Functional Characterization Of The Phospholipase D Gene Family In Fusarium Graminearum

Fusarium head blight(FHB)is an important disease of cereal crops,whose main pathogen is Fusarium graminearum.Phospholipase D(PLD)is an important phospholipid hydrolase that plays critical roles in various biological processes in eukaryotic cells.However,little is known about its functions of PLD in plant pathogenic fungi.In this study,three PLD genes were identified from the Fusarium graminearum genome,and the PLD knockout vector was constructed by Split-marker PCR.?plds and ?pld1-C were obtained by homologous recombination and protoplast transformation.In order to reveal characterizing function of FgPLD in the growth and pathogenicity of Fusarium graminearum,the related phenotypic changes of FgPLD mutants were further studied.Deletion of FgPLD1(?pld1),but not deletion of FgPLD2 and FgPLD3,affected hyphal growth,conidiation,and perithecium formation.In comparison with the wild type strain,colony diameter of the ?pld1 mutant was reduced by approximately 58.23 %;The ?pld1 mutant also showed a distinct colony morphology with shorter aerial hyphae and more hyphae branches than that of the wild type strain.The conidiation of the ?pld1 mutant was reduced by approximately 68.71 % compared to the wild type strain;Additionally,when incubated in YEPD medium,approximately 64.72 % of the ?pld1 conidia germinated at 6 h post incubation(hpi),indicating that FgPLD1 plays an important role in conidiation and conidium germination.The ?pld1 mutant failed to form perithecium,indicating that it is defective in female fertility.Disease index scores for the ?pld1 mutant showed approximately 95 % reduction in virulence compared with the wild type strain;Analysis of DON production in the infested wheat seeds showed that the ?pld1 mutant produced significantly less DON compared with the wild type strain;Further analysis of the expression levels of TRI5 and TRI6 showed that the relative expression levels of TRI5 and TRI6 were significantly lower in the ?pld1 mutant than that in the wild type strain.The phenotype of ?pld2 and ?pld3 mutants in colony growth,asexual and sexual reproduction and pathogenicity were not significantly different from wild type strains.Meanwhile,the phenotype of ?pld1-C in colony growth,asexual and sexual reproduction and pathogenicity was restored to the level of the wild type strain.To dissection of PX domain of FgPLD1,we deleted only PX domain of FgPLD1,named after ?pld1px mutant.?pld1px had similar phnotype of ?pld1,including hyphal growth,conidiation,and pathogenicity,indicating that the PX domian is important for functions of FgPLD1.In conclusion,FgPLD1,but not other two FgPLDs,is involved in regulation of hyphal growth,sexual and asexual reproduction,and plant infection.Furthermore,the PX domain of FgPLD1 is important for entile protein functions.