Verification And Preliminary Functional Analysis Of FgATG8 Interacting Proteins From Fusarium Graminearum

Fusarium head blight(FHB)is a global and devastating fungal disease caused by Fusarium graminearum.FHB not only causes a large decrease in wheat production,but also poses a threat to human and animals health because of the fungal toxins such as deoxynivalenol(DON)produced in the process of infecting wheat heads.Wheat is second only to rice in the world and is also one of the main grain crops in our country.High quality and high yield of wheat play an important role in our food security.Autophagy is a highly conserved lysosome/vacuole degradation pathway in eukaryotes,which plays a crucial role in maintaining cell homeostasis.Autophagy is closely related to appressorium development,mycelium formation and infection of plant pathogens at the early stage of infection,and also plays a very important role in the vegetative growth,sporulation,spore germination,infection structure formation and pathogenicity of filamentous fungi.The autophagy process of plant pathogenic fungi is similar to that of yeast.As the core protein of autophagy,ATG8 is almost involved in the whole process of autophagy,and plays an important role in regulating the formation of autophagosomes and the selection of degradation substrates.Previous studies have shown that FgATG8 deficiency decreases the pathogenicity of host wheat,but how does it regulate the pathogenicity of Fusarium graminearum against wheat?And the interaction genes and interaction networks need further study.In this study,FgATG8 was cloned from the cDNA of Fusarium graminearum wild type strain(PH-1),and pGBKT7_FgATG8 vector was constructed.Nine candidate Fusarium graminearum interaction genes were obtained by screening Fusarium graminearum yeast two-hybrid library.By constructing the pGADT7 plasmid of the candidate interaction gene,and carrying out the yeast double-hybrid test with pGBKT7_FgATG8,four Fusarium graminearum genes interacting with FgATG8 were preliminatively identified,whose numbers were Fg2-2,Fg2-3,Fg2-8 and Fg2-9,respectively.At the same time,knockout and complement tests of the above 4 genes were carried out using Split-PCR method,and multiple transformational strain materials were successfully obtained.The growth rate test,pressure response test,conidial yield analysis,spore morphology observation,mycelia marginal branching identification,and pathogenicity analysis of host wheat were also carried out for each mutant.The results showed that compared with PH-1 of Fusarium graminearum wild type standard strain,there was no significant difference in mycelial growth of the 4 gene knockout;The knockout of candidate gene Fg2-2 was more sensitive to Congo Red stress,while the knockout of candidate gene Fg2-9 was less sensitive to Congo Red stress and H2O2 stress.The spore morphology of all mutants was not significantly different from that of PH-1,but the spore production of candidate gene Fg2-2 decreased and that of candidate gene Fg2-8 increased.At the same time,candidate genes numbered Fg2-3,Fg2-8 and Fg2-9 had more mycelial marginal branches than PH-1.The virulence of candidate genes Fg2-8 and Fg2-9 against host wheat was significantly enhanced(P<0.05),suggesting that candidate genes were involved in the process of pathogen infection of wheat.In conclusion,in this study,four genes interacting with Fusarium graminearum autophagy gene FgATG8 were obtained by screening Fusarium graminearum two-hybrid library.Split-PCR method was further used to carry out knockout and complement tests on the four candidate genes,and the growth rate of each mutant was measured in laboratory phenotype and field pathogenicity analysis.The results indicated that only two of the four candidate interaction genes were involved in the pathogenic process of Fusarium graminearum.This study provides research materials and basis for further analysis of the interaction network of Fusarium graminearum FgATG8 and its interaction mechanism in filamentous fungi.