Project description:This SuperSeries is composed of the following subset Series: GSE8517: Magnaporthe oryzae gene expression during biotrophic invasion of rice using version 2 of the Agilent Magnaporthe grisea Array (G4137B). GSE8518: Rice gene expression during biotrophic invasion by the rice blast fungus Magnaporthe oryzae using the Agilent Rice Array (G4138A). Keywords: SuperSeries Refer to individual Series
Project description:The rice blast fungus Magnaporthe oryzae grows inside living host cells. Cytological analyses by live-cell imaging have revealed characteristics of the biotrophic invasion, particularly the extrainvasive hyphal membrane (EIHM) originating from the host plasma membrane and a host membrane-rich structure, biotrophic interfacial complex (BIC). Here, we observed rice subcellular changes associated with invasive hyphal growth using various transformants expressing specifically localized fluorescent proteins. The invasive hyphae did not penetrate across but were surrounded by the host vacuolar membrane together with EIHM even after branching. High-resolution imaging of BICs revealed that the host cytosol was accumulated at BIC with aggregated EIHM and a symplastic effector, Pwl2, in a punctate form. The vacuolar membrane did not aggregate in but closely surrounded the BIC. A good correlation was observed between the early collapse of vacuoles and damage of invasive hyphae in the first-invaded cell. Furthermore, a newly developed, long-term imaging method has revealed that the central vacuole gradually shrank until collapse, which was caused by the hyphal invasion occurring earlier in the neighboring cells than in the first-invaded cells. These data suggest that M. oryzae may suppress host vacuole collapse during early infection stages for successful infection.
Project description:To cause plant diseases, pathogenic micro-organisms secrete effector proteins into host tissue to suppress immunity and support pathogen growth. Bacterial pathogens have evolved several distinct secretion systems to target effector proteins, but whether fungi, which cause the major diseases of most crop species, also require different secretory mechanisms is not known. Here we report that the rice blast fungus Magnaporthe oryzae possesses two distinct secretion systems to target effectors during plant infection. Cytoplasmic effectors, which are delivered into host cells, preferentially accumulate in the biotrophic interfacial complex, a novel plant membrane-rich structure associated with invasive hyphae. We show that the biotrophic interfacial complex is associated with a novel form of secretion involving exocyst components and the Sso1 t-SNARE. By contrast, effectors that are secreted from invasive hyphae into the extracellular compartment follow the conventional secretory pathway. We conclude that the blast fungus has evolved distinct secretion systems to facilitate tissue invasion.
Project description:The blast fungus Magnaporthe oryzae threatens global food security through the widespread destruction of cultivated rice. Foliar infection requires a specialized cell called an appressorium that generates turgor to force a thin penetration hypha through the rice cuticle and into the underlying epidermal cells, where the fungus grows for the first days of infection as a symptomless biotroph. Understanding what controls biotrophic growth could open new avenues for developing sustainable blast intervention programs. Here, using molecular genetics and live-cell imaging, we dismantled M. oryzae glucose-metabolizing pathways to reveal that the transketolase enzyme, encoded by TKL1, plays an essential role in facilitating host colonization during rice blast disease. In the absence of transketolase, Δtkl1 mutant strains formed functional appressoria that penetrated rice cuticles successfully and developed invasive hyphae (IH) in rice cells from primary hyphae. However, Δtkl1 could not undertake sustained biotrophic growth or cell-to-cell movement. Transcript data and observations using fluorescently labeled histone H1:RFP fusion proteins indicated Δtkl1 mutant strains were alive in host cells but were delayed in mitosis. Mitotic delay could be reversed and IH growth restored by the addition of exogenous ATP, a metabolite depleted in Δtkl1 mutant strains. We show that ATP might act via the TOR signaling pathway, and TOR is likely a downstream target of activation for TKL1. TKL1 is also involved in controlling the migration of appressorial nuclei into primary hyphae in host cells. When taken together, our results indicate transketolase has a novel role in mediating--via ATP and TOR signaling--an in planta-specific metabolic checkpoint that controls nuclear migration from appressoria into primary hyphae, prevents mitotic delay in early IH and promotes biotrophic growth. This work thus provides new information about the metabolic strategies employed by M. oryzae to enable rice cell colonization.
Project description: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.
Project description:BackgroundRice blast fungus Magnaporthe oryzae is one of the most devastating pathogens in rice. Avirulence genes in this fungus share a gene-for-gene relationship with the resistance genes in its host rice. Although numerous studies have shown that rice blast R-genes are extremely diverse and evolve rapidly in their host populations, little is known about the evolutionary patterns of the Avr-genes in the pathogens.ResultsHere, six well-characterized Avr-genes and seven randomly selected non-Avr control genes were used to investigate the genetic variations in 62 rice blast strains from different parts of China. Frequent presence/absence polymorphisms, high levels of nucleotide variation (~10-fold higher than non-Avr genes), high non-synonymous to synonymous substitution ratios, and frequent shared non-synonymous substitution were observed in the Avr-genes of these diversified blast strains. In addition, most Avr-genes are closely associated with diverse repeated sequences, which may partially explain the frequent presence/absence polymorphisms in Avr-genes.ConclusionThe frequent deletion and gain of Avr-genes and rapid non-synonymous variations might be the primary mechanisms underlying rapid adaptive evolution of pathogens toward virulence to their host plants, and these features can be used as the indicators for identifying additional Avr-genes. The high number of nucleotide polymorphisms among Avr-gene alleles could also be used to distinguish genetic groups among different strains.
Project description:Eukaryotic pathogens of humans often evade the immune system by switching the expression of surface proteins encoded by subtelomeric gene families. To determine if plant pathogenic fungi use a similar mechanism to avoid host defenses, we sequenced the 14 chromosome ends of the rice blast pathogen, Magnaporthe oryzae. One telomere is directly joined to ribosomal RNA-encoding genes, at the end of the approximately 2 Mb rDNA array. Two are attached to chromosome-unique sequences, and the remainder adjoin a distinct subtelomere region, consisting of a telomere-linked RecQ-helicase (TLH) gene flanked by several blocks of tandem repeats. Unlike other microbes, M.oryzae exhibits very little gene amplification in the subtelomere regions-out of 261 predicted genes found within 100 kb of the telomeres, only four were present at more than one chromosome end. Therefore, it seems unlikely that M.oryzae uses switching mechanisms to evade host defenses. Instead, the M.oryzae telomeres have undergone frequent terminal truncation, and there is evidence of extensive ectopic recombination among transposons in these regions. We propose that the M.oryzae chromosome termini play more subtle roles in host adaptation by promoting the loss of terminally-positioned genes that tend to trigger host defenses.
Project description:The hemibiotrophic fungus Magnaporthe oryzae produces specialized biotrophic invasive hyphae (IH) that alter membrane structure and defense responses in invaded rice cells. IH successively invade live neighbor cells, apparently through plasmodesmata. Understanding fungal and rice genes that contribute to biotrophic invasion has been a challenge because so few plant cells have encountered IH at the earliest infection stages. Using a rice sheath inoculation method, we successfully enriched for infected tissue RNA that contained ~20% fungal RNA at a point when most IH were still growing in first-invaded rice cells. The RNAs were analyzed using the whole-genome M. oryzae oligoarray and a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included the PWL2 avirulence gene and genes encoding hypothetical secreted proteins. The IH-specific secreted proteins are candidate effectors, proteins that the fungus secretes into live host cells to control cellular processes. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease. Keywords: Disease state analysis
Project description:The hemibiotrophic fungus Magnaporthe oryzae produces specialized biotrophic invasive hyphae (IH) that alter membrane structure and defense responses in invaded rice cells. IH successively invade live neighbor cells, apparently through plasmodesmata. Understanding fungal and rice genes that contribute to biotrophic invasion has been a challenge because so few plant cells have encountered IH at the earliest infection stages. Using a rice sheath inoculation method, we successfully enriched for infected tissue RNA that contained ~20% fungal RNA at a point when most IH were still growing in first-invaded rice cells. The RNAs were analyzed using the whole-genome M. oryzae oligoarray and a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included the PWL2 avirulence gene and genes encoding hypothetical secreted proteins. The IH-specific secreted proteins are candidate effectors, proteins that the fungus secretes into live host cells to control cellular processes. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease. Keywords: Disease state analysis
Project description:Fungi are a rich source of natural products with biological activities. In this study, we evaluated viral effects on secondary metabolism of the rice blast fungus Magnaporthe oryzae using an isolate of APU10-199A co-infected with three types of mycoviruses: a totivirus, a chrysovirus, and a partitivirus. Comparison of the secondary metabolite profile of APU10-199A with that of the strain lacking the totivirus and chrysovirus showed that a mycotoxin tenuazonic (TeA) acid was produced in a manner dependent on the mycoviruses. Virus reinfection experiments verified that TeA production was dependent on the totivirus. Quantitative reverse transcription PCR and RNA-sequencing analysis indicated the regulatory mechanism underlying viral induction of TeA: the totivirus activates the TeA synthetase gene TAS1 by upregulating the transcription of the gene encoding a Zn(II)2-Cys6-type transcription factor, TAS2. To our knowledge, this is the first report that confirmed mycovirus-associated regulation of secondary metabolism at a transcriptional level by viral reinfection. Because only treatment with dimethyl sulfoxide has been reported to trigger TeA production in this fungus without gene manipulation, our finding highlights the potential of mycoviruses as an epigenomic regulator of fungal secondary metabolism.