Study On The Functions Of WprA And WprB In Aspergillus Flavus

Aspergillus flavus,a common agricultural pathogenic fungus,is notorious for producing the carcinogenic aflatoxin B1 which is the most toxic secondary metabolites ever found in nature.A.flavus infections can occur before,during and after harvest and leads to aflatoxins contamination of crops or agricultural products,poses a serious threat to food safety and human health.The fungal specific transcription factors containing WOPR box domain have been documented recently.They belong to a new class of sequence specific DNA binding protein family and play diverse functional roles in fungi.In this study,we identified two genes,wprA and wprB,which putatively encode two WOPR box containing proteins in Aspergillus flavus by bioinformatic analysis.We constructed the wprA and wprB deletion and over-expression strains respectively by the homologous recombination and we found that they played similar but different roles in the cells.Compared with wild type strain,deletion of wprA or wprB significantly reduced the growth of aerial hyphae,promoted the conidiation and accelerated the germination of conidia.While overexpression of wprA or wprB could result in the decrease of conidia production and delaying its germination.Loss of wprA blokced the sclerotia formation and the biosythesis of AFB1,whereas the absence of wprB seriously affected the AFB1 production and blokced the sclerotia formation.Then we analyzed the effects of wprA and wprB on the response to various environmental stimuli and several antifungal drugs in A.flavus.And we found that deletion of wprA or wprB made the strain more sensitive to hydrogen peroxide,but less to menadione sodium bisulfite and amphoericin B.In addition,wprB deletion strain could respond to the stimulation of sodium dodecyl sulfate rather than triton X100.One thing to be noted that both wprA and wprB deletion strains exhibited lower tolerance to high temperature.For further study on the function of wprA and wprB in response to heat shock stimulus,geldanmycin,a specific inhibitor of heat shock protein Hsp90,was added into the medium during incubation with high temperature.As the results showed that the growth of wprA and wprB deletion strains were restrained while wprA and wprB over-expression strains were obviously better than the wild type strain,we proposed that hsp90 participated in the response of wprA and wprB to heat shock stress.In order to verify the results obtained on artificial synthetic media,seed infection experiments were performed to mimic the natural process for fungi-host interaction.We found ΔwprA strain colonized faster on the surface of the peanut or maize seeds with less aerial phyphae than wild type strain,and consequently produced more conidia and less aflatoxin.It’s consistent with our previous datas on artificial synthetic media.However,the results of seed infection by AwprB strain were not completely coincident with our expectations.The ability of colonization on seeds and conidiation did not change distinctly comparing with the wild type strain,expect the decrease of the aerial hyphae and aflatoxin production,However,the wprB over-expression strain produced more conidia than wild type strain.In conclusion,we identified two putetive WOPR box domain containing proteins encoding genes wprA and wprB and preliminarily studied their functions in A.flavus.Our results demonstrated that wprA and wprB do be involved in regulation of the life processes including the hyphal growth phenotype,conidiation,conidia germination,sclerotia formation,aflatoxin production,stresses response and pathogenesis.These results not only build foundations for futher study on the cellular roles of wprA and wprB in regulating the morphological development and secondary metabolism of A.flavus,but also provide a novel approach to control the contamination of this fungus.