Cloning And Functional Characterization Of Two Tal Genes From Xanthomonas Oryzae PV. Oryzae Strain OS198

Rice bacterial leaf blight is one of the most important bacterial diseases on rice. It is caused by gram-negative bacteria Xanthomonas oryzae pv. oryzae. TALEs are secreted by type III secretion system and they are encoded by tal genes. Now the research of tal genes mainly focused on several sequenced strains which were from Philippines, Japan and South Korea. avr/pthC8a, an avirulence gene from C8, was the only reported tal gene from Chinese strain. Discovering new tal genes is the basis to reveal the mechanism of pathogenic, provides theoretical basis and genetic resources for further studying the genetic engineering of Xoo.OS 198 is the representative of China strain of race 6 of Xoo. It displayed low virulence on different varieties of rice. To understand the molecular genetic mechanisms of pathogenicity of OS 198, two library clones of pUF369 and pUF377 were screened for tal gene cloning and function analysis.We first isolated the 3.2 kb band from pUF369 and the 2.6 kb band from pUF377 and cloned the fragments to pZW respectively, a backbone vector for tal gene cloning. The two tal gene clones were then linearized and ligated to the vector pHMl to produce the constructs of pHZ369, pHZ377 respectively.In vitro Tn5 insertion was adoppted to mutant the tal genes. And the primers from both ends of the Tn5 were used for sequencing the repeat regions of 34 amino acids. The full tal gene sequence of pzw369-3.2 was named as talR26.5 （NCBI login number for KJ192237.1）. It is 4452 bp in length, encoding 1483 amino acids. TalR26.5 has 26.5 repeats and displayed 99% identity with the TAL effectors AvrXa7-3M of Philippines strain PXO357 and YP₄51895 of Japan strain MAFF 311018. These three TALEs have same repeat regions. The tal gene in pzw377-2.6 was named as talR20.5 （NCBI login number for KJ192238）. It is 3819 bp in length, encoding 1272 amino acids with 20.5 repeats of 34 amino acid in the repeat region and has a high homology as 98%with YP₄51156 of Japan strain MAFF 311018, but the repeat variable diresidues were not identical.pHZ369, pHZ377 were introduced into PXO99A to obtained the recombinants PXO99A/pHZ369 and PXO99A/pHZ377. When inoculated in rice, PXO99A/pHZ369 showed reduced virulence in IRBB8, IRBB14 and IRBB214 compared with PXO99A. PXO99A/pHZ377 displayed reduced virulence in rice IRBB50. But compared with OS 198, the two strains showed significant increased phenomenon in both lesion length and the bacterial growth in leaves. The expression of rice susceptible gene Os8N3 was investigated by semi-quantitative RT-PCR 24 h after inoculation PXO99A/pHZ369 and PXO99A/pHZ377, we found that the expression of Os8N3 was reduced by infection of PXO99A/pHZ369 and PXO99A/pHZ377 compared with PXO99A.After inoculated in tobacco leaves, the PXO99A/pHZ369, PXO99A/pHZ377 enhanced hypersensitive response and increased the number of cell death on tobacco compared with PXO99AIn summary, talR26.5 and talR20.5 of OS 198 play avirulence role and have the role of inducing plant resistance after pathogen infection.