Disruption And Characterization Of Trehalose Synthase Genes In Fusarium Graminearum

Fusarium head blight (FHB) or scab caused by species of Fusarium is an important disease on small grain cereal crops, prevalently in the areas with warm temperature and high humidity worldwide. In China, epidemics of FHB occur frequently in the middle and downstream regions of the Yangtze River and in the Heilongjiang province in the northeastern region. Fusarium Head blight not only causes the severe grain yield loss, but also causes the harvested grain contaminated with several mycotoxins that contribute a potential problem for human and farm animals. Nowadays, although great efforts have been made in studying the mechanism of plant induced disease resistance and identifying some antigens, we still lack knowledge about the molecular plant and F.graminearum interaction system. Identification related genes can help us to find an effective way to figure out the problem. The metabolism pathway of trehalose synthase is the potential target for anti-fungus drug, because we have not found trehalose in any mammal nowadays. Besides, trehalose plays an important role in spores, which can be used to clarify the pathogen-plant interaction system.In this research, we used Blast P to design and synthesize the homologous fragments of trehalose synthase, then constructed gene knock-out vector, via Agrobacterium mediated transformation to get the mutants of?tps1 and?tps2. The mutants were identified with Southern blot, PCR and RT-PCR. The results indicated that, both TPS1 and TPS2 are successfully knocked out. The phenotype of tps1 shows no difference compared with the wild type, while the?tps2 appears less airborne filaments and grows rather slowly on PDA. Then we used different media to analyze the antibiotic stress of the mutants. It demonstrates that tps2 is much more sensitive than both the wild type and tpsl. After that the utilization of carbon and nitrogen source was test.?tps2 grows better on media when selected fructose rather than glucose as the sole carbon source. And the nitrate reductase is not affected by either TPS1 or TPS2. Moreover, the pathogenicity of of TPS1 and TPS2 were analyzed. Inoculation test, spore productivity test were used to assay the function of TPS1 and TPS2. We found out the wild type had the longest lengths of lesion with wheat coleoptiles inoculation experiment. However, in the experiment of floret inoculation,?tps1 shows hypervirulence phenotype. It indicates that different negatively regulated feedback loops are operating during the infection process of plant host species. Further more, both the?tps1 and?tps2 produce fewer spores compared with the wild type. Finally, we use semi-quantitative PCR to test the transcription of TPS1 and TPS2. Both genes'expression show no difference when changing temperature or carbon, nitratesource for a period of time.