灰霉病菌双组分组氨酸激酶信号途径上五个关键基因的功能研究
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摘要
双组分组氨酸激酶(以下简称TCHK)信号途径在真菌体内参与多种生命活动,但目前,人们对灰霉病菌的TCHK信号途径还缺乏深入的了解。已有研究表明,二甲酰亚胺类杀菌剂(以下简称DCFs)通过激活TCHK信号途径而抑制灰霉病菌生长,但该药剂具体的作用位点尚不清楚,因此,研究灰霉病菌TCHK信号途径对于了解DCFs的作用机制有重要的理论和现实意义。
本课题利用农杆菌介导的真菌转化系统、通过基因敲除和互补的方法研究了灰霉病菌TCHK途径上五个重要元件BOS1、BRRG-1、BOS4、BOS5和BOS2的生物学功能,结果发现：1) BRRG-1、BOS4、BOS5和BOS2的敲除突变体均不形成气生菌丝和分生孢子；BOS5敲除突变体的菌丝严重扭曲畸形、多分枝、顶端膨大。2)药剂敏感性测定发现,只有BOSl基因敲除突变体对DFCs表现抗性,而其它四个基因敲除突变体对该类药剂表现更加敏感；另外,BOS1上点突变可导致病菌对DFCs产生抗性。3)渗透压敏感性测定发现,所有五个元件的敲除突变体对NaCl和KCl产生的离子型渗透压力表现敏感,而对山梨醇等中性渗透压力的敏感性无改变。4)氧化压力敏感性测定发现,除BOS1以外其他四个元件的敲除突变体对H202均表现敏感。5)致病性测定表明,灰霉病菌TCHK信号途径上的三个促分裂原活化蛋白(以下简称MAP)激酶BOS4、BOS5和BOS2与致病性密切相关,这些基因缺失突变体能正常侵染穿透寄主细胞但不能形成病斑。6)蛋白杂交结果显示,在正常培养条件下,BRRG-1、BOS4 (?)口BOS5敲除突变体内均检测不到BOS2磷酸化,表明这些组分位于BOS2的上游。
本研究结果表明,灰霉病菌TCHK信号途径参与菌体致病性、产孢、生长发育等多种生命活动；BOS1应该是DFCs的靶标位点；此外,由于BOS4、BOS5和BOS2在病菌致病中发挥重要作用,它们可能成为潜在的新药靶。
Two-component histidine kinase (TCHK) signaling pathway is involved in a variety of life activities of fungi, but to date, little is known about the TCHK in Botrytis cinerea, which causes grey mold in more than 200 economically important crops. Previous studies have showed that the dicarboximides fungicides (DCFs) act as antifungal compounds, in part, through activation of the TCHK in B. cinerea. However, the target of DCFs remains unclear. In this study, thus, we investigated functions of the five important TCHK components:BOS1, BRRG-1, BOS4, BOS5 and BOS2 in Botrytis cinerea using a gene knockout and complementary strategy. Results of this study showed that 1) BRRG-1, BOS4, BOSS and BOS2 knockout mu-tants could not produce aerial hyphae and conidia. The BOS5 deletion mutant formed seriously distorted hyphal and swelling tips with more branches.2) The disruption mutant of BOS1 showed increased resistance to DCFs, while mutants of other four elements exhibited more sensitive to these fungicides compared to the wild type strain. Additionally, mutations in BOS1 resulted to resistance to DCFs.3) Sensitivity to osmotic stress showed that all five gene deletion mutants revealed increased sensitiv-ity to ionic osmotic stresses produced by NaCl and KC1, but not by the neutral os-motic stress mediated by sorbitol.4) Sensivity to oxidative stress indicated that the mutants of BRRG-1, BOS4, BOS5 and BOS2 became more sensitive to oxidative stress generated by H2O2 except that of BOS1as compared to the wild-type strain.5) Inoculation on cucumber and rapeseed leaves showed that the three mitogen-activated protein (MAP) kinases BOS4, BOS5 and BOS2 in TCHK pathway were essential for the pathogenicity. These gene disruption mutants could penetrate the host surface but couldn't form lesions.6) Western blotting analysis showed that the phosphorylation of BOS2 protein was not detected in BRRG-1, BOS4 and BOS5 deletion mutants un-der normal cultural condition, suggesting that these elements were located at the BOS2 upstream of TCHK signaling pathway.
Results of this study indicate that 1) the TCHK signaling pathway is involved in regulating vegetative differentiation, pathogenicity, and the sensitivity to osmotic and oxidative stresses in B. cinerea; 2) BOS1 could be the target of DCFs, meanwhile be-cause BOS4, BOS5 and BOS2 are important virulence determinants in B. cinerea, they could become potential targets for the development of new antifungal com-pounds.
本课题利用农杆菌介导的真菌转化系统、通过基因敲除和互补的方法研究了灰霉病菌TCHK途径上五个重要元件BOS1、BRRG-1、BOS4、BOS5和BOS2的生物学功能,结果发现：1) BRRG-1、BOS4、BOS5和BOS2的敲除突变体均不形成气生菌丝和分生孢子；BOS5敲除突变体的菌丝严重扭曲畸形、多分枝、顶端膨大。2)药剂敏感性测定发现,只有BOSl基因敲除突变体对DFCs表现抗性,而其它四个基因敲除突变体对该类药剂表现更加敏感；另外,BOS1上点突变可导致病菌对DFCs产生抗性。3)渗透压敏感性测定发现,所有五个元件的敲除突变体对NaCl和KCl产生的离子型渗透压力表现敏感,而对山梨醇等中性渗透压力的敏感性无改变。4)氧化压力敏感性测定发现,除BOS1以外其他四个元件的敲除突变体对H202均表现敏感。5)致病性测定表明,灰霉病菌TCHK信号途径上的三个促分裂原活化蛋白(以下简称MAP)激酶BOS4、BOS5和BOS2与致病性密切相关,这些基因缺失突变体能正常侵染穿透寄主细胞但不能形成病斑。6)蛋白杂交结果显示,在正常培养条件下,BRRG-1、BOS4 (?)口BOS5敲除突变体内均检测不到BOS2磷酸化,表明这些组分位于BOS2的上游。
本研究结果表明,灰霉病菌TCHK信号途径参与菌体致病性、产孢、生长发育等多种生命活动；BOS1应该是DFCs的靶标位点；此外,由于BOS4、BOS5和BOS2在病菌致病中发挥重要作用,它们可能成为潜在的新药靶。
Two-component histidine kinase (TCHK) signaling pathway is involved in a variety of life activities of fungi, but to date, little is known about the TCHK in Botrytis cinerea, which causes grey mold in more than 200 economically important crops. Previous studies have showed that the dicarboximides fungicides (DCFs) act as antifungal compounds, in part, through activation of the TCHK in B. cinerea. However, the target of DCFs remains unclear. In this study, thus, we investigated functions of the five important TCHK components:BOS1, BRRG-1, BOS4, BOS5 and BOS2 in Botrytis cinerea using a gene knockout and complementary strategy. Results of this study showed that 1) BRRG-1, BOS4, BOSS and BOS2 knockout mu-tants could not produce aerial hyphae and conidia. The BOS5 deletion mutant formed seriously distorted hyphal and swelling tips with more branches.2) The disruption mutant of BOS1 showed increased resistance to DCFs, while mutants of other four elements exhibited more sensitive to these fungicides compared to the wild type strain. Additionally, mutations in BOS1 resulted to resistance to DCFs.3) Sensitivity to osmotic stress showed that all five gene deletion mutants revealed increased sensitiv-ity to ionic osmotic stresses produced by NaCl and KC1, but not by the neutral os-motic stress mediated by sorbitol.4) Sensivity to oxidative stress indicated that the mutants of BRRG-1, BOS4, BOS5 and BOS2 became more sensitive to oxidative stress generated by H2O2 except that of BOS1as compared to the wild-type strain.5) Inoculation on cucumber and rapeseed leaves showed that the three mitogen-activated protein (MAP) kinases BOS4, BOS5 and BOS2 in TCHK pathway were essential for the pathogenicity. These gene disruption mutants could penetrate the host surface but couldn't form lesions.6) Western blotting analysis showed that the phosphorylation of BOS2 protein was not detected in BRRG-1, BOS4 and BOS5 deletion mutants un-der normal cultural condition, suggesting that these elements were located at the BOS2 upstream of TCHK signaling pathway.
Results of this study indicate that 1) the TCHK signaling pathway is involved in regulating vegetative differentiation, pathogenicity, and the sensitivity to osmotic and oxidative stresses in B. cinerea; 2) BOS1 could be the target of DCFs, meanwhile be-cause BOS4, BOS5 and BOS2 are important virulence determinants in B. cinerea, they could become potential targets for the development of new antifungal com-pounds.
引文
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