Identification And Functional Analysis Of Unique Genes In Isolate Guy11 Of Magnaporthe Oryzae

Rice(Oryza sativa)is a staple food crop for more than 50%population all over the world,with the large proportion of rice consumers in developing countries including China.Rice blast caused by filamentous ascomycete fungus Magnaporthe oryzae is one of most destructive rice diseases,leading to significant yield losses world-widely and posing huge challenges to global food security especially for China.To date,one of the most effective and economical methods of controlling rice blast is the utilization of appropriate resistant cultivars which contain major resistant genes against rice blast.However,due to the selection pressure from the host,avirulence gene is highly instable.M oryzae is able to overcome the resistance cultivars within 1-3 years of their first deployment in the field.So far there are more than 20 rice blast resistance genes have been cloned,while the cloned avirulence genes are still very limited.Cloning novel avirulence genes would help us know better about the mutational mechanism of the avirulence gene and interaction mechanism with the resistance gene which will be useful for controlling the rice blast in the near future.In this study,we re-sequenced the genomes of M.oryzae field isolates:Guy11 and FJ81278 due to their significant difference in the pathogenicity.Guy11 is avirulent to the resistant rice with resistance gene Pid3,while FJ81278 is virulent to the resistance gene Pid3.We postulated that Guy11 carries avirulence gene AvrPid3 which is absent in FJ81278.A total of 0.87Mb sequences,including 37 isolate-unique secreted proteins which presented in the genome of Guy11 rather than FJ81278 were identified through comparative genomics.Uniqueness validation was conducted by PCR amplification and sequencing.Consistent with the known effector features,the majority of these proteins are small,cysteine-rich,specifically expressed at the early stage of infectious process,and possessed presence or absence polymorphisms among different field isolates.Among these unique genes,some of them had been reported involved in the infectious process of M oryzae.Such as Ung31 could cause programmed cell death in the rice protoplast and tobacco(Nicotiana benthamiana)leaves.Meanwhile,due to the different origin of Guy11 and FJ81278,unique genes owned the diverse distribution patterns in the strains isolated from different rice varieties(Oryza sativa ssp.indica and japonica).According to the association analysis,we identified Ung30 which was a member of sequence-unrelated but structurally conserved fungal MAX(Magnaporthe Avrs and ToxB like)effectors family showed partial association with the function of avirulence protein AvrPid3.Genetic complementation demonstrated AvrPid3 was not obtained after a series of transformation so far.In the end,we did not find any Guy11 unique gene could trigger programmed cell death in N.benthamiana leaves by Agrobacterium-mediated transient expression system to date.In conclusion,our comparative genomics approach is effective to generate a list of unique genes which could encode candidate effectors.Fully understanding the function of these unique genes will help us learn more about infectious process of M.oryzae and provide scientific support for better control of the rice blast disease.