Cloning And Functional Analysis Of Arginine Synthetic And Metabolic Pathway Genes In Ustilago Esculenta

Jiaobai,is China's second largest aquatic vegetable,for rich nutrition,delicious taste and high medicinal value.Ustilago esculenta is a parasitic fungus of Zizania latifoli,which inhibits host flowering and induces enlargement of its stem base to form an edible gall called Jiaobai.Studies have shown that U.esculenta is similar to Ustilago maydis and has two types of yeast and mycelium,and is a typical dimorphic fungus.The fungal dimorphism transformation,ie,the transition between yeast and mycelia,is the key to the pathogenicity of the dimorphic fungi.Previous studies found that arginine is an important factor that promotes the infection of most pathogenic fungi from the yeast type to the mycelia type.our group previous study on the U.esculenta found that exogenous arginine significantly inhibited mycelial formation,which was contrary to its positively regulated role in other fungi reported before,indicating a different mechanism of arginine involved in regulation of fungal dimorphism.In this study,genes for arginine synthetic and metabolic pathways were cloned and identified.In vitro fusion experiments?in vitro culture promotes the transformation of yeast from to mycelial form?,microscopic observation,and fluorescence quantitative PCR were performed.The biological function was studied to determine the role and mechanism of arginine in the growth,virulence and dimorphism of U.esculenta.First,we cloned the genomic sequences and cDNA sequences of the arginine synthesis pathway genes UeArg1,UeArg2,UeArg3,UeArg4,UeArg5,6,UeArg7,UeArg8 and the arginine metabolic pathway gene UeArginase.The comparative analysis of the basic information,protein structure and homology of these genes showed that there is an intact arginine synthesis and metabolic regulation system in U.esculenta.It has been reported that CO₂ produced by metabolism of arginine acts on the cAMP-PKA pathway and affects the dimorphism conversion of fungi.Therefore,we further studied the arginine metabolic pathway gene UeArginase.The expression pattern analysis revealed that:UeArginase is fused in the T-type strain.During the process,the expression decreased continuously,and the expression level was significantly lower than the basal level during the dimorphism transition period.In vitro fusion assay results showed that the gene-deleted strain of UeArginase was similar to the result of exogenously added excess arginine that a delay appeared during fusion process.Meanwhile,the HPLC test results showed that the endogenous arginine decreased during the fusion process,while the UeArginase gene-deleted strain and exogenously added arginine all increased the endogenous arginine.Therefore,we speculate that in the smut black fungus,it may be arginine itself,rather than the arginine metabolic pathway involved in dimorphic transformation.Further,we conducted a preliminary study on the function of the arginine synthesis pathway gene.The expression patterns of UeArg1,UeArg2,UeArg3,UeArg4,UeArg5,6,UeArg7 and UeArg8 in the fusion process of T-strains were analyzed.The results showed that UeArg1 had the highest expression at the 24 h of the key time point of dimorphism transformation;The expression of UeArg3 and UeArg4 gradually decreased during the 12-48 h of dimorphism transition;UeArg2 and UeArg7 showed a gradually increasing expression during the 12-48 h transition of dimorphism;UeArg8was in the second type.The expression level of UeArg5,6 did not change significantly after the state transition was completed.However,the expression level of UeArg5,6 did not change significantly during the dimorphism transition.Knockout was further performed.In vitro fusion,artificial inoculation,and microscopic observations showed that the UeArg1-deleted strain became shorter and thicker and the growth rate became slower,but it did not affect its dimorphic transformation on YEPS medium.The deletion of UeArg2 and UeArg7 genes did not affect the haploid growth and in vitro fusion ability of the strains;multi-site budding of UeArg4-deleted strains;deletion of UeArg3,UeArg5,6 genes resulted in shorter and a slower growth rate and a weakened fusion ability;UeArg8-deficient strains have slower growth rate and enhanced in vitro fusion ability.Infection experiments revealed that,except for the UeArg4 gene-deleted strain showing a good infective state,the deletion of other genes all weakened the pathogenicity of the strain.Based on the above results,we found that UeArg3,UeArg5,6,and UeArg8 have a significant effect on the haploid growth,in vitro fusion process and pathogenicity of U.esculenta.Previous studies have shown that these three genes are arginine feedback regulatory genes.Therefore,we further studied the physiological functions of the feedback regulatory pathways UeArg3,UeArg5,6,and UeArg8 and their roles in the dimorphism of U.esculenta.The morphological and physiological functions of UeArg3,UeArg5,6,and UeArg8 were studied on YEPS,YEPS+0.2%L-Arginine,CM,and CM+0.2%L-Arginine.The haploid growth test results showed that the growth rates of UeArg3,UeArg5,6,UeArg8 gene-deficient strains were slowed down to varying degrees compared with the control.In CM medium,the difference between the growth rate of UeArg3,UeArg5,6,UeArg8 and the control was significantly higher than that on YEPS medium;after the addition of arginine exogenously,the growth rate was significantly different on YEPS medium but in CM,the difference in the medium is small.The observation of haploid microscopic morphology showed that the haploid strains of UeArg3 and UeArg5,6 gene deletion strains were shorter and thicker than the control,while the haploid morphology of UeArg8 gene deletion strains on the four media had no significant difference from the control.In vitro fusion assays and the expression analysis of Uekpp2,Uekpp6,UePkaC,and UePrf1,which are key genes in the MAPK and PKA pathways were carried out.Results showed that during this process,the UeArg3-deleted strains were weaker in dimorphism on the different media than the controls,but the critical time points for their growth?eg,24 h on YEPS?,Uekpp2,Uekpp6,UePkaC and UePrf1 all showed high expression,indicating that deletion of this gene does not affect the MAPK and PKA pathways;the length of hyphae of UeArg5,6-deleted strains was significantly reduced compared to the control,and Uekpp2,Uekpp6,UePkaC and UePrf1 all showed low expression;while the fusion time of UeArg8-deficient strain was earlier and the hyphal length was significantly longer than that of the control,and Uekpp2,Uekpp6,UePkaC,UePrf1 showed a continuous high expression trend.Therefore,we hypothesize that UeArg5,6 and UeArg8 may regulate the dimorphic transformation of U.esculenta through the MAPK and PKA pathways.The above results preliminarily confirmed the role of arginine synthesis and metabolic pathway genes in the haploid growth,morphogenesis,and fusion process of U.esculenta.It is a basic material for further studies on the function and mechanism of arginine anabolic metabolism in the interaction of U.esculenta and Z.latifolia.