Functional Analysis Of Tal4 Gene Of Xanthomonas Oryzae Pv. Oryzae Strain PXO99~A

Transcription activator-like effectors (TALEs) encoded by tal genes are secreted into plant cells through the type III secretion system (T3SS) of gram-negative plant pathogenic bacteria, ta.14 is one of nineteen tal genes in Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99A, but its function is unknown.In this study, the function of tal4 gene was exploited by measuring the virulence in rice, the stress tolerance to temperature, salt and acid, and the sensitivity against bismerthiazol of the tal4-deleted mutant compared with the wild type.tal4 gene knock-out mutant PXO99?tal4 was generated from PXO99A based on double crossover homologous recombination. The cosmid pUZtal4 containing the tal4 coding sequence was introduced into PXO99?tal4 to construct the complementation strain C-?tal4. PXO99A, PXO99?tal4 and C-?tal4 were grown in liquid medium to compare the growth rates under the stress conditions of different temperatures, salt contents and pH values. The sensitivity of the mutant to bismerthiazol was measured both with plate assay and in rice leaf sprayed with bismerthiazol 24 h before and post inoculation. Real-time Polymerase Chain Reaction (qPCR) was taken to analysis the expression of two pesticide resistance related genes, ribosomal RNA 16S (rrs) and 30S ribosomal subunit protein S12 (rpsL).Two weeks after inoculation, neither the lesion length nor the bacterial growth in leaves among the three strains showed notable difference, which indicated that missing tal4 did not affect the virulence of PXO99A in rice (IR24). No effect on the growth of PXO99?tal4 under the stress condition of temperature, salt, acidity and alkaline compared to that of the wild type strain, it showed that ta14 did not play a significant role in resistance to environmental adversity.PXO99?tal4 showed significantly reduced resistance to bismerthiazol in either plate assay or in the rice plant assay. The minimum inhibitory concentration (MIC) of bismerthiazol to PXO99?tal4 is 70?g/mL, lower than 110?g/mL to PXO99A; the inhibitory concentration EC50 of bismerthiazol to PXO99?ttal4 was 22.1% lower than that to PXO99A (P<0.05). The introduction of tal4 coding sequence to PXO99?tal4 could recover the deficiency to PXO99A level. The appearance of the lesion phenotype on bismerthiazol sprayed leaves and the lesion length caused by the inoculation of PXO99A and FXO99?Atal4 showed no notable changes, while the bacterial growth in leaves of PXO99?tal4 showed a significant reduction (P<0.05) on sprayed rice even when the bismerthiazol concentration was only 10 ?g/mL. There was no difference exist between spraying bismerthiazol before or past Xoo inoculation.qPCR detection results revealed that the expression of rrs of PXO99?tal4 was reduced significantly compared with that in PXO99A, its expression was only 38% of PXO99A (P<0.01). While there was no significant expression difference of rpsL between VXO99?tal4 and PXO99A. rrs gene knock-out mutant PXO99?rrs was generated from PXO99A based on double crossover homologous recombination. The mutant showed increased sensitivity to bismerthiazol compared with PXO99A.These results showed above indicated that tal4 gene has no effect on the virulence of PXO99A in rice, but it plays an important role in resistance to bismerthiazol. tal4 gene may affect the expression of rrs to impact the resistance to bismerthiazol of PXO99A. This is the first report that the tal gene play roles in resistance to pesticide bismerthiazol.