding the molecular basis of plant responses to pathogen-associated molecular patterns (PAMPs) is an active area of research in the field of plant-microbe interactions. A growing number of plant genes involved in various steps of PAMP-triggered immunity (PTI) pathways and microbial factors involved in the elicitation or suppression of PTI have been identified. These studies have largely relied on Arabidopsis thaliana and, therefore, most of the PTI assays have been developed and optimized for that model plant system. Although PTI is a conserved feature among plants, the response spectra vary across different species. Thus, there is a need for robust PTI assays in other pathosystems, such as those involving Solanaceae plant-pathogen interactions, which include many economically important plants and their diseases. We have optimized molecular, cellular, and whole-plant methods to measure PTI responses in two widely studied solanaceous species, tomato (Solanum lycopersicum) and Nicotiana benthamiana. Here, we provide detailed protocols for measuring various PTI-associated phenotypes, including bacterial populations after pretreatment of leaves with PAMPs, induction of reporter genes, callose deposition, activation of mitogen-activated protein kinases, and a luciferase-based reporter system. These methods will facilitate limited genetic screens and detailed characterization of potential PTI-related genes in model and economically important Solanaceae spp.-pathogen interactions.

Induction of chlorosis, ROs generation and cell death by a toxin isolated from Pyricularia oryzae.

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.

Systematic functional analysis of calcium-signalling proteins in the genome of the rice-blast fungus, Magnaporthe oryzae, using a high-throughput RNA-silencing system.

We developed an RNA-silencing vector, pSilent-Dual1 (pSD1), with a convergent dual promoter system that provides a high-throughput platform for functional genomics research in filamentous fungi. In the pSD1 system, the target gene was designed to be transcribed as a chimeric RNA with enhanced green fluorescent protein (eGFP) RNA. This enabled us to efficiently screen the resulting transformants using GFP fluorescence as an indicator of gene silencing. A model study with the eGFP gene showed that pSD1-based vectors induced gene silencing via the RNAi pathway with slightly lower efficiency than did hairpin eGFP RNA-expressing vectors. To demonstrate the applicability of the pSD1 system for elucidating gene function in the rice-blast fungus Magnaporthe oryzae, 37 calcium signalling-related genes that include almost all known calcium-signalling proteins in the genome were targeted for gene silencing by the vector. Phenotypic analyses of the silenced transformants showed that at least 26, 35 and 15 of the 37 genes examined were involved in hyphal growth, sporulation and pathogenicity, respectively, in M. oryzae. These included several novel findings such as that Pmc1-, Spf1- and Neo1-like Ca(2+) pumps, calreticulin and calpactin heavy chain were essential for fungal pathogenicity.

Transcriptional control and protein specialization have roles in the functional diversification of two dicer-like proteins in Magnaporthe oryzae.

Quantitative RT-PCR and overexpression studies of two Dicer-like proteins, MoDcl1 and MoDcl2, in Magnaporthe oryzae indicated that the functional diversification of the MoDcl1 and MoDcl2 proteins in RNA-mediated gene silencing pathways was likely to have arisen from both transcriptional control and protein specialization.