The Molecular Mechanisms Of Pathogenicity Variation Of Xanthomonas Oryzae Pv. Oryzae

Rice is a most important food crop in China. The bacterial blight (BB) caused by Xanthomonas oryzae pv.oryzae(Xoo) is one of the most serious bacterial pathogens in the rice in China. The pathogenicity variation of Xoo often results in BB even for the disease-resistant rice, which could bring about tremendous yield reduction and economic loss. Two new mutants of Xoo with variable pathogenicity, HuB05-8and HAN08-1, were studied for understanding the molecular mechanisms why they could infect the rice contained gene Xa21with broad-spectrum resistance to BB. In our present experiment, we obtained DNA sequences in genes related BB in HuB05-8and HAN08-1by PCR amplification and DNA sequencing techniques. After compared with PXO99A by using bioinformatics methods, we found the differences in pathogenic genes. Moreover, changes in pathogenicity of experimental microbial strains were observed by constructing vector and transferring DNA sequences of the pathogenic genes into the receptor. The pathogenicity related gene expression was then observed by RT-PCR and the genetic regulation was revealed. Following are the main results.(1) According to the genetic sequences of sulfurated protein efflux by Xoo publicated by NCBI, we designed the primers, then amplified and sequenced target DNA.9genes related pathogenicity were obtained in HuB05-8and HAN08-1.(2) The base A responsible for coding positively charged Lys was changed into the base G for negatively charged Glu in Ax21of both HuB05-8and HAN08-1. In addition, a few bases in raxA and raxB were changed, including some synonymous mutations. Besides, There were no different in genetic sequences in the two genes.(3) raxA, raxB, Ax21genes in PXO99A were inserted into plasmid pEASY, cloned in vector PHM1, and expressed in HuB05-8and HAN08-1. The complementation test indicates that raxA and raxB gene variations influenced little, if any, on pathogenicity variation, whereas a single base variation in Ax21gene behaved otherwise. So, raxA and raxB genes could encode probably the type I secretion system (TOSS) and could not change its function. However, a single base variation in Ax21change the gene expression and function of protein encoding. As a result, PAMP (Pathogen-associated molecular pattern) could not be recognized by PRR-XA21(Pattern recognition receptor) to stimulate disease-resistant response in rice.(4)There are eight genes required for Ax21activity. Our experiment shows that cell population size had less effect on expression of raxA, raxB and raxC gene encoding the type I secretion system (TOSS), similar to the expression of raxST, raxP and raxQ which encode enzymes involved in sulfation, for these genes belong to constitutive expression. Whereas the expression of raxH/raxR gene encoding two-component regulatory (TCSs) could strikingly increase at low or high cell densities, so raxH/raxR are hypothesized to function in the perception and regulation of Ax21.(5) Under the condition of Xoo PXO99A, HuB05-8, HAN08-1cell density consistency (OD600=1.0), the expression of Ax21, raxHR, raxSTAB genes in HuB05-8and HAN08-1were obviously lower than PXO99A, whereas there were no difference among raxPQ, raxC gene expression.This result indicates that the pathogenicity variation of HuB05-8, HAN08-1was involved in decreased expression of Ax21, raxHR and raxSTAB, for the lower Ax21expression could reduce rice defence response which is induced by recognition of pathogen-associated molecular pattern (PAMP) Ax21by XA21at the cell surface. several bases variations in raxA and raxB genes of HuB05-8and HAN08-1may probably affect transcription efficiency, and two-component regulatory system raxH/raxR are involved in regulation and secretion of Ax21.