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Characterization of RNAi genes and their impact on sRNA biogenesis, growth and development of the rice blast fungus, Magnaporthe oryzae

ABSTRACT: RNAi is a conserved mechanism in eukaryotic organisms and has been the subject of recent attention pertaining to many animal and plant models. But research related to fungal systems is limited, especially in plant pathogenic models. The present study was undertaken to examine the involvement of RNAi components on the biogenesis of small RNAs and their impact on growth and pathogenicity of the plant pathogenic model organism Magnaporthe oryzae. Through genetic and genomic analyses, we demonstrate that an individual Dicer, an RdRP and an Argonaute are essential for the generation of sRNAs. Collectively, they are involved in biogenesis of sRNAs matching to coding regions, repeats and intergenic regions. The loss of a particular Argonaute made the fungus less virulent on barley leaves and was also essential for sRNA production. Furthermore, one specific RdRP that was not essential to sRNA generation nonetheless reduced fungal virulence. In total, ten deletion mutants were constructed in each of the putative sRNA-generating genes, as well as a double deletion mutant in the two Dicers, and comparison of sRNA and RNA-seq profiles of each mutant yielded new insights into the involvement of sRNAs in transcriptional regulation and intergenic regions in M. oryzae. Together, these data support that RNAi machinery in M. oryzae is not only involved in generation of sRNAs, but is involved in regulating various developmental processes including fungal growth and virulence.

ORGANISM(S): Pyricularia oryzae

PROVIDER: GSE81640 | GEO | 2017/05/22

SECONDARY ACCESSION(S): PRJNA322180

REPOSITORIES: GEO

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Project description:The rice blast fungus, Magnaporthe oryzae is a destructive pathogen of rice and other related crops, causing significant yield losses worldwide. Endogenous small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs) are critical components of gene regulation in many eukaryotic organisms. Recently several new species of sRNAs have been identified in fungi. This fact along with the availability of genome sequence makes M. oryzae a compelling target for sRNA profiling. We have examined sRNA species and their biosynthetic genes in M. oryzae, and the degree to which these elements regulate fungal stress responses. To this end, we have characterized sRNAs under different physiological stress conditions, which had not yet been examined in this fungus. The resulting libraries are composed of more than 37 million total genome matched reads mapping to intergenic regions, coding sequences, retrotransposons, inverted, tandem, and other repeated regions of the genome with more than half of the small RNAs arising from intergenic regions. The 24 nucleotide (nt) size class of sRNAs was predominant. A comparison to transcriptional data of M. oryzae undergoing the same physiological stresses indicates that sRNAs play a role in transcriptional regulation for a small subset of genes. Support for this idea comes from generation and characterization of mutants putatively involved in sRNAs biogenesis; our results indicate that the deletion of Dicer-like genes and an RNA-Dependent RNA Polymerase gene increases the transcriptional regulation of this subset of genes, including one involved in virulence. Various physiological stressors and in planta conditions alter the small RNA profile of the rice blast fungus. Characterization of sRNA biosynthetic mutants helps to clarify the role of sRNAs in transcriptional control. Examination of small RNA populations of eight mycelial as well as In planta tissues of M. oryzae strain 70-15. Mycelia grown in complete medium (CM), minimal medium (MM), carbon starved medium (CS: MM lacking carbon source), nitrogen starved medium (NS: MM lacking nitrogen source) and a reactive oxygen species-rich environment generated by amendment of Paraquat (PQ) to the CM. In planta tissues include mock inoculated rice leaves (LMg0), 72 hours post-inoculation in rice leaves (LMg72) and 96 hours post-inoculation in rice leaves (LMg96).

Project description:Heterochromatic DNA domains play important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. In organisms ranging from fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway. Typically, sRNAs, originating from long noncoding RNAs transcribed from DNA repeats, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and tri-methylation (H3K9me2 and H3K9me3, respectively) and heterochromatin formation. H3K9me is in turn required for recruitment of RNAi to chromatin and promotes sRNA generation. However, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast homolog of mammalian SUV39H H3K9 methyltransferases, we designed active site mutations, which allow H3K9me2 catalysis but block the transition to H3K9me3. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing (CTGS). Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct heterochromatin states and uncover a mechanism for formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in RNAi-mediated genome defense.

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Characterization of RNAi genes and their impact on sRNA biogenesis, growth and development of the rice blast fungus, Magnaporthe oryzae

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