Creation Of T-DNA Insertion Mutants For Fusarium Graminearum And Identification Of Its Key Genes Responsible For Metabolizing Salicylic Acid

Fusarium head blight （FHB） is one of the most destructive diseases to wheat. It caused by several Fusarium spp., and Fusarium graminearum is the most important one. Pathogenesis of the Fusarium graminearum is still unclear. Functional analysis of genes of the F. graminearum can help to understand the mechanism of wheat disease resistance to FHB, and to develop new technologies to reduce the hazard of FHB. The main results were listed as follows:1. T-DNA mutatants of F. graminearum were created by using Agrobacetrium tumaefeiens-mediated transformation （ATMT）. Though the optimized method, the transformation efficiency in averge was 70-80 transformants per 1.0 X 106 conidia. In total, we acquired 365 T-DNA mutants. The growth of these mutants was analyzed on both the normal SNA media and the SNA media supplemented with salicylic acid （SA）. Pathogenesis of these mutants was elevated by point inoculation of spores into flowering spike of wheat in greenhouse. There are 6 mutants grown slowly on normal SNA plates; eight mutants showed reduced pigment on the normal SNA plates; three mutants did not grow on the SNA media with 2.0 mM SA, at which wild type fungus can grow; two mutants significantly decreased their virulence on wheat. Among them, △Ma334 grown slowly and reduced pigment on normal SNA media; △Ma208 grown slowly on normal SNA medium and couldn’t grow on the SNA media with 2.0 mM salicylic acid; △Ma39 grown slowly on normal SNA media and significantly decreased its virulence on wheat.The mutants were verified by PCR amplification of part of the HPH gene sequence, using their genomic DNA as templates. T-DNA flanking sequences were obtained by TAIL-PCR. PCR products were purified and sequenced, Genome locations of T-DNA in F. graminearum genome were identified by blasting the newly obtained flanking sequence against its genome information.In AMa39, T-DNA was inserted at 542464 bp in supercontig 3.4. It was located within the gene FGSG₀6605.3. This gene is the probable beta-glucosidase 1 precursor. FGSG₀6605.3 was probably hindered from normal expression due to the insertion, resulting in slow growth on normal SNA media and decreased virulence on wheat.In △Ma247, T-DNA was inserted at 1547302 bp in supercontig 3.6. It was located within the gene FGSG₁3581.3. This gene is a conserved hypothetical protein. FGSG 13581.3 was probably hindered from normal expression due to the insertion, resulting in reduced pigment production.In AMa256, T-DNA was inserted at 2133337 bp in supercontig 3.3. It was located at 819 bp upstream of FGSG₁2730.3 and 1339 bp upstream of the gene FGSG 05397.3. They are both conserved hypothetical protein. It is probably that FGSG 12730.3 and FGSG 05397.3 were hindered from normal expression due to the insertion, resulting in reduced pigmentation.In AMa319, T-DNA was inserted at 1360744 bp in supercontig 3.6. It was located at downstream of FGSG₀9387.3, which is annonated as an uncharacterized protein. It is probably that the uncharacterized protein was hindered from normal expression due to the insertion, resulting in slow growth on the normal SNA media.2. Gene expression of F. graminearum on the plates using either SA or glucose as sole carbon source were compared by using RNA-seq, to identified the key genes involved in SA methobilism. There were 173 genes exhibited significant difference in expression. Among them,66 were up-regulted when using SA as carbon source. Compared with previous study, we verified the important of the gene FGSG₀9063 in SA metabolism. We found some other gene related to the aromatic hydrocarbon metabolism and had significantly different expression as well. These genes may be associated with SA metabolism. Some genes had the most significantly different expression, while their functions remain unclear. Futher research is needed to clarify the function of genes differently expressed.