Identification And Characterization Of MAP Kinase FvMK1 And Transcription Factor FvST12 In Fusarium Verticillioides

The filamentous ascomycete Fusarium verticillioides (teleomorph Gibberella moniliformis) is one of the most ubiquitous pathogens of maize(Zea mays). It can infect different maize tissues and cause severe ear and stalk rots. These diseases often cause significant yield losses and tremendous economic damages worldwide. Moreover, infected corn kernels are usually contaminated with fumonisins and other mycotoxins. Mycotoxins produced by F. verticillioides are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. MAP Kinases have been implicated in the regulation of various development and plant infection processes in filamentous fungi. However, the function of FUS3/KSS1 ortholog MAPK in F. verticillioides is not clear. In this study, we identified and characterized the FvMKl (Fusarium verticillioides MAP Kinase 1) gene and its downstream transcription factor gene FvST12 in F. verticillioides wild type strain Fv7600 by using the reverse genetic approaches. Our results are as followings:(1) The F. graminearum GPMK1 gene was used to search for its ortholog in the F. verticillioides genome sequence by BlastP approach. One predicted gene, FVEG₀5063.2, was identified to be highly similar to GPMK1, and named FvMK1. It shares 98% identity with Gpmkl of F. graminearum. The FvMK1 gene encodes a 355-amino acid polypeptide with a TEY dual phosphorylation site and conserved serine/threonine protein kinase domains. The Phylogenetic relationship showed FvMK1 gene is highly conserved with its orthologs in filamentous fungi.(2) The FvMK1 gene replacement constructs generated with the double-joint PCR approach were transformed into protoplast of the wild type strain Fv7600. Hygromycin-resistant transformants were isolated, then screened by PCR and confirmed by Southern blot. Southern blot result showed that the FvMKl gene was only single copy in F. verticillioides genome. The Fvmkl mutant was significantly reduced in vegetative growth rate and aerial hyphae production in different media. The Fvmkl mutant was reduced about 50% in conidiation when grew on the tested agar plates. Although it was reduced in conidiation, the percentage of macroconidia was significantly increased in the Fvmkl mutant. The FvMK1 gene has no effect on the conidia germination.(3) The plant infection test results indicated that the FvMK1 MAP kinase gene was critical for the virulence on corn in F. verticillioides. The Fvmkl mutant was defective in colonizing and spreading in the corn pith. No stalk rot or ear rot symptoms were observed beyond the inoculation sites. The complemented transformant can extensively colonized corn stalks and ears, and caused severe discoloration in these corn tissues. FvMKl can also functionally complement the pmkl mutant in Magnaporthe oryzae although F. verticillioides does not form appressoria for plant infection.(4) The FvMK1 positively regulated the production of fumonisin B1 and the expression of FB1 biosynthetic genes in F. verticillioides. The FB1/ergosterol ratio in corn kernels inoculated with the Fvmkl mutant was only half to that of the wild type.In the Fvmkl mutant, the expression level of FUM1 was reduced about 12-fold in comparison with that of the wild type or complemented transformant according to the qRT-PCR results. The expression level of FUM8 was reduced about three times in the Fvmkl mutant.(5) The FvMK1 gene was not essential for sexual reproduction in F. verticillioides. Although the mutant was female sterile, it had no obvious defects in mating as the male. On carrot agar plates, black perithecia and ascospores were produced by the mutant as efficiently as the ectopic and wild type strains after fertilization.(6) The FvMK1-eGFP fusion proteins assays showed that the FvMK1 appears to be constitutively expressed in F. verticillioides and FvMkl protein may localize to the nucleus when it is activated. The FvMkl protein phosphorylation levels was regulated by its upstream Gbb1 protein.(7) The F. oxysporum FoST12 transcription factor was used to search for its ortholog in the F. verticillioides genome sequence by BlastP approach. One predicted gene, FVEG₀5267.3, was identified to be highly similar to FoST12, and named FvST12. It shares 98% identity with FoStl2 of F. oxysporum. The FvST12 gene contained STE Homedomain and C2H2 Zinc Finger domain.(8) The target gene FvST12 in F. verticillioides was knockout by using the protoplast transformation approach. Southern blot results showed that FvST12 gene was only single copy in F. verticillioides genome. Compared with wild type, Fvst12 deletion mutant has normal growth rate, condiation, colony morphology, and stress response tested in this study.(9) The FvST12 gene was important to the plant infection in F. verticillioides. Fvstl2 mutant strain was significantly reduced the virulence on corn stalk and ear. The complemented transformant can restore the defects on plant infection. Gene expression results indicated that FvST12 gene has continuous and high expression levels in the plant infection stage.(10) The FvST12 gene should be one of the downstream transcription factors of MAPK gene FvMK1 in F. verticillioides. The major function of this transcription factor seems involved in the regulation of the plant infection in F. verticillioides. Also, there might be some other transcription factors, which were regulated by FvMKl, involving in the process of vegetative growth and conidia production in F. verticillioides.