Identification And Preliminarily Functional Analysis Of Sumoylation Pathway Enzymes In Aspergillus Flavus

Aspergillus flavus is a common plant pathogenic fungus,which can infects the seeds of oil crops and agricultural products.When growing on the seeds,the fungus produces toxic and carcinogenic aflatoxins,severely threatens food safety and human health.So far,sumoylation modification has been proved to be involved in regulating various life programs in eukaryotic organisms.Previously studies in our laboratory demonstrated that sumoylation can affect the morphogenesis and aflatoxin biosynthesis in A.flavus.The SumO protein of A.flavus is different from the previously reported SUMO family members,which expressed as a natively mature form and can directly enter sumoylation cycle without proteolytic activation.The purpose of this study is to identify the components of the sumoylation pathway,and to pave the way for the subsequent research on sumoylation in A.flavus.We identified four putative sumoylation pathway enzymes encoding genes,including E1(AosA-UbaB),E2(UbcI)and E3(SizA),in A.flavus by bioinformatic analysis.Gene deletion strains were obtained by PCR-mediated gene disruption technology and verified by Southern blot analysis.Then the complementary strains were constructed to compare the pleiotropic phenotypes with deletants and the wild type strain,such as radical growth,development,toxin production and pathogenicity.In addition,HA-tagged SumO protein was expressed in wild type and four putative enzyme gene deletion strains for analyzing the sumoylation status and modification level of total protein in vivo by Western blot.Compared to the wild type strain,the four gene deletion strains were more sensitive to high temperature,oxidant and DNA damage related drugs.The production of conidia and aflatoxin B1 decreased significantly in the deletion strains.Besides that,they produced more sclerotia than wild type strain when incubated at 29?,but less at 37?.When infected the peanut and maize seeds,the four gene deletion strains were crippled in their ability to colonize and sporulate on host seeds,and decreased aflatoxin B1 contamination in seeds was observed.All above were similar with the phenotypes of sumO gene deletion strain,suggesting that aosA,ubaB,ubcI and sizA may play roles in the same pathway with sumO.In the strains expressing HA-SumO,HA-SumO monomer and SumO modified proteins with different molecular weights can be detected.However,sumoylated proteins obviously decreased in the absence of each putative enzyme gene.This result suggested that the four putative enzyme encoding genes,aosA,ubaB,ubcI and sizA are bona fide sumoylation pathway genes in A.flavus.In conclusion,the putative sumoylation pathway enzyme encoding genes aosA,ubaB,ubcI and sizA were identified and functionally studied in A.flavus.Morphological and in vivo biochemical analyses demonstrated that they are bona fide sumoylation pathway genes and participate in regulating a variety of life programs in A.flavus,such as stress response,sporulation,sclerotia formation,aflatoxin biosynthesis and pathogenicity.Taken together,these results not only provide a preliminary understanding of the sumoylation pathway in A.flavus,but also facilitate further study of the intact sumoylation cycle and its molecular mechanism on regulating fungal biology for controlling A.flavus contamination.