| Ustilago esculenta,a pathogenic basidiomycete fungus,infects Zizania latifolia and induces swelling or enlargement of the tissue near the base of the host plant,forming edible galls named jiaobai.There are three phenotypes in the field,named male Jiaobai,grey Jiaobai and white Jiaobai.The previous studies found that U.esculenta in white Jiaobai existed in the form of mycelia-teliospore(M-T)type,while teliospore(T)is the major type in grey Jiaobai.Studies suggest that dimorphism of U.esculenta from yeast to hyphae is relate to the formation of Jiaobai.However,it is unknow that whether there are differences between the fungi hosted in the white Jiaobai and grey Jiaobai.In this study,we first isolated compatible haploid strains from grey Jiaobai(T-type strain)and white Jiaobai(MT-type strain),named Ue T14(CCTCC AF 2015016),Ue T55(CCTCC AF 2015015),UeMT10(CCTCC AF 2015020)and UeMT46(CCTCC AF 2015021)respectively,by single spore separation operation under micromanipulator.And succeed in the mating and mycelium growing with the compatible haploid strains in vitro.Meanwhile,the life cycle of U.esculenta was cleared through constructed strains overexpressing nuclear localization EGFP fusion protein and the artificial inoculation technology,showing its morphologic changes in different phase.Further research showed that there were many differences between T and MT strains,and the T type stains had stronger environment adaptability.Specifically,On the different carbon source and nitrogen source medium,the growth rate of T haploid strain was faster.The fusion and growth of mycelium were affected by different nutrition condition and physical-chemical condition and the compatible haploid strains of T-type could mate and form filament on various nitrogen and carbon source medium.At the same time,T strains had stronger adaptability to the changes of various conditions such as p H,hydrogen peroxideand hydrophobic.Genome data analysis between T and MT strains showed that abandent genes related to pathogenicity were mutated in MT strains.Previous research declared that the pathogenicity of smut fungi were related to its dimorphism conversion(haploid converted to mycelia),and mating type genes were responsible for the regulation of dimorphism,where mycelia growth was regulated by b genes.So we did further research on the function of b genes in U.esculenta.Fisrt,the b mating type loci and b genes in the four haploid strains of U.esculenta were cloned separately,and the bioinformatics analysis results showed that similar to Ustilago.maydis,there were two b genes named b-East(bE)and b-West(bW)in one b mating type loci existed in one strain,and bE and b W gene have a constant regions in 3' region,and the 5' region is variable.The b1 locus was cloned from Ue T14 strain,including bE1 and bW1 genes;The b2 locus was existed in Ue T55 and UeMT46 strains,including bE2 and bW2 genes;The b3 locus was cloned from UeMT10 strain,including bE3 and bW3 genes.Next,different b gene mutation strains were constructed by PEG mediated protoplast transformation method and the mating experiment assays showed that b gene mutation had no obvious influence on haploid strains fusion progress,but inhibited the the hyphal growth progress.In general,it is similar to U.maydis the that U.esculenta grow as hyphae only the haploid strains have a pair of bE and bW genes.So further more,the upstream and downstream genes of b genes(Kpp2,Prf1,Kpp6,Rbf1,Clp1)were cloned and there expression profiling during the mating progress of the tested strains were analyzed by q RT-PCR.The results showed that Kpp2 Prf1 and Kpp6 were continuously expressed during the mating and hyphal growth progress,while b genes were upregulated obviously after mating.And the expression of Rbf1 and Clp1 genes had no obvious change during the assays without haphae formation.All of them indicated that similar to U.maydis,b genes were regulated by Kpp2,Prf1,and Kpp6,and control hyphal growth through Rbf1 and Clp1,but the details needs further researches. |
PDF Full Text Request