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Mutagenesis of 18 Type III Effectors Reveals Virulence Function of XopZ_PXO99 in Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae depends on a type III secretion system (T3SS) to translocate effectors into host cells for its ability to cause bacterial blight of rice. All type III (T3) effectors with known function in X. oryzae pv. oryzae belong to a family of transcription activator-like (TAL) effectors. However, other, non--TAL-related effector genes are present in the genome, although their role in virulence and their mode of action have yet to be elucidated. Here, we report the generation of mutants for 18 non-TAL T3 effector genes and the identification of one that contributes to the virulence of strain PXO99^A. XopZ_PXO99 encodes a predicted 1,414-amino-acid protein of unknown function. PXO99^A contains two identical copies of the gene due to a duplication of 212 kb in the genome. Strains with knockout mutations of one copy of XopZ_PXO99 did not exhibit any visible virulence defect. However, strains with mutations in both copies of XopZ_PXO99 displayed reduced virulence in terms of lesion length and bacterial multiplication compared with PXO99^A. The introduction of one genomic copy of XopZ_PXO99 restores the mutant to full virulence. Transient expression of XopZ_PXO99 in Nicotiana benthamiana leaves suppresses host basal defense, which is otherwise induced by a T3SS mutant of PXO99^A, suggesting a role for XopZ_PXO99 in interfering with host innate immunity during X. oryzae pv. oryzae infection. XopZ_PXO99-related genes are found in all Xanthomonas spp. whose genomic sequences have been determined, suggesting a conserved role for this type of effector gene in pathogenesis of Xanthomonas spp. Our results indicate that XopZ_PXO99 encodes a novel T3 effector and contributes virulence to X. oryzae pv. oryzae strains for bacterial blight of rice.