Mechanisms Of MoWhi2 Involved In Appressorium Formation And Mitophagy In Regulaion Of The Pathogenicity Of Magnaporthe Oryzae

Rice blast is one of the most serious diseases during rice production.With the gradually deepening research on rice and Magnaporthe oryzae,the rice-M.oryzae has become a model system for studying the interaction between plants and pathogenic fungi.Studying the molecular mechanism of the rice blast fungus pathogenicity can not only deepen the understanding of the pathogenic mechanism of phytopathogenic fungi,but also has guiding significance for the green control of rice blast.In M.oryzae,the appressorium and mitophagy are crucial for the infection of M.oryzae,but the molecular mechanisms regulating appressorium formation and mitophagy remain unclear.In this study,MoWhi2 was regarded as the research object to reveal the mechanism by which MoWhi2 regulated appressorium formation and mitophagy in M.oryzae.Analyzing the molecular mechanism of MoWhi2-mediated infection and pathogenesis will provide an theoretical basis for the control of rice blast.(1)MoWhi2—Mo Psr1 regulates the number of appressoria and is involved in the pathogenesis in M.oryzae.Appressorium formation is very important for M.oryzae to invade rice.In the early stage,we obtained a mutant strain that produced multiple appressoria by screening a T-DNA inserted mutant library.We successfully amplified the flanking sequence by a Hi Tail-PCR method and identified it by sequencing.The locus was MGG＿11241,which is highly homologs to the yeast gene WHI2(General stress response protein),thus named Mo WHI2.Then,the Mo WHI2 gene was deleted,and it was found that the Mo WHI2 gene plays important role in mycelial growth,sporulation and pathogenicity.Similar to the phenotypes of the T-DNA insertion mutant,conidia of the Mo WHI2 deletion strain also form multiple appressoria under on the hydrophobic surface.To futher reveal the functions of MoWhi2 in regulating the number of appressoria,then the candidate interactor Mo Psr1 was obtained by screening the c DNA library of M.oryzae via a yeast two-hybrid method with MoWhi2 as a bait protein.The interaction between MoWhi2 and Mo Psr1 were verified by the yeast two-hybrid and coimmunoprecipitation methods.Subcellular localization showed that both were co-located on the plasma membrane and punctate structures.It was observed that conidia of the deletion of Mo WHI2 or Mo PSR1 mutants formed multiple appressoria on the hydrophobic surface,and had abnormal division of nuclei during appressorium formation and markedly prolonged autophagy within conidia.In contrast,rapamycin,an inhibitor of TOR,can restore the single appressorium phenotype of the ΔMowhi2 or ΔMopsr1 strains.It was indicated that MoWhi2 or Mo Psr1 is involved in the appressorium formation by negatively regulating TOR signaling pathway.In addition,the study found that the c AMP contents of the mutant strains ΔMowhi2 or ΔMopsr1 were significantly increased.Therefore,we speculated that MoWhi2 or Mo Psr1 regulates appressorium formation by negatively regulating c AMP accumulation.Further analysis found that the appressoria from ΔMowhi2or ΔMopsr1 could not effectively penetrate the rice surface,and the spread of infected hypha was slowed down in the ΔMowhi2 or ΔMopsr1 strains.In summary,the experimental results showed that MoWhi2 or Mo Psr1 regulate appressorium formation by negatively regulating TOR and c AMP signaling pathways,thereby controlling the pathogenicity in M.oryzae.(2)MoWhi2—Mo Uth1 mediates mitophagy and regulates pathogenicity in M.oryzae.In addition to regulating appressorium formation,MoWhi2 was also required for mitophagy.In yeast,Whi2 is involved in mitophagy,but the mechanism remain unclear.Studies had shown that the deletion of Mo WHI2 inhibited the mitophagy under nitrogen starvation conditions.To analyze the role of MoWhi2 in regulating mitophagy,by screening the c DNA library of M.oryzae,the candidate protein Mo Uth1 was obtained to interact with MoWhi2.And the interaction between MoWhi2 and Mo Uth1 was verified by the yeast two-hybrid DUAL membrane system and the co-immunoprecipitation method.The Mo UTH1 gene deletion strains showed obvious defects in growth,sporulation and pathogenicity.Similar to ΔMowhi2,mitophagy was blocked in ΔMouth1 strain under nitrogen starvation conditions.Further study revealed that mitophagy in foot cells was disrupted in theΔMowhi2 or ΔMouth1 strains,resulting in a decrease in the number of conidia and abnormal morphology.The mitophagy in the invasive hyphae was restrained,resulting in a decrease in the spread of invasive hyphae.In addition,it was found that MoWhi2 or Mo Uth1 was not required for macroautophagy,pexophagy,and CVT pathway.These results suggested that MoWhi2 or Mo Uth1 is involved in mitophagy,promotes the production of conidia and the invasive hyphae growth,and then affecting the virulence of M.oryzae.(3)Mo Uth1 is the first identified mitophagy receptor in M.oryzae.To study the mechanisms of Mo Uth1 in mitophagy,the protein Mo Atg8 was found to interact with Mo Uth1 by screening the c DNA library of M.oryzae via a yeast two-hybrid assay.Then,the interaction between Mo Uth1 and Mo Atg8 was verified further by a yeast two-hybrid DUAL membrane system and the co-immunoprecipitation method.Mo Uth1 was predicted to contain 6 AIM domains by i LIR website.AIM domains mutation study indicated that Mo Uth1 interacts with Mo Atg8 via the 1/4/6 AIM domains.Laser confocal observation and biochemical experiments showed that Mo Uth1 exists in the outer membrane of mitochondria.Upon nitrogen starvation,Mo Uth1 accompanied mitochondria to enter the vacuole for degradation,while the gene deletion mutant of Mo ATG8 inhibited the degradation of Mo Uth1.Subcellular localization revealed that Mo Uth1 and Mo Atg8 colocalize to autophagosomes after nitrogen starvation treatment,and the 1/4/6 AIM point mutation of Mo Uth1 affected Mo Uth1 and Mo Atg8 interaction and colocalization.In addition,m Cherry-Mo Atg8-marked autophagosomes co-localized with Mito-GFP labelled mitochondria upon SD-N treatment for 4 hours,however,this co-localization was disrupted in the ΔMouth1/m Cherry-Mo ATG8/Mito-GFP strains.The results suggested that Mo Uth1 is involved in mitophagy by recruiting Mo Atg8 to mitochondria.All these results suggested that Mo Uth1 may be involved in mitophagy as a mitophagy receptor.Further study found that the gene deletion mutant of Mo WHI2 led to the inhibition of the degradation of Mo Uth1 under nitrogen starvation treatment,indicating that MoWhi2 regulates Mo Uth1-mediated mitophagy.Taken together,MoWhi2 participates in the appressorium formation through regulating the c AMP and TOR signaling pathways,while MoWhi2 regulates mitophagy with the mitophagy receptor Mo Uth1 in M.oryzae.This study demonstrated the molecular mechanism of rice blast fungus regulating appressorium formation and mitophagy.The results deepened the understanding of the pathogenic mechanism of M.oryzae,and provided an theoretical basis for the control of rice blast.