HAG Effector Evolution in Pyricularia Species and Plant Cell Death Suppression by HAG4.

Seventy host-adapted gene (HAG) effector family members from Pyricularia species are found in P. oryzae and three closely related species (isolates LS and 18-2 from an unknown Pyricularia sp., P. grisea, and P. pennisetigena) that share at least eight orthologous HAG family members with P. oryzae. The genome sequence of a more distantly related species, P. penniseti, lacks HAG genes, suggesting a time frame for the origin of the gene family in the genus. In P. oryzae, HAG4 is uniquely found in the genetic lineage that contains populations adapted to Setaria and Oryza hosts. We find a nearly identical HAG4 allele in a P. grisea isolate, suggesting transfer of HAG4 from P. grisea to P. oryzae. HAG4 encodes a suppressor of plant cell death. Yeast two-hybrid screens with several HAG genes independently identify common interacting clones from a rice complementary DNA library, suggesting conservation of protein surface motifs between HAG homologs with as little as 40% protein sequence identity. HAG family orthologs have diverged rapidly and HAG15 orthologs display unusually high rates of sequence divergence compared with adjacent genes suggesting gene-specific accelerated divergence. The sequence diversity of the HAG homologs in Pyricularia species provides a resource for examining mechanisms of gene family evolution and the relationship to structural and functional evolution of HAG effector family activity. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Evolution and Regulation of a Large Effector Family of Pyricularia oryzae.

Plant pathogen effectors play important roles in parasitism, including countering plant immunity. However, investigations of the emergence and diversification of fungal effectors across host-adapted populations has been limited. We previously identified a gene encoding a suppressor of plant cell death in Pyricularia oryzae (syn. Magnaporthe oryzae). Here, we report the gene is one of a 21-member gene family and we characterize sequence diversity in different populations. Within the rice pathogen population, nucleotide diversity is low, however; the majority of gene family members display presence-absence polymorphism or other null alleles. Gene family allelic diversity is greater between host-adapted populations and, thus, we named them host-adapted genes (HAGs). Multiple copies of HAGs were found in some genome assemblies and sequence divergence between the alleles in two cases suggested they were the result of repeat-induced point mutagenesis. Transfer of family members between populations and novel HAG haplotypes resulting from apparent recombination were observed. HAG family transcripts were induced in planta and a subset of HAGs are dependent on a key regulator of pathogenesis, PMK1. We also found differential intron splicing for some HAGs that would prevent ex planta protein expression. For some genes, spliced transcript was expressed in antiphase with an overlapping antisense transcript. Characterization of HAG expression patterns and allelic diversity reveal novel mechanisms for HAG regulation and mechanisms generating sequence diversity and novel allele combinations. This evidence of strong in planta-specific expression and selection operating on the HAG family is suggestive of a role in parasitism.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Emergence of a hybrid PKS-NRPS secondary metabolite cluster in a clonal population of the rice blast fungus Magnaporthe oryzae.

Secondary metabolites (SMs) are crucial for fungi and vary in function from beneficial antibiotics to pathogenicity factors. To generate diversified SMs that enable different functions, SM-coding regions rapidly evolve in fungal genomes. However, the driving force and genetic mechanism of fungal SM diversification in the context of host-pathogen interactions remain largely unknown. Previously, we grouped field populations of the rice blast fungus Magnaporthe oryzae (syn: Pyricularia oryzae) into three major globally distributed clades based on population genomic analyses. Here, we characterize a recent duplication of an avirulent gene-containing SM cluster, ACE1, in a clonal M. oryzae population (Clade 2). We demonstrate that the ACE1 cluster is specifically duplicated in Clade 2, a dominant clade in indica rice-growing areas. With long-read sequencing, we obtained chromosome-level genome sequences of four Clade 2 isolates, which displayed differences in genomic organization of the ACE1 duplication process. Comparative genomic analyses suggested that the original ACE1 cluster experienced frequent rearrangement in Clade 2 isolates and revealed that the new ACE1 cluster is located in a newly formed and transposable element-rich region. Taken together, these results highlight the frequent mutation and expansion of an avirulent gene-containing SM cluster through transposable element-mediated whole-cluster duplication in the context of host-pathogen interactions.

Magnaporthe oryzae CK2 Accumulates in Nuclei, Nucleoli, at Septal Pores and Forms a Large Ring Structure in Appressoria, and Is Involved in Rice Blast Pathogenesis.

Magnaporthe oryzae (Mo) is a model pathogen causing rice blast resulting in yield and economic losses world-wide. CK2 is a constitutively active, serine/threonine kinase in eukaryotes, having a wide array of known substrates, and involved in many cellular processes. We investigated the localization and role of MoCK2 during growth and infection. BLAST search for MoCK2 components and targeted deletion of subunits was combined with protein-GFP fusions to investigate localization. We found one CKa and two CKb subunits of the CK2 holoenzyme. Deletion of the catalytic subunit CKa was not possible and might indicate that such deletions are lethal. The CKb subunits could be deleted but they were both necessary for normal growth and pathogenicity. Localization studies showed that the CK2 holoenzyme needed to be intact for normal localization at septal pores and at appressorium penetration pores. Nuclear localization of CKa was however not dependent on the intact CK2 holoenzyme. In appressoria, CK2 formed a large ring perpendicular to the penetration pore and the ring formation was dependent on the presence of all CK2 subunits. The effects on growth and pathogenicity of deletion of the b subunits combined with the localization indicate that CK2 can have important regulatory functions not only in the nucleus/nucleolus but also at fungal specific structures such as septa and appressorial pores.

A Magnaporthe Chitinase Interacts with a Rice Jacalin-Related Lectin to Promote Host Colonization.

The genome of rice blast fungus (Magnaporthe oryzae) encodes 15 glycoside hydrolase 18 family chitinases. In this study, we characterized the function of an M. oryzae extracellular chitinase, MoChi1, and its interaction with a host protein, OsMBL1, a jacalin-related Mannose-Binding Lectin (MBL) in rice (Oryza sativa). Deletion of MoChi1 resulted in reduced aerial hyphal formation and reduced virulence in rice by activating the expression of defense-responsive genes. We confirmed MoChi1 interaction with rice OsMBL1 in vitro and in vivo. OsMBL1 was induced by pathogen-associated molecular patterns and M. oryzae infection. Overexpression of OsMBL1 led to activation of rice defense-responsive genes and a chitin-induced reactive oxygen species burst, thereby enhancing resistance to M. oryzae Knockdown of OsMBL1 enhances susceptibility of rice plants to M. oryzae Furthermore, MoChi1 suppressed chitin-induced reactive oxygen species in rice cells and competed with OsMBL1 for chitin binding. Taken together, our study reveals a mechanism in which MoChi1 targets a host lectin to suppress rice immunity.

Population genomic analysis of the rice blast fungus reveals specific events associated with expansion of three main clades.

We examined the genomes of 100 isolates of Magnaporthe oryzae (Pyricularia oryzae), the causal agent of rice blast disease. We grouped current field populations of M. oryzae into three major globally distributed groups. A genetically diverse group, clade 1, which may represent a group of closely related lineages, contains isolates of both mating types. Two well-separated clades, clades 2 and 3, appear to have arisen as clonal lineages distinct from the genetically diverse clade. Examination of genes involved in mating pathways identified clade-specific diversification of several genes with orthologs involved in mating behavior in other fungi. All isolates within each clonal lineage are of the same mating type. Clade 2 is distinguished by a unique deletion allele of a gene encoding a small cysteine-rich protein that we determined to be a virulence factor. Clade 3 isolates have a small deletion within the MFA2 pheromone precursor gene, and this allele is shared with an unusual group of isolates we placed within clade 1 that contain AVR1-CO39 alleles. These markers could be used for rapid screening of isolates and suggest specific events in evolution that shaped these populations. Our findings are consistent with the view that M. oryzae populations in Asia generate diversity through recombination and may have served as the source of the clades 2 and 3 isolates that comprise a large fraction of the global population.

WD40-repeat protein MoCreC is essential for carbon repression and is involved in conidiation, growth and pathogenicity of Magnaporthe oryzae.

Carbon catabolite repression (CCR) is a common regulatory mechanism used by microorganisms to prioritize use of a preferred carbon source (usually glucose). The CreC WD40-repeat protein is a major component of the CCR pathway in Aspergillus nidulans. To clarify the function of the CreC ortholog from Magnaporthe oryzae in regulating gene expression important for pathogenesis, MoCreC was identified and genetically characterized. The vegetative growth rate of the MoCreC deletion mutant on various carbon sources was reduced. The MoCreC mutant produced fewer conidia and with about 60% of conidia having septation defects. Appressorium formation was impaired in the MoCreC mutant. Although some appressoria of the mutant could penetrate the leaf surface successfully, the efficiency of penetration and invasive growth of infection hyphae was reduced, resulting in attenuated virulence toward host plants. The CCR was defective as the mutant was more sensitive to allyl alcohol in the presence of glucose, and 2-deoxyglucose was unable to fully repress utilization of secondary carbon sources. qRT-PCR results indicated that the genes encoding cell wall degradation enzymes, such as ß-glucosidase, feruloyl esterase and exoglucanase, were upregulated in MoCreC mutant. Taken together, we conclude that MoCreC is a major regulator of CCR and plays significant roles in regulating growth, conidiation, and pathogenicity of M. oryzae.

Directional Selection from Host Plants Is a Major Force Driving Host Specificity in Magnaporthe Species.

One major threat to global food security that requires immediate attention, is the increasing incidence of host shift and host expansion in growing number of pathogenic fungi and emergence of new pathogens. The threat is more alarming because, yield quality and quantity improvement efforts are encouraging the cultivation of uniform plants with low genetic diversity that are increasingly susceptible to emerging pathogens. However, the influence of host genome differentiation on pathogen genome differentiation and its contribution to emergence and adaptability is still obscure. Here, we compared genome sequence of 6 isolates of Magnaporthe species obtained from three different host plants. We demonstrated the evolutionary relationship between Magnaporthe species and the influence of host differentiation on pathogens. Phylogenetic analysis showed that evolution of pathogen directly corresponds with host divergence, suggesting that host-pathogen interaction has led to co-evolution. Furthermore, we identified an asymmetric selection pressure on Magnaporthe species. Oryza sativa-infecting isolates showed higher directional selection from host and subsequently tends to lower the genetic diversity in its genome. We concluded that, frequent gene loss or gain, new transposon acquisition and sequence divergence are host adaptability mechanisms for Magnaporthe species, and this coevolution processes is greatly driven by directional selection from host plants.

Retromer Is Essential for Autophagy-Dependent Plant Infection by the Rice Blast Fungus.

The retromer mediates protein trafficking through recycling cargo from endosomes to the trans-Golgi network in eukaryotes. However, the role of such trafficking events during pathogen-host interaction remains unclear. Here, we report that the cargo-recognition complex (MoVps35, MoVps26 and MoVps29) of the retromer is essential for appressorium-mediated host penetration by Magnaporthe oryzae, the causal pathogen of the blast disease in rice. Loss of retromer function blocked glycogen distribution and turnover of lipid bodies, delayed nuclear degeneration and reduced turgor during appressorial development. Cytological observation revealed dynamic MoVps35-GFP foci co-localized with autophagy-related protein RFP-MoAtg8 at the periphery of autolysosomes. Furthermore, RFP-MoAtg8 interacted with MoVps35-GFP in vivo, RFP-MoAtg8 was mislocalized to the vacuole and failed to recycle from the autolysosome in the absence of the retromer function, leading to impaired biogenesis of autophagosomes. We therefore conclude that retromer is essential for autophagy-dependent plant infection by the rice blast fungus.

The exocyst complex: delivery hub for morphogenesis and pathogenesis in filamentous fungi.

Regulated by several small GTPases, the octameric exocyst complex directs the docking and tethering of exocytic vesicles to the destined plasma membrane sites, providing the precise spatiotemporal control of exocytosis. Although the exocyst components are well conserved among various fungal species, the mechanisms for the regulation of its assembly and activity are diverse. Exocytosis is crucial for the generation of cell polarity as well as the delivery of effector proteins in filamentous fungi, and thus plays an important role for fungal morphogenesis and pathogenicity on plant hosts. This review focuses on current findings about the roles of the exocyst complex in the morphogenesis and pathogenesis of filamentous fungi.

Rab GTPases are essential for membrane trafficking-dependent growth and pathogenicity in Fusarium graminearum.

Rab GTPases represent the largest subfamily of Ras-related small GTPases and regulate membrane trafficking. Vesicular transport is a general mechanism that governs intracellular membrane trafficking along the endocytic and exocytic pathways in all eukaryotic cells. Fusarium graminearum is a filamentous fungus and causes the devastating and economically important head blight of wheat and related species. The mechanism of vesicular transport is not well understood, and little is known about Rab GTPases in F. graminearum. In this study, we systematically characterized all eleven FgRabs by live cell imaging and genetic analysis. We find that FgRab51 and FgRab52 are important for the endocytosis, FgRab7 localizes to the vacuolar membrane and regulates the fusion of vacuoles and autophagosomes, and FgRab8 and FgRab11 are important for polarized growth and/or exocytosis. Furthermore, both endocytic and exocytic FgRabs are required for vegetative growth, conidiogenesis, sexual reproduction, as well as pathogenesis and deoxynivalenol metabolism in F. graminearum. Thus, we conclude that Rab GTPases are essential for membrane trafficking-dependent growth and pathogenicity in F. graminearum.

Neurospora crassa ASM-1 complements the conidiation defect in a stuA mutant of Aspergillus nidulans.

Aspergillus nidulans StuA and Neurospora crassa ASM-1 are orthologous APSES (ASM-1, PHD1, SOK2, Efg1, StuA) transcription factors conserved across a diverse group of fungi. StuA and ASM-1 have roles in asexual (conidiation) and sexual (ascospore formation) development in both organisms. To address the hypothesis that the last common ancestor of these diverse fungi regulated conidiation with similar genes, asm-1 was introduced into the stuA1 mutant of A. nidulans. Expression of asm-1 complemented defective conidiophore morphology and restored conidia production to wild type levels in stuA1. Expression of asm-1 in the stuA1 strain did not rescue the defect in sexual development. When the conidiation regulator AbaA was tagged at its C-terminus with GFP in A. nidulans, it localized to nuclei in phialides. When expressed in the stuA1 mutant, AbaA::GFP localized to nuclei in conidiophores but no longer was confined to phialides, suggesting that expression of AbaA in specific cell types of the conidiophore was conditioned by StuA. Our data suggest that the function in conidiation of StuA and ASM-1 is conserved and support the view that, despite the great morphological and ontogenic diversity of their condiphores, the last common ancestor of A. nidulans and N. crassa produced an ortholog of StuA that was involved in conidiophore development.

Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice.

Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression, massively parallel signature sequencing, and sequencing by synthesis to examine transcriptome profiles of infected rice leaves. A total of 6,413 in planta-expressed fungal genes, including 851 genes encoding predicted effector proteins, were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. Four of them induced cell death in Nicotiana benthamiana. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in planta-expressed cell death-inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.

Mesophilic and thermophilic conditions select for unique but highly parallel microbial communities to perform carboxylate platform biomass conversion.

The carboxylate platform is a flexible, cost-effective means of converting lignocellulosic materials into chemicals and liquid fuels. Although the platform's chemistry and engineering are well studied, relatively little is known about the mixed microbial communities underlying its conversion processes. In this study, we examined the metagenomes of two actively fermenting platform communities incubated under contrasting temperature conditions (mesophilic 40°C; thermophilic 55 °C), but utilizing the same inoculum and lignocellulosic feedstock. Community composition segregated by temperature. The thermophilic community harbored genes affiliated with Clostridia, Bacilli, and a Thermoanaerobacterium sp, whereas the mesophilic community metagenome was composed of genes affiliated with other Clostridia and Bacilli, Bacteriodia, γ-Proteobacteria, and Actinobacteria. Although both communities were able to metabolize cellulosic materials and shared many core functions, significant differences were detected with respect to the abundances of multiple Pfams, COGs, and enzyme families. The mesophilic metagenome was enriched in genes related to the degradation of arabinose and other hemicellulose-derived oligosaccharides, and the production of valerate and caproate. In contrast, the thermophilic community was enriched in genes related to the uptake of cellobiose and the transfer of genetic material. Functions assigned to taxonomic bins indicated that multiple community members at either temperature had the potential to degrade cellulose, cellobiose, or xylose and produce acetate, ethanol, and propionate. The results of this study suggest that both metabolic flexibility and functional redundancy contribute to the platform's ability to process lignocellulosic substrates and are likely to provide a degree of stability to the platform's fermentation processes.

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma.

BACKGROUND: Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma. RESULTS: Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei. CONCLUSIONS: The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

acon-3, the Neurospora crassa ortholog of the developmental modifier, medA, complements the conidiation defect of the Aspergillus nidulans mutant.

Aspergillus nidulans and Neurospora crassa are ascomycetes that produce asexual spores through morphologically distinct processes. MedA, a protein with unknown function, is required for normal asexual and sexual development in A. nidulans. We determined that the N. crassa ortholog of medA is acon-3, a gene required for early conidiophore development and female fertility. To test hypotheses about the evolutionary origins of asexual development in distinct fungal lineages it is important to understand the degree of conservation of developmental regulators. The amino acid sequences of A. nidulans MedA and N. crassa ACON-3 shared 37% identity and 51% similarity. acon-3 is induced at late time points of conidiation. In contrast, medA is constitutively expressed and MedA protein localizes to nuclei in all tissue types. Nonetheless, expression of acon-3 using its native promoter complemented the conidiation defects of the A. nidulans ΔmedA and medA15 mutants. We conclude that the biochemical activity of the medA orthologs is conserved for conidiation.

Temporal and spatial regulation of gene expression during asexual development of Neurospora crassa.

In this study we profiled spatial and temporal transcriptional changes during asexual sporulation in the filamentous fungus Neurospora crassa. Aerial tissue was separated from the mycelium to allow detection of genes specific to each tissue. We identified 2641 genes that were differentially expressed during development, which represents ∼25% of the predicted genes in the genome of this model fungus. On the basis of the distribution of functional annotations of 1102 of these genes, we identified gene expression patterns that define key physiological events during conidial development. Not surprisingly, genes encoding transcription factors, cell wall remodeling proteins, and proteins involved in signal transduction were differentially regulated during asexual development. Among the genes differentially expressed in aerial tissues the majority were unclassified and tended to be unique to ascomycete genomes. This finding is consistent with the view that these genes evolved for asexual development in the Pezizomycotina. Strains containing deletions of several differentially expressed genes encoding transcription factors exhibited asexual development-associated phenotypes. Gene expression patterns during asexual development suggested that cAMP signaling plays a critical role in the transition from aerial growth to proconidial chain formation. This observation prompted us to characterize a deletion of the gene encoding a high-affinity cAMP phosphodiesterase (NCU00478). NCU00478 was determined to be allelic to aconidiate-2, a previously identified genetic locus controlling conidiation.

Structure and dynamics of the microbial communities underlying the carboxylate platform for biofuel production.

The carboxylate platform utilizes a mixed microbial community to convert lignocellulosic biomass into chemicals and fuels. While much of the platform is well understood, little is known about its microbiology. Mesophilic (40 degrees C) and thermophilic (55 degrees C) fermentations employing a sorghum feedstock and marine sediment inoculum were profiled using 16S rRNA tag-pyrosequencing over the course of a 30-day incubation. The contrasting fermentation temperatures converted similar amounts of biomass, but the mesophilic community was significantly more productive, and the two temperatures differed significantly with respect to propionic and butyric acid production. Pyrotag sequencing revealed the presence of dynamic communities that responded rapidly to temperature and changed substantially over time. Both temperatures were dominated by bacteria resembling Clostridia, but they shared few taxa in common. The species-rich mesophilic community harbored a variety of Bacteroidetes, Actinobacteria, and gamma-Proteobacteria, whereas the thermophilic community was composed mainly of Clostridia and Bacilli. Despite differences in composition and productivity, similar patterns of functional class dynamics were observed. Over time, organisms resembling known cellulose degraders decreased in abundance, while organisms resembling known xylose degraders increased. Improved understanding of the carboxylate platform's microbiology will help refine platform performance and contribute to our growing knowledge regarding biomass conversion and biofuel production processes.

Functional analysis of an alpha-1,2-mannosidase from Magnaporthe oryzae.

Identification of enzymes that are expressed during host colonization and characterization of their biochemical properties are prerequisite to understanding their role in the pathogen-host interaction. Nine alpha-1,2-mannosidase homologs were identified in the analysis of the Magnaporthe oryzae genome. Endoplasmic reticulum localized alpha-1,2-mannosidases play an important role in protein glycosylation. However, several members of the alpha-1,2-mannosidase gene family are predicted to be secreted. The biological role of such extracellular enzymes in host colonization has not been defined. Here, we characterized a secreted alpha-1,2-mannosidase of M. oryzae, MGG_00994.6, and found that the mature polypeptide is a glycoprotein capable of hydrolyzing alpha-1,2 linked mannobiose. The gene is expressed during growth in vitro and during colonization on rice plants, however, deletion of the gene did not affect pathogenicity. Five other members of the alpha-1,2-mannosidase of M. oryzae were expressed with a pattern similar to MGG_00994.6, suggesting the potential for functional redundancy. These results form the basis for additional studies on the role of this gene family in the rice blast fungus and its interaction with rice.

Gene Ontology annotation of the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: Magnaporthe oryzae, the causal agent of blast disease of rice, is the most destructive disease of rice worldwide. The genome of this fungal pathogen has been sequenced and an automated annotation has recently been updated to Version 6 http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/MultiDownloads.html. However, a comprehensive manual curation remains to be performed. Gene Ontology (GO) annotation is a valuable means of assigning functional information using standardized vocabulary. We report an overview of the GO annotation for Version 5 of M. oryzae genome assembly. METHODS: A similarity-based (i.e., computational) GO annotation with manual review was conducted, which was then integrated with a literature-based GO annotation with computational assistance. For similarity-based GO annotation a stringent reciprocal best hits method was used to identify similarity between predicted proteins of M. oryzae and GO proteins from multiple organisms with published associations to GO terms. Significant alignment pairs were manually reviewed. Functional assignments were further cross-validated with manually reviewed data, conserved domains, or data determined by wet lab experiments. Additionally, biological appropriateness of the functional assignments was manually checked. RESULTS: In total, 6,286 proteins received GO term assignment via the homology-based annotation, including 2,870 hypothetical proteins. Literature-based experimental evidence, such as microarray, MPSS, T-DNA insertion mutation, or gene knockout mutation, resulted in 2,810 proteins being annotated with GO terms. Of these, 1,673 proteins were annotated with new terms developed for Plant-Associated Microbe Gene Ontology (PAMGO). In addition, 67 experiment-determined secreted proteins were annotated with PAMGO terms. Integration of the two data sets resulted in 7,412 proteins (57%) being annotated with 1,957 distinct and specific GO terms. Unannotated proteins were assigned to the 3 root terms. The Version 5 GO annotation is publically queryable via the GO site http://amigo.geneontology.org/cgi-bin/amigo/go.cgi. Additionally, the genome of M. oryzae is constantly being refined and updated as new information is incorporated. For the latest GO annotation of Version 6 genome, please visit our website http://scotland.fgl.ncsu.edu/smeng/GoAnnotationMagnaporthegrisea.html. The preliminary GO annotation of Version 6 genome is placed at a local MySql database that is publically queryable via a user-friendly interface Adhoc Query System. CONCLUSION: Our analysis provides comprehensive and robust GO annotations of the M. oryzae genome assemblies that will be solid foundations for further functional interrogation of M. oryzae.