Effects Of Protein Kinase A Core Catalytic Subunit FpCpk1 On Growth And Pathogenicity Of Fusarium Pseudograminearum

Wheat is one of the most important crops in the world.Fusarium Crown Rot(FCR)is a common soil-borne disease in wheat caused by a variety of pathogens worldwide,which mainly include Fusarium pseudograminearum,F.graminearum,and F.oxysporum.The Huang-Huai-Hai River Plain is the largest winter wheat producing region in China.F.pseudograminearum in the Huang-Huai-Hai River Plain of China is reported to be one of the main pathogens causing FCR.Like F.graminearum,F.pseudograminearum can also produce many toxin compounds during the infection of wheat,which not only affects the grain yield and quality,but also poisons humans and animals in serious cases.Due to the lack of resistant cultivars,it is difficult to control the disease at present.Therefore,the research on the pathogen of FCR is increasingly urgent,especially the study of the pathogenic genes in pathogenesis.Similar to other filamentous fungi,the F.pseudograminearum genome contains two genes encoding the catalytic subunits of the cyclic adenosine-dependent protein kinase A(PKA)ortholog,FpCpkl and FpCpk2.In F.graminearum,the Cpk2 knockout mutant has similar growth and pathogenicity to wild type.In order to clarify the CPK1 ortholog gene of the cAMP-PKA signaling pathway in F.pseudograminearum,we used the spilt-marker strategy to construct a knockout cassette containing hygromycin phosphotransferase(HYG),combined with PEG-mediated protoplast transformation approach.Transformants with hygromycin-resistance were screened by PCR and confirmed by Southern blot.We obtained FpCpkl clean knockout mutants.Compared with the wild type,the colony growth rate of the FpCpk1 knockout mutant decreased significantly with the colony diameter about 1/6 of the wild type after 3d culturing.The sporulation amount was also significantly lower than the wild type,which was 250-fold lower.The pathogenicity of the mutant to barley and wheat coleoptiles was significantly reduced,and the expansion of hyphae was significantly slower than the wild type.The histopathological examination showed that the cells invaded by the mutant infectious hyphae became obviously brown or dark brown,which indicates the deletion of FpCPKl causes strong host defense response likely the generation of ROS.Under the fluorescence microscope,the green fluorescent protein(GFP)signal was observed in the nucleus and cytoplasm of spores,germ tubes and hyphae at different vegetative stages,indicating that the FpCpkl protein is localized in the nucleus and cytoplasm.Based on the above results,we propose that the FpCPKl gene in cAMP-dependent PKA pathway plays an important role in F.pseudograminearum on fungal development and pathogenesis with overcoming host defense response to infect and colonize the host.