Functional analysis of tal4 of Xanthomonas oryzae pv. oryzae strain PXO99A in resistance to bismerthiazol.

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. tal4 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 dug 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 wildtype. tal4 gene knock-out mutant PXO99▵ tal4 was generated from PXO99A based on double crossover homologous re- combination. The cosmid pHZtal4 containing the tal4 coding sequence was electroporated into PXO99▵ tal4 to construct the comple-mentation 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 PXO99▵ tal4 to bismerthiazol was measured both with plate assay and in rice leaf sprayed with bismerthiazol 24 h before and past inoculation. Real-time polymerase chain reaction(qPCR)was taken to analyze the expression of two pesticide resistance related genes of ribosomal RNA 16S( rrs) and 30S ribosomal subunit protein S12(rpsL). Neither the lesion length nor the bacterial growth in rice leaves showed notable difference two weeks past inoculation of the three strains,which indicated that missing tal4 did not affect the virulence of PXO99A in rice(variety of IR24). There was 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 tal4 did not play a 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 con- centration(MIC) of bismerthiazol to PXO99▵ tal4 is 70 μg.mL-1 , lower than 110μg.mL-1 to PXO99A. The median effect concentration(EC50 )of bismerthiazol to PXO99▵ tal4 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. The lesion length in bismerthiazol sprayed leaves caused by the inoculation of PXO99Aand PXO99▵ tal4 showed no notable changes, but the bacterial growth inside the leaves infected by PXO99▵ tal4 showed a significant reduction( P<0. 05) even when the applied concentration of bismerthiazol was at 10μg.mL-1 . There existed no difference between the treatments 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, and its expression was only 38% of PXO99A(P<0. 01). There was no obvious difference of the expression of rpsL between PXO99▵ tal4 and PXO99A. tal4 gene has no effect on the virulence of PXO99A in rice, but it plays an important role in resistance to bismerthiazol. This is the first report that the TALE proteins play a role in resistance to pesticide bismerthiazol. [ABSTRACT FROM AUTHOR]