The Mechanism Study Of PKA Signal Pathway Gene UePkaC In Polar Growth In Ustilago Esculenta

Ustilago esculenta,an endophytic fungus,could infect Zizania latifolia,to suppress host flowering and trigger host stem swollen called Jiaobai.U.esculentaas a typical dimorphism fungus could transform from yeast-like form to dikaryotic filament form under different environment.The infectivity and pathogenicity of U.esculenta is closely related to the dimorphism transition.Previous studies proved that the dimorphism transition was co-regulated by the MAPK signal pathway and PKA signal pathway.PKA holoenzyme without kinase activity is composed of two catalytic subunits(Pka C)and two regulatory subunits(Pka R).Cyclic Adenosine monophosphate(c AMP)could induce PKA holoenzyme to change its conformation and liberate active catalytic subunit after the combination of c AMP and inactive PKA regulatory subunits.This study is mainly focused on the influence of the PKA signaling pathway on the growth of U.esculenta,spread infection and the dimorphism transition.In this study,it was found that the filamentous lost its ability of polar growth and the haploid strain could not split in time after the germination and there was a phenomenon of multi budding after adding c AMP.The high concentration of c AMP could influence the filamentous growth of the compatible haploids.The PKA catalytic subunit of U.esculenta Ue Pka C has high homology with the PKA catalytic subunit of Ustilago maydis and sporisorium reilianum.After knock out of Ue Pka C in wild type strains by homologous recombination,it was found that Ue Pka C could not affect the growth rate of U.esculenta,but the deletion of Ue Pka C resulted in haploid more longer.The Ue Pka C gene was significantly up-regulated at the early stage of fusion,and the deletion of Ue Pka C gene in any of the two haploidcould cause the two haploid unable to fuse.The infection experiment also showed that the deletion of Ue Pka C caused that the two haploids couldn't form the dikaryotic filament to infect plant.The result shows that Ue Pka C gene involved in the fusion process.Maybe it can regulate the expression of pheromone gene that the gene can control the growth of conjugation tube.The above results showed that the PKA signal pathway may be involved in the the mating of U.esculenta haploid and the process of polar growth.F-actin is a helical polymer of actin monomer which closely related to the polar growth of fungi.Previous studies had proved that PKA could directly phosphorylate actin in vitro,but still lack the experimental support in vivo.We had cloned actin genein U.esculenta and named it Ue Actin.What's more,we forecasted two phosphorylation sites of Ue Pka C gene on Ue Actin gene.We constructed a overexpression strain Ue T14::Ue Actin and there point mutation overexpression strains Ue T14::Ue ActinT204 I,Ue T14::Ue ActinT205 I,Ue T14::Ue ActinT204I&T205I,respectively.Compared the point mutation overexpression strains with overexpression strains,there were no obvious difference in phenotype.We used immunofloresence assay to clear the subcellular location of Ue Actin.Compared Ue T14::Ue Actin with Ue T14?Ue Pka C::Ue Actin,the fluorescence signal of was Ue Actin always distributed evenly in cell.We inferred that Ue Pka C gene may not phosphorylated Ue Actin gene to regulated polar growth in U.esculenta.But how Ue Pka C gene regulates the Ue Actin gene still needs more studies to make it clear.