Research On The Mechanism Of Weak Pathogenicity Of PY20-1 And AY20-17 Strains In Fusarium Pseudograminearum

In recent years,Fusarium crown rot（FCR）has posed a serious threat to the security of food production in the Huanghuai wheat growing region.Fusarium pseudograminearum is the dominant fungus,displaying the clearly differentiated pathogenicity in nature.In this study,40 F.pseudograminearum strains with different pathogenicity were isolated from the collected samples in the fields to investigate the mechanism of weak pathogenicity for the weak pathogenic strains.The colony morphology of 40 strains was first observed and PCR amplification with specific primers was used to further confirm all 40 strains as F.pseudograminearum and to determine their mating type.Sexual reproduction experiments identified that 89 crosses with the vary ability in fertility did not produce mature ascospores in the laboratory condition,while over 30% of the crosses field-induced sexual produced mature ascospores and ascospores in the field-induced manner.The possibility of sexual reproduction under field conditions is predicted.A rapid determination of the pathogenicity of F.pseudograminearum isolates on wheat coleoptiles using fungal agar disks and strains with a highly significant decrease in pathogenicity compared to WZ-8A were phenotypically determined.The screening results showed that mycelial growth rate was not related to pathogenicity.The spore morphology of the weakly pathogenic strain AY20-17 was defect in conidial morphology.The stress tolerance of strains PY20-1 and AY20-17 was similar to that of the highly pathogenic strains,and therefore,PY20-1 and AY20-17 were selected for further investigation in the study.Both PY20-1 and AY20-17 were able to form appressoria infection structures on cellophane,but could not penetrate the cellophane,while the infection of AY20-17 was blocked between the cells of the wheat coleoptiles and could not penetrate the host cell wall,whereas could go through the intercellular space,probably due to the decreased pressure in the infectious hyphae tips.The infectious structure of PY20-1 was abnormal.Compared with the previous research results of our group,the weak pathogenic strains PY20-1 and AY20-17 had similar defects in the infestation structure with the deletion mutant of the FpPMK1 gene,and the expression of the FpPMK1 gene was significantly downregulated during the infestation of wheat by the two weak pathogenic strains,suggesting that the weak pathogenic mechanism is related to the MAP kinase pathway.A total of 51 differentially phosphorylated proteins were identified by proteomic analysis.Seventeen differentially phosphorylated proteins were screened by yeast two-hybrid,of which only one directly interacted with FpPmk1.The protein encoded by FPSE₀9517 is proline-and arginine-rich,was named FpMPR1（F.pseudograminearum MAPK-activated proline arginine 1）and was predicted to encode 571 amino acids with a molecular mass of65.857 k Da.Mass spectrometry analysis showed that S115,S116 and T119 of FpMpr1 were differentially phosphorylated.To verify the function of the FpMPR1 gene,the target gene FpMPR1 was knocked out in wild-type WZ-8A and phenotypically similar ΔFpmpr1-1 and ΔFpmpr1-2 were identified as single-copy replacement in situ.The ΔFpmpr1-1 and ΔFpmpr1-2 knockout mutants showed a highly significant decrease in colony growth rate as well as the significantly reduced pathogenicity compared to the wild type.FM4-64 staining showed that the number of vesicles in the mutant conidia and germ tubes was significantly reduced,indicating that FpMpr1 is required for vesicle formation.Quantitative PCR results showed that the expression level of this gene was significantly decreased in AY20-17.Therefore,FpMpr1 is a virulence factor associated with the pathogenicity of F.pseudograminearum.In conclusion,based on the previous studies of our group,this study identified the phenotypes of PY20-1 and AY20-17,which had morphological mutations,while PY20-1had a normal phenotype.The two isolates showed similarity to the highly pathogenic strains in terms of stress performance.Two isolates with defective appressoria infectious structure were the main cause of low pathogenicity;it was clear that expression of the mitogenactivated MAP kinase gene FpPMK1 positively down-regulated the low pathogenicity of the strains,and FpPmk1 achieved colonization by regulating the phosphorylation of the downstream target FpMpr1.The elucidation of the weak pathogenicity mechanism of F.pseudograminearum field isolates will provide a theoretical basis for the effective prevention and control of wheat crown rot,as well as potential targets for the development of novel drugs.