Functional Characterization Of Fofmk1Gene In Fusarium Oxysporum F.Sp.Cubense

Fusarium wilt of banana caused by Fusarium oxysporum f.sp.cubense (FOC) is considered to be one of the most destructive diseases of banana, and it is widely spread in all the banana growing regions. For lack of effective fungicides and availability of resistant varieties, the disease become a serious threat to banana productions. The pathogenesis of Foc is poorly understood so that development of the effective control strategies is hindered. Study on the pathogenicity genes of Foc would provide insights into the pathogenesis, and help us to make some novel control strategies in future.finkl encoding a mitogen-activated protein kinase(MAPK) of F. oxysporum that belongs to the yeast and fungal extracellular signal-regulated kinase (YERK1) subfamily. To understand its role in pathogenesis of Foc, the DNA fragments from the upstream and downstream of open read frame were amplified by PCR, respectively. They were cloned into the pCT74to construct the vector for homologous recombination, then was transferred into Foc B2strain by the PEG-Mediated Protoplast Transformation. At last, several transformants with seletcion marker of GFP and hph cassette were obtained. Among them, three transformants were confirmed to be the Fofmkl gene-deletiong mutants(ΔFofmkl) by PCR with four pairs of the gene-specific primers and Southern blot. Targeted mutants of Fusarium oxysporum f.sp.cubense carrying an inactivated copy of Fofmkl have slight reduction in growth rate and defect in colony morphology, but show normal microconidia production and germination of conidia as well as morphology of conidia and hypha. The ΔFofmkl strains displayed dramatically reduced in pathogenicity on banana plants. Knockout mutants lacking Fofmkl are deficient in multiple virulence-related functions, including root adhesion and penetration of cellophane membranes,, invasive growth. Additionally, the activity of pectinase in ΔFofmkl mutant reduced by44%, compared with the wild-type strain, but have no change in cellulase activity. We found several pathogenicity-related genes of Fmkl mutants also show drastically reduced expression levels of Foc-pll encoding pectate lyase and Foc-835encoding the cell wall-degrading enzyme by qRT-PCR. Complement of ΔFofmkl mutant with the original Fofmkl gene, the pathogenicity and biologic characteristics of complementary strain were recovered.We preliminarily elucidated functions of Fofmkl which is a pathogenicity fact in the pathogen of fusarium wilt of banana. These results facilitate the further studies on the pathogenesis of Foc concerning physicochemical properties, protein interaction and analysis functions of Fofmkl in detail.