tal4基因在水稻白叶枯病菌PXO99～A对噻枯唑抗性中的作用

Functional analysis of tal4 of Xanthomonas oryzae pv. oryzae strain PXO99～A in resistance to bismerthiazol

类似转录激活子(transcription activator-like effector,TALE)是植物病原细菌通过Ⅲ型泌出系统(T3SS)分泌到寄主细胞中的效应蛋白,其编码基因为tal基因。tal4基因是水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)PXO99A中19个tal基因中的一个,但其功能未知。本研究通过测定tal4基因缺失突变菌株在寄主水稻上的毒力,对温度、盐、酸碱等胁迫的反应以及对杀菌剂的敏感性来挖掘tal4基因的功能。通过同源交换,对PXO99A中的tal4基因进行敲除,获得了tal4基因缺失突变菌株PXO99Δtal4。将携带tal4编码基因的黏粒pHZtal4电击转化到PXO99Δtal4感受态细胞中,获得回补菌株C-Δtal4。用液体培养法测定了PXO99A、PXO99Δtal4及C-Δtal4在不同温度、盐和酸碱胁迫下的生长情况。在NA平板及寄主水稻上进行PXO99Δtal4对噻枯唑敏感性检测,并通过实时定量PCR检测了菌株中与抗药性相关的核糖体RNA 16S(ribosomal RNA 16S,rrs)和30S核糖体蛋白S12(30S ribosomal subunit protein S12,rpsL)基因的表达情况,从转录水平探究tal4基因在菌株抗药性中的作用机制。对苗期水稻的接种结果表明,PXO99Δtal4在水稻上形成的病斑长度及在水稻叶片中的定殖能力与PXO99A相比无明显变化,tal4基因的缺失不影响水稻白叶枯病菌在水稻上的毒力。PXO99Δtal4在温度、盐和酸碱等胁迫条件下的生长能力与PXO99A一致。在喷施噻枯唑的水稻上和含噻枯唑的NA平板上,PXO99Δtal4对噻枯唑的敏感性较PXO99A明显增强。噻枯唑对PXO99Δtal4的最低抑制浓度(MIC)为70μg·mL-1,显著低于对PXO99A的最低抑制浓度(110μg·mL-1),而噻枯唑对PXO99Δtal4的抑制中浓度EC50值为PXO99A的77.9%。tal4基因可以恢复PXO99Δtal4对噻枯唑的敏感性至PXO99A水平。实时荧光定量PCR测定发现,PXO99Δtal4中rrs基因的表达量显著下降,仅为PXO99A的38%,而rpsL基因的表达量与PXO99A没有显著差异。上述结果表明:PXO99A中tal4基因的缺失对病原菌的毒力没有明显影响,但在病原菌对杀菌剂的抗性中起一定的作用。本研究首次发现,水稻白叶枯病菌效应蛋白基因tal参与了病原菌对杀菌剂噻枯唑的抗药性。

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 PXO99Δtal4 was generated from PXO99Abased on double crossover homologous recombination. The cosmid pHZtal4 containing the tal4 coding sequence was electroporated into PXO99Δtal4 to construct the complementation strain C-Δtal4. PXO99A,PXO99Δtal4 and C-Δtal4 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 PXO99Δtal4 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 30Sribosomal 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 PXO99Ain rice（variety of IR24）. There was no effect on the growth of PXO99Δtal4 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. PXO99Δtal4 showed significantly reduced resistance to bismerthiazol in either plate assay or in the rice plant assay. The minimum inhibitory concentration（MIC） of bismerthiazol to PXO99Δtal4 is 70 μg·mL-1,lower than 110 μg·mL-1to PXO99A. The median effect concentration（EC50） of bismerthiazol to PXO99Δtal4 was 22. 1% lower than that to PXO99A（P0. 05）. The introduction of tal4 coding sequence to PXO99Δtal4 could recover the deficiency to PXO99A. The lesion length in bismerthiazol sprayed leaves caused by the inoculation of PXO99Aand PXO99Δtal4 showed no notable changes,but the bacterial growth inside the leaves infected by PXO99Δtal4 showed a significant reduction（P0. 05） even when the applied concentration of bismerthiazol was at 10 μg·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 PXO99Δtal4 was reduced significantly compared with that in PXO99A,and its expression was only 38% of PXO99A（P0. 01）. There was no obvious difference of the expression of rpsL between PXO99Δtal4 and PXO99A. tal4 gene has no effect on the virulence of PXO99Ain 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.