| Rice blast fungus(Pyricularia oryzae syn.Magnaporthe oryzae)is one of the most destructive pathogenic fungi in rice production,leading to significant yield losses world-widely and huge economic losses every year.At present,a series of breakthroughs have been obtained in the understanding its pathogenesis,however,the mechanism underlying the interaction between the fungus and its rice host is still largely obscure.It is known that Effectors play crucial and attractive roles in the interaction between the fungus and rice,cloning and functional analysis of novel effectors will not only help to understand the interaction mechanism involved,but also provide a theoretical basis for continuous control of rice blast disease in rice production.Recently,numerous related research indicated that isolate-unique secreted proteins may be a reservoir of novel effectors.Therefore,in this study,we obtained 37 Guyll-unique secreted proteins as candidate effectors through bioinformatics analysis of re-sequenced genomes of the field isolates Guyll and FJ81278.Then we detected the dynamic expression patterns of all 37 candidate genes by qRT-PCR,and most candidate genes specifically expressed during the early stage of infection,similar to the expression pattern of AvrPib and AvrPi9,indicating that there may be a large number of candidate effectors among these Guy11-unique secreted proteins.Then,we attempted to screen for the effectors which could inhibit cell death by using the Agrobacterium-mediated transient expression system in tobacco,and currently no related functional candidate has been identified.We also conducted gene function analysis by knockout the candidate genes through homologous recombination approach.So far,we have obtained knockout mutants of two candidate genes G03 and G20,and phenotypic analysis showed that,compared with the wild type strain Guyll,the deletion of G03 did not cause any defects in the vegetative growth and pathogenicity,while ΔG20 mutants exhibited obvious defects in vegetative growth and pigment production,but the pathogenicity was normal.In addition,through subcellular localization observations,we found that G30 could accumulate in the biotrophic interfacial complex(BIC)during the rice leaf sheath infection process,indicating that it may be a potential cytoplasmic effector,and G16 was found to localize in plant cell membrane and nucleus when conducting transient expression assays in tobacco leaves,indicating that it may be transferred into host nucleus to play a currently unknown role.Our study provides a system to indentify specific gene functions in the large amount isolates from the rice field. |
