Mapping Of Avr-Pid2 And Deep-sequencing Analysis Of The Magnaporthe Oryzae Isolate FJ81278

Rice blast is one of the most important diseases of rice in the world. It seriously affects stability of crop production and food safety. Developing resistant varieties has been proved to be the most effective and economical way to control this disease. But the resistance could be easily lost within several years after being cultivated in the field, because the prevailing of the virulent isolates which were originally avirulent. The interaction between rice and Magnaporthe oryzae corresponds to―gene for gene‖relationship. The loss or mutation of avirulence genes (AVR genes) may cause the generation of new pathogen races and the loss of host resistance. Therefore, the study of AVR genes is important for understanding the pathogenesis of M. oryzae and can shed light on the mechanisms involved in interaction and co-evolution of hosts and pathogens. It also facilitates the rapid and effective deployment of R genes in rice cultivation.The isolate FJ81278 is a fertile strain of predominant virulence of M.oryzae in Fujian province, China. The progenies of FJ81278 and GUY11 were randomly isolated and their virulences were tested on rice cultivar Pid2 and TP309. The results showed that FJ81278 possesses Avr-Pid2, and the segregation ratio between avirulence and virulence on Pid2 in the progenes fits 1:1, which suggested that Avr-Pid2 in FJ81278 is an independent locus. Two markers based on SSR and PATE (Present Absent Transposon Element) were found to link with Avr-Pid2. The genetic distance between Avr-Pid2 and the marker Propizt and ms7-27 is estimated to be 6.1cM and 13cM respectively. These two markers delimited Avr-Pid2 in a range of 420 kb in chromosome 7, which provides a solid basis for cloning Avr-Pid2.To facilitate the Avr gene cloning, deep-sequencing was also applied to sequence the genome and transcriptome of FJ81278 in this study. By comparing genome sequences of FJ81278 and GUY11, 678 unique genes were identified in the genome sequence of FJ81278. Out of these 678 genes, 161 genes encode proteins with signal peptides. We identified 9759 annotated transcripts and 3706 novel transcripts that were expressed in at least one of the hypha, conidium and appressorium. A substantial number of alternative splicing (AS) events were found in transcripts data, and 13% of the Magnaporthe oryzae genes experience AS events, among which retention of introns was shown to be the major AS event. Besides, 2470 SNPs and 1007 InDels were also identified from the transcripts data. AgriGO was used for functional annotation and enrichment analysis of the expressed genes. The unigenes were functionally categorized into 31 GO categories belonging to biological process, molecular function or cellular components. The analysis of low expressed transcripts showed that they were mainly involved in interaction and stress responds. Deep sequencing will facilitate the identification of novel genes and their expression patterns in FJ81278 during different life stages, provide remarkable insight into the level and tissue specificity of gene expression and contribute to our understanding of fungal pathogenesis, evolution and interaction with hosts.Most of the cloned AVR genes are small novel secreted proteins generally lacking homology to known proteins. In total, 21 specific genes were served as candidates for functional complementation in virulent strain GUY11 and association analysis using field strains in Fujian province. The results showed that the pathogenicities of most mutants were as normal as GUY11, which indicated that these 21 genes might not be the corresponding AVR genes. The association analysis suggested that no distinct association was identified between DNA polymorphism and AVR phenotype. More specific genes and distincted phylogeny strains should be analyzed in future.Most of the 21 specific genes lack typical conserved domains except for MoHsbA, where hydrophobic surface binding protein A (HsbA) domain was found. The amino acid sequence and the gene expression pattern of MoHsbA at different developmental stages were analyzed. The results showed that the most likely signal peptide cleavage site in MoHsbA located between 18A and 19A, and MoHsbA might be a hydrophobic protein. Quantitative RT-PCR results showed that MoHsbA expressed in all the developmental stages, and reached to the highest transcript level at the appressorium period, indicating that MoHsbA might be involved in appressorium development. Taking together, our study may contribute to further functional study of HsbA in Magnaporthe oryzae.