Molecular analyses of avrBs3-like virulence factors from the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae

Each strain of Xanthomonas oryzae pv. oryzae has multiple copies of avrBs3 related genes. Mutations in specific avrBs3 related genes in strains PX099, T7174 and KX085 exhibited severely impaired virulence on susceptible rice plants. The genes responsible to the virulence in the respective strains were cloned and sequenced. AvrBs3-like genes pthXO1 and pthXO2 were identified as virulence factors for PX099 and T7174, respectively. The virulence factor in KX085 was functionally similar to avrXa7 from PXO86. AvrXa7 contributes to virulence of PX086 on susceptible rice varieties and avirlence on Xa7-containing plants. pthXO1 and pthXO2 had no recognizable avirulence specificity. However, avrXa7 , pthXO1 and pthXO2 were interchangeable for virulence activity. The proteins of the avrBs3 family have striking structural features. The middle third of the protein consists of varying number of 34 amino acid repeats and determines the specificity of each protein. The carboxyl terminus of the protein contains three nuclear localization signals (NLS) and an acidic transcriptional activation domain (AD). AvrXal0 and AvrXa7 require the AD for their avirulence activities. The loss of avirulence activity due to the mutations in the acidic transcriptional activation domain was restored by addition of the AD from the herpes simplex, viral protein VP 16. The AD was also required for the virulence activity of AvrXa7. However, the VP16 domain could not substitute for the endogenous domain in virulence assays. The NLS motifs were required for avirulence and virulence activities of AvrXa7, and the requirement could be compensated by the NLS motif from the T-antigen of SV40 virus. In a yeast assay system the NLS motifs could direct the full-length AvrXa7 into the cell nuclei and mutations of NLS motifs severely reduced the nuclear localization. In gel shift assays, AvrXa7 bound double-stranded DNA with a preference for dA/dT rich sequences. The results indicate that Xanthomonas oryzae pv. oryzae injects the products of avrBs3-related genes as virulence factors to host cell nuclei and have the potential to interact with the host DNA and transcriptional machinery as part of their mode-of-action. The results also suggest that the host defensive recognition mechanisms are targeted to the virulence factor site-of-action.