Effect Of Thiamin On Sensitivity Of Xanthomonas Oryzae Pv. Oryzae To Bismerthiazol And Function Of Thiamin Synthesis Genes

Thiamin, as vitamin B1, was a necessary cofactor for all organisms. Thiamin and bismerthiazol both had active group of thiazole ring, and bismerthiazol had a good inhibition to Xanihomonas oryzae pv. oryzae in vitro and vivo. In this study, because of thiamin and bismerthiazol with similar chemical structure, we were from the following three aspects to explore how the thiamin and its synthesis key genes of Xoo effected on sensitivity of Xoo to bismerthiazol, and the function of thiamin biosynthesis.1. Effect of thiamin on sensitivity of Xoo to bismerthiazolTo understand thiamin whether related to mechanism of bismerthiazol, and how to effect on sensitivity of Xoo to bismerthiazol, we assayed the antagonistic action between them in vitro and vivo. As the result thiamin could reverse the bacteriostasis to ZJ173, wild-type strain of Xoo, of bismerthiazol, but falled short of wild-type level in vitro. However, the inhibition was 54.93% with 30mMol thiamin, and was 75.62% and 85.43% with 100 μg/ml and 300 μg/ml bismerthiazol, respectly. But when the thiamin and bismerthiazol were combined, the inhibition rate was 28.40% and 49.39%, respectively. Therefore, thiamin was an antagonistic action of sensitivity of Xoo to bismerthiazol. The thiamin biosynthesis related genes was up-regulated after treatment of bismerthiazol in vitro. In a word, the result indicated that mechanism of bismerthiazol could relate to thiamin biosynthesis pathway.2. Cloning and functional studies of thiD, thiE, and thiG of thiamin biosynthesis genes in XooThiamin and its form thiamin pyrophosphate (TPP) are essential cofactors in carbohydrate metabolic pathways. In this study, we found that the thiamin biosynthesis key genes what were associated with pathogenicity in Xoo. Three genes related to thiamin biosynthesis (.thiD, thiE, and thiG) were required for the full virulence in rice. Sequence analysis revealed that thiD, thiE, and thiG were highly conversed in Xanthomonas spp. (>80% identity). The deletion of thiD, thiE, or thiG, which compared to wild-type strain ZJ173 in Xoo dramatically reduced extracellular polysaccharide (EPS) production and affected biofilm formation. The mutations of thiE and thiG had effect on pyruvate metabolism, when the two deletion mutants compared to wild-type strain ZJ173 the accumulate pyruvate was increased. The expression of thiG was down-regulated in △thiE, the expression of thiE was also down-regulated in △thiG. The result demonstrated that the thiamin biosynthesis key genes was essential for pathogenicity of Xoo, and effected energy metabolism of bacterial. In addition, the expression of thiE and thiG was regulated by each other.3. Effect of thiamin synthesis key genes on sensitivity of Xoo to bismerthiazolThiamin was an antagonistic action of sensitivity of Xoo to bismerthiazol, and previous studies had shown that thiamin synthesis related genes were the source of drug targets. The research results were that bismerthiazol supplied thiazol to △thiE and △thiG and promoted growth, and thiamin supplied exogenous nutrients to △thiD, △thiE, and △thiG to promote growth in vitro. The inhibitory action of 0.1mMol or 30mMol of thiamin and 300μg/mL of bismerthiazol to △thiE were significantly decreased, and were 17.87%, 54.97%, and 16.7% respectively. Therefore, we deduced that the target of bismerthiazol was thiamin synthesis related genes thiE.