Identification and Functional Analysis of the 5-Oxoprolinase Genes in Fusarium oxysporum
[Objective]5-Oxoprolinase(OXP)is one of the six core enzymes in the γ-glutamine cycle.The objective of this study is to characterize the OXP gene of Fusarium oxysporum,clarify its relationship with the pathogenicity of F.oxysporum,and to provide a theoretical basis for the analysis of the molecular mechanism of F.oxysporum pathogenicity and the prevention and control of cotton wilt.[Method]Bioinformatics methods were used to identify the FoOXPs from the genome of F.oxysporum and analyze their gene structure,domains of the encoded proteins,chromosomal localization and evolutionary relationships.The phenotypes of F.oxysporum after FoOXP2 mutation were analyzed by using knockout mutant and complementary strain and the pathogenicity differences between the mutant and wild-type strains on cotton seedlings were detected.Furthermore,the disease grade rate and disease index on cotton were investigated by using host-induced gene silencing(HIGS)technology.The fungal biomass in cotton and expression of FoOXP2 in cotton stems transformed with interfering fragments of the FoOXP2 were examined by real-time quantitative PCR(qRT-PCR).[Result]A total of two FoOXPs(FoOXP1 and FoOXP2)were identified in the F.oxysporum genome with coding sequence lengths of 4 080 and 3 921 bp,encoding 1 359 and 1 306 amino acids,respectively.Their protein molecular weights are 14.90 and 14.07 kDa,and theoretical isoelectric points are 5.73 and 5.30,respectively.The FoOXP1 protein is located in the mitochondria and the FoOXP2 protein is located in the cytoskeleton.FoOXP1 and FoOXP2 locate on chromosome JH657921 and chromosome JH657938,do not form gene clusters,with a sequence similarity of 52.00%.Phylogenetic analysis revealed that FoOXP1 and FoOXP2 belonged to two subgroups.Compared with the wild-type strain,the FoOXP2 knockout mutant strain showed significantly lower spore production and spore germination,loss of penetration ability,and increased tolerance to CR and sorbitol,but was more sensitive to cell wall stress(SDS and CFW),oxidative stress(H2O2),and osmotic stress(NaCl and KCl).Moreover,the pathogenicity on cotton was significantly reduced.The results of the HIGS assay illuminated that after 14 and 21 d of inoculation,the incidence of FoOXP2 silenced cotton plants was significantly reduced,whose disease indexes(17.3 and 40.2)were significantly lower than those of the control(28.2 and 77.1).The expression of FoOXP2 and fungal biomass were significantly lower than those of the control.[Conclusion]The FoOXP2 positively regulates the pathogenicity of F.oxysporum and may play an important role in host-pathogen interactions.
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