AvrRxo1 Regulates Rice Resistance To Bacterial Leaf Streak By Interfering The Synthesis Of Vitamin B6

Rice bacterial leaf streak (BLS), which is caused by Xanthomonas oryzae pv. oryzicola (Xoc), results in considerable yield loss, especially in southern and central China. Untill now, there is no effective method to prevent BLS. There is no major genes for resistance to BLS have been reported in rice. It is reported that Xoc may contain one or more effectors, which are dependent on type three secretation system, to suppress the resistance mediated by R genes in rice. Analysing the molecular mechanism of suppressors will explain the absense of R gene against Xoc and will contribute to prevent the accurance of BLS.To clone the suppressor of Xoc, we constructed a genomic cosmid DNA library of Xoc strain RS105 and then transformed the library cosmids into the Xoo strain PXO99A one by one. After incubating on the resistant rice line IRBB21, some clones were found to induce longer lesions. And one of the clones contains fragment which has only one type three effector gene, avrRxol. Moreover, the lesion length caused by PXO99A (pHM1:avrRxo1) on IRBB21 was signaficantly longer than that of PXO99A (pHM1), suggesting that the AvrRxo1 has the suppressor function. In addition, compared with the wild type strain RS105, the mutant RS105△avrRxo1 has weaker pathogenicity with shorter lesion length and less bacterial density. The complementation strain with plasmid-borne full-length avrRxol gene ORF1 and ORF2 largely restored virulence of RS105△avrRxo1 towards the wild-type level, suggesting that the AvrRxo1 plays essential role in the process of Xoc infection of rice.To find out the molecular mechanism of AvrRxo1 in Xoc invasion of rice, we screened the AvrRxo1-interact proteins from rice. One of the AvrRxol-interact proteins is OsPDX1.2, which has two homology, OsPDX1.1 and OsPDX1.3, with highly similar amini acid sequence. The yeast two-hybrid experiment results show that, OsPDX1.1 and OsPDX1.3 can also interact with AvrRxo1. The interaction between AvrRxol and OsPDXl proteins were validated via bimolecular fluorescence complementation experiments and Co-immunoprecipitation in vivo. We also found that, the OsPDXl proteins can interact with each other to assemble into homologous and heterologous dimer. And AvrRxo1 can also interact with the members of AtPDX1 protein faminly and SNZ1, which is the homologous of PDX1 in yeast. Taken together, the interaction of AvrRxol-PDX1 is conserved in rice (a monocot), Arabidopsis (a dicot) and yeast (a fungus)Compared with the wild type strain, the OsPDX1-overexpressing transgenic plants were more resistant to Xoc,while the OsPDX1-suppresing transgenic plants were more susceptible to Xoc. At the same time, the knockout mutants of OsPDX1.1 and OsPDX1.2 were more susceptible to Xoc, suggesting that the OsPDXl genes are positive regulating the resistance to Xoc. And injection with RS105 or the complementation strain will decrease the level of OsPDXl proteins in rice. However, when being injected with RS105△avrRxo1, there will be no effects on the level of OsPDX1 proteins, compared with the control. These resusts indicated that AvrRxo1 may have protease activity. To confirm the results, we tested the protease activity of AvrRxo1 in cell-free system and used prokaryotic expression and purification system. And in Arabidopsis, induced expression of avrRxol gene could decrease the level of PDX1 proteins. In addition, we found that the degradation of OsPDXl proteins mediated by AvrRxo1 is dependent on ATP. These results indicated that dependent on ATP, AvrRxo1 interacts with and degrades the PDX1 proteins.In funga and plants, PDX1 proteins play roles in the biosynthesis of vitamin B6. we found that transient expression of avrRxol could decrease the level of vitamin B6 in NB. Further research identified that injected with RS105 or the complementation strain could significantly decrease the level of vitamin B6 in leaves of rice, while, in comparion with control, injected with RS105△avrRxol could induce no significant difference in the level of vitamin B6. And we also found that vitamin B6 treatment could protect rice against Xoc. The above data indicated that AvrRxol interacts with and degrades the OsPDXl proteins, decreaes the level of vitamin B6, to contribute to pathogenicity of Xoc.AvrRxol can be recognized by Rxol and triggered hypersensitive response in maize and rice. We analysised that the expression pattern of OsPDXl genes and found that the expression of OsPDXl.3 in the Rxol-transgenic plants was much higher than that of the wild-type control. And we also found that transient coexpression of Rxol and avrRxol in Nb could induce the expression of GFP driven by the promoter of OsPDXl.3. In contrast, it is failed to induce GFP expression when only using AvrRxo1 or Rxo1. Rxo1 was identified to interact with AvrRxo1 and promote AvrRxol protein entering the nucleus. Bioinformation analysis results show that there is a cis acting element, named S000465, which play essencial role in the expression of OsPDXl.3 induced by AvrRxo1. S000465 element fused with 35S mini basal promoter to drive GFP gene could also be induced by AvrRxol. We found that AvrRxol can interact with S000465 in Y1H and EMSA analysis. Taken together, the results suggest that, when there is Rxol gene, AvrRxol protein changed its subcellular localization, induced the expression of OsPDX1.3, and promote the biosynthesis of vitamin B6 to protect rice against Xoc.AvrRxo1 was found to be toxic to yeast cells and the toxicity can be weakened by vitaminB6. AvrRxol was fused with fluorescent protein at its N- and C-terminus, respectively. And we found that AvrRxo1-GFP was not toxic to plant cells, while YFP-AvrRxo1 inhibited the elongation of root and induce cell death in Arabidopsis. Subcellular localization analysis showed that AvrRxol-GFP localized in the nucleus, but YFP-AvrRxo1 localized in membrane. Vitamin B6 was found to promote the root elongation of YFP-AvrRxol transgenic Arabidopsis plants. These data suggest that the toxic function of AvrRxo1 is associated with its interaction with PDX1 and the membrane localization is required for toxicity of AvrRxo 1.