Functional Characterization Of AMT1 Genes In Fusarium Graminearum

Fusarium graminearum is the major agent of Fusarium head blight(FHB) disease, which can infect small grains, maize and so on. This pathogen can not only cause yield and quality losses but also produce mycotoxins which can contaminate the grains and are hazardous to animals, thus making the grain unfit for food or feed. In the last decade, outbreaks of FHB have been reported in the whole world, which led to severe losses of crop yield and contamimation of mycotoxins. However, it also spurred the basic research of F.graminearum which has become one most studied plant pathogen on pathogenicity, population genetics, evolution and genomics. As more and more chemical fungicides were supplied in fields, resistant strains had appeared in some places which increased the potential risk of re-emergence of FHB. Because of the huge destruction of FHB, the difficulty of breeding resistant cultivars, scarcity of resistance resources and so on, it is urgent to study the pathogenic mechanism of F.graminearum .Based on the strategy of reverse genetics, we germinated amt1 mutant by technique of gene knockout and speculated functions of AMT1 gene through observing the phenotypes of the mutant. In this study, we employed the method of split PCR to construct the knockout cassette containing Hygromycin-resistant gene hph. We got the Hygromycin-resistant transformants by PEG-mediated transformation of protoplasts of PH-1 and homologous recombination in vivo. In order to obtain the true amt1 deletion mutant, we screened the transformants by PCR with 4 pairs of primers and confirmed by southern blot.We observed the phenotypes of amt1 deletion mutant, including growth rate and morphology of colony on CM and PDA plates, conidiation, sexual reproduction, germination and so on, and we also did infection assays on wheat and maize. We concluded that the lacking of AMT1 gene leads to reduction of growth rate, fewer aerial hyphae and slower germination, however, it did not influence sexual and asexual reproduction. Interestingly, the amt1 mutant is significantly reduced in virulence and the toxin level was about 48.6% of that of the wild-type in infested wheat kernels.We found several pathogenicity-related genes by qRT-PCR, whose levels were about 14%-44% of wild type. The reduction of these genes expression besides DON may be main reasons for decreased virulence of amt1 mutant. To confirm the function of AMT1 gene, we cloned a AMT1 gene from wild type into vector pHZ100 and transformed it into amt1 deletion mutant. We observed that all defects were complemented. We also demonstrated that the Amt1 protein localized mainly in necleus by transforming AMT1-GFP fusion in amt1 mutant. Given the known effect of HMT1 gene in yeast on nuclear transport of hnRNPs, we tested whether AMT1 gene influence nuclear transport of putative Hrp1 and Nab2 orthologs in F. graminearu by expressing GFP-tagged FgHrp1 and FgNab2 proteins in F. graminearu strains with and without AMT1 gene. We concluded that the AMT1 can facilitate export of FgHrp1, but not FgNab2 in F. graminearum.We preliminarily investigated functions of AMT1, and further studies must be carried out in the fields of physicochemical properties, protein interaction and so on to indentify detailed functions.