Transcriptome Analysis Of Rice Blast Fungus

Magnaporthe oryzae causes the rice blast disease,which is one of the most devastating crop diseases worldwide.Although the whole genome sequence of M.oryzae is publicly available,the fungal transcriptome remains incomplete.Therefore,it is required to provide a global view of M.oryzae transcriptome.In order to offer a global view of M.oryzae transcriptome,the author carried out three aspects of analyses on three sets of RNA-Seq data from conidia and vegetative hyphae of two field isolates,P131 and Y34,whose whole genomes were sequenced previously.The first analysis was to reveal how many genes are expressed by the genome of M.oryzae.With a hybrid strategy that integrated genome guided and genome-independent assembles,the author identified 19,418 transcripts from 16,192 genes.This new annotation included 3000 more genes and 6000 more transcripts as compared with the MG8 annotation.The author also identified 80 P131-specific and 140 Y34-specific genes that mainly encode small proteins.Secondly,the author also evaluated importance of alternative splicing in the transcriptome of M.oryzae,and found that 2,358 genes had multiple isoforms of transcritps,which could be generated by 3,479 alternative splicing events.537 alternative transcripts from 249 genes generated distinct proteins.2,112 transcripts from 938 genes contained premature termination codon(PTC),which has been recently reported to trigger the nonsense-mediated decay(NMD)for cell differentiation.Notably,approximately half of the alternative splicing events varied significantly between conidia and hyphae.Expression levels of several genes encoding splicing factors were found to be different between conidia and hyphae,implying that the splicing machinery plays important roles in regulating the alternative splicing.About hundreds of novel protein-coding genes were newly identified,most of which were found to be M.oryzae-specific.More importantly,transcripts of approximate 3,000 novel genes may be long non-coding RNAs(IncRNAs),many of which were specifically expressed in conidia or in vegetative hyphae,Moreover,in more than 150 Agrobacterium tumerfaciens-mediated transformation mutants,the T-DNA was found to insert into loci for IncRNAs.These results suggest that IncRNAs may play important role in regulating conidiation and vegetative hyphae growth of M.oryzae.In addition,the author evaluated expression levels of individual genes and found that 2,108 genes were differentially expressed between conidia and hyphae.Among them,291 and 302 genes were highly expressed in conidia and hyphae,respectively.373 genes were expressed specifically in conidia,whereas 1,142 genes were expressed specifically in hyphae.Some previously reported conidiation-associated and pathogenicity-related genes were included in these differentially expressed genes,suggesting that differential expression of genes is important for vegetative mycelium growth,conidiation,and pathogenesis.In summary,our results enriched contents of M.oryzae transcriptome and will be useful for further understanding of M.oryzae transcriptome.In a previous study of our laboratory,a pathogenicity-essential gene PCG1 was found to encode a novel splicing factor.Deleting PCG1 resulted in reduced splicing efficiency of about one tenth of total introns in M.oryzae.To reveal why splicing efficiency of these introns is affected,the author tried to identify features in the introns in this study.By comparing the unaffected and affected introns,the author found that there are multiple branch point mimic motifs between the 5'splicing site and the branch point in the affected introns.Mutation of the branch point mimic motifs could increase splicing efficiency of the introns in the PCG1 deletion mutant,suggesting that the branch point mimic motifs may interfere in the recognition and binding of the authentic branch point by the spliceosome.