Multi-omic Analyses And Characterization Of Novel Pathogenicity-related Genes Of Magnaporthe Oryzae

Rice blast,caused by Magnaporthe oryzae,is one of the most destructive causal agents during rice cultivation severely threaten the yield of rice crops.Recently,the management of rice blast relies on the frequent application of resistant rice cultivars.However,the resistant cultivars often lose their resistance to the pathogen because of easy variation of the pathogen races.Thus,the control of the rice blast disease is still a durable problem,and the molecular mechanisms of the fungi development and its pathogenicity would be crucial.The functional analysis of effectors will provide new insight into the biological characteristic and molecular pathological mechanism of M.oryzae and leading to the prospects for sustainable control of rice blast.In this paper,we applied genomics and transcriptomic tools to investigate the field isolate 98-06.In this paper,we resequenced one field isolate 98-06 collected from Jiangsu Province,which contained 12 AVR genes and showed aggressive infection of susceptible rice cultivars.We constructed the genomic Illumina paired-end libraries of 98-06 isolate using a whole-genome shotgun approach.The sequencing data(135-fold coverage)was placed into scaffolds,providing a perfect assembly.Scaffolds of isolate 98-06 were aligned with the assembled genome of 70-15,and 98-06 contained 1.5 Mb of isolate-specific sequences dispersed throughout its genome.Blocks of the reverse-alignment sequences and chromosomal breakpoints were found at the telomeres that may enable host colonization.Further more,we also analyzed gene prediction,gene clusters,repeated sequences and transposons.The cloned avirulence genes of M.oryzae were mostly less than 200 amino acids in length and were more cysteine rich than the total proteome.We initially distinguished 645 small candidate effector proteins with an average cysteine content of 3.11%of 1732 secreted proteins.Based on the genome of 98-06,the interaction transcriptome of the M.oryzae 98-06 and rice based on RNA-seq at at six stages:mycelial stage,and conidial infection at 0,8,24,48,and 72 h.Millions of reads were aligned to the reference genome of M.oryzae and rice,and almost all genes of both 98-06 and O.sativa(11,078 and 26,452 respectively)were transcribed.The alignment gene expression data was performed for differential expression analysis.The evaluation of pathogenesis-related gene families and effectors revealed two major expression patterns,respectively.The pathogenicity-related pattern represens high expression throughout the infection process,with the exception of sudden downregulation at 24 hpi,and the effectors pattern shows very high expression level only during infection.These results indicate that the key expression patterns represent the typical pathogen-host interaction and can be used to explore novel pathogenicity genes and effectors.Genes related to the SA,JA,and ET pathways showed that SA pathway mainly upregulated expression in response to M.oryzae,indicating that the SA pathway is involved in rice defense mechanisms.In our study,five of these 15 identified effectors were clustered into a same expression pattern.Based on these observations,we further explored 133 candidate avirulence genes with similar expression patterns of Avr-Pik/km/kp and Avr-Pizt from those 645 candiate effectors.Of them,6 unique genes in isolate 98-06 that coded for putative secreted protein were finally identified.Based on the analysis of genome and transcriptome,the candidate effectors were characterized from the 98-06 isolate.PEX6 and PEX9 were unique to 98-06,PEX12 possessed a nuclear localization domain and was present in P131 and Y34,PEX17 and PEX18 were unique to 98-06 and were predicted to share functional domain.The three effectors were all significantly up-regulated during infection,whereas there were no significant differences in transcript levels of PEX17 and PEX18 between the infection and mycelial stages.PEX6,PEX9,PEX17,and PEX18 were revealed to show presence/absence polymorphisms in 42 field isolates.Furthermore,disruption of PEX6 resulted in phenotype associated with impaired conidiogenesis and pathogenicity,suggesting that some isolate-unique genes are involved in pathogen propagation and virulence.The putative effector proteins Pex6 and Pex12 accumulated exclusively in the biotrophic interfacial complex(BIC).Pex9,another isolate-specific effector,was localized mainly in primary hypha and in the BIC.In addition,Pex6,Pex9,and Pex12 could suppress BAX-mediated programmed cell death in Nicotiana benthamiana leaves as effectors,and Pex12 localizes to the nucleolus in N.benthamiana cells.These derived transformants showed virulence against the resistant rice strains IRBLkh-K3,IRBLz5-CA,IRBLsh-S,IRBL1-CL,IRBL7-M,IRBL20-IR24,IRBLta2-Re,and the susceptible strain LTH,suggesting that they did not receive new AVR genes.However,pathogenicity tests of Guy 11 transformed with PEX12 indicated that Pex12 could suppress ETI and facilitate the invasive growth in planta.The finding that Pex12 effector is capable of inhibiting the ETI in rice is the first in M.oryzae.It may be precisely because Pex12 effector present in large amounts isolates of M.oryzae,so ETI suppression of rice caused Pib,Pi 12,Pill resistant variety failure in the field.Identification Pexl2 effector will greatly facilitate the molecular mechanisms of effectors inhibition of plant immune system,and provide a target for the new blast resistance agents.Based on the results above,this study characterizes novel effectors through systemically assaying genome and interaction transcriptome of a dominant field isolate.Three novel isolate-specific effectors were all reminiscent of the telomeric position,revealing that the isolate-specific genes beyond the "core" genome may act as a source for M.oryzae adaptation to environment.Our data provides genetic and molecular insights into the evolutionary mechanism of M.oryzae infection.