Analysing The Molecular Mechanism Of Interaction Between Xa13 Promoter And Plant Pathogen In Rice

Multiple bacteria strains of Xanthomonas can cause plant disease, such as Xanthomonas oryzae pv. Oryzicola (Xoc), X. campestris pv. Vesicatoria (Xcv) as well as the pathogen causing bacterial blight X. oryzae pv. Oryzae (Xoo). One type of effector secreted by Xanthomonase named TAL (Transcription activator-like) can directly bind the UPT (up-regulated by TAL effectors) box in the promoter of host target genes and induce the expression of them for pathogenesis. Rice Xa 13 gene, which promoter harbors a UPT box, UPTPthXol, plays a pivotal role in the race-specific pathogenicity caused by Xanthomonas oryzae pv. oryzae(Xoo) strain PXO99. PXO99 causes rice disease by inducing Xa13. Here we revealed the molecular mechanism of interaction between UPT box in the Xa13 promoter and the TAL effector PthXol. We also explored the role of plant transcription factor TFIIAy5 in the interaction.Analysis of a series of truncated promoter showed that the UPTPthXol box in the Xal3 promoter is the only cis-element that can be induced by Xoo strain PXO99, while lacking of the UPT box in the xa13 promoter made them can not be induced by PXO99. The binding capacity of probe containing the complete UPTPthXol box with the total protein of PXO99 was significantly higher than probes from the corresponding region of different recessive xal3 promoters. EMSA (Electrophoretic mobility shift assay) analysis of site-mutated UPTPthXol box probe to PXO99 total proteins showed that the 5’end second, third and forth nucleotides of UPTPthXol box were critical for its binding ability, while the sixth nucleotide had lillte contribution to the binding. According to the result, we deduced that different nuleotides in the UPTPthXol box did not has the same contribution to the binding. Analysis of site-mutated Xal3 promoter revealed the the loss of binding ability to PXO99 proteins also resulted in the loss of faculty of promoter induced by PXO99. This suggested the binding of UPTPthXol box to TAL effector was the foundation of its induction. The promoter activity of a serious site-truncated promoter showed the integrality of UPTPthXol box in the Xal3 promoter not only influenced its binding ability but also was the key factor of Xal3 induction by PXO99. And the 3’flanking sequence of UPTPthXol box also contributed to Xa13 induction by PXO99.In order to figure out the effect of TFIIAy5 on PthXol induced expression of Xa13, we inoculated plant containing TFIIAy5 and its recessive allele xa5 with PXO99 and PX071 respectively, and observed changes in expression levels of Xal3. These two strains both cantein effector PthXol. The result proved that the mutated TFIIAy5 significantly reduced Xal3 induction by PthXol. In order to confirm this result, we overexpressed TFIIAγ5 as well as suppressed it in Xa13-containing Japonica variety Zhonghua 11 by Agrobacterium-mediated transformation. The result showed that overexpressed TFIIAy5 increased the expression of Xal3, especially after PXO99 inoculation, while the effect of suppressed TFIIAy5 was similar to xa5, which significantly decreased the Xa13 induction by PXO99. These suggest TFIIAγ5 can help PthXol induce Xal3 expression. Analysis the binding ability of UPT box to nuclear proteins from different plants in vivo, we found that the binding ability of UPT box to proteins from xa5-containing plant was much lower than that from TFIIAγ5-containing plant. Also, the binding ability of UPT box to proteins from transgenic plants was remarkably different with proteins from wide type Zhonghuall, while mutated UPT boxes had almost the same binding ability to both transgenic plants and wide type. These results suggest the expression level change of TFIIAy5 or the structural change of TFIIAy5 will both influence the binding ability of UPT box to plant nuclear proteins, which may lead to changes in the induction level of Xa13 by PthXol.In conclusion, UPT box in the Xa13 promoter is the key cis-element that contribute to recognition and induction of Xa13 by PthXol. And the rice transcription factor TFIIAγ5 act as a positive regulator in this process.