Autophagy-Related Gene FpAtg3 Involves in Growth and Pathogenicity of Fusarium pseudograminearum
[Background]Fusarium crown rot(FCR),caused by Fusarium pseudograminearum,is one of the newly-occurred destructive diseases in the main wheat-growing region of China.Autophagy is an evolutionarily conserved cellular process that regulates the growth,development and infection of different phytopathogenic fungi.However,the role of autophagy in F.pseudograminearum is still unknown.[Objective]To clarify the role of the autophagy gene FpAtg3 in the growth and pathogenicity of F.pseudograminearum,understand the mechanisms of the fungal infection,and to provide a theoretical basis for FCR prevention.[Method]The known Atg3 proteins of different fungi were downloaded from NCBI,and the MEGA 5.05 was used to construct the phylogenetic tree of Atg3 proteins.The Split-PCR approach was used to generate FpAtg3 gene-replacement constructs,and then the FpAtg3 deletion mutants(ΔFpAtg3)were constructed by polyethylene glycol(PEG)-mediated protoplast fungal transformation,and obtained by hygromycin resistance screening and PCR detection.The sequence of FpAtg3 and its native promoter was amplified and fused in pKNTG vector.The plasmid of pKNTG-FpAtg3 was then introduced into ΔFpAtg3 protoplasts for the complementation assay.The hyphal growth and colony morphology of the wild type,ΔFpAtg3 and the complementary(FpAtg3-C)strains were assayed on PDA,CM and MM plates.The hyphal blocks were cultured on PDA plates for hyphal morphology and hyphal fusion test;hyphal blocks were introduced into liquid CMC medium to assess conidiation and conidia morphology;conidia were cultured on Petri dishes to explore conidial anastomosis tube(CAT)-mediated fusion.The hyphal blocks were inoculated on wheat coleoptiles and barley leaves to explore pathogenicity,and the pot-culture experiment was performed for FCR assay.Congo red,sodium dodecyl sulphate(SDS)and H2O2 reagents were added to PDA plates to determine the F.pseudograminearum responses to cell wall,cell membrane and oxidative stresses,respectively.[Result]Atg3 homologous proteins from different fungi were highly conserved and consistent with the direction of species evolution.The FpAtg3 had a close relationship with Atg3 of Fusarium graminearum and Fusarium oxysporum.The ΔFpAtg3 and FpAtg3-C strains were obtained.The phenotypic measurements showed that compared to wild type and FpAtg3-C strains,ΔFpAtg3 exhibited significantly reduced colony growth rates and aerial hypha,curved hypha,reduced conidiation,shorter in length and fewer in septa;ΔFpAtg3 also showed significantly reduced hyphal fusion rate and CAT-mediated fusion rate.The pathogenicity of ΔFpAtg3 on wheat coleoptiles and barley leaves was significantly reduced comparing to that of the wild type and FpAtg3-C strains.The reduced pathogenicity of ΔFpAtg3 was further examined on FCR.Furthermore,ΔFpAtg3 displayed more sensitive to cell wall,cell membrane and oxidative stress than that of the wild type and FpAtg3-C strains.[Conclusion]Autophagy-related gene FpAtg3 plays important roles in growth,conidiation,hyphal fusion,pathogenicity and response to abiotic stresses of F.pseudograminearum.
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