西瓜噬酸菌Ⅲ型分泌系统hrcQ基因功能分析
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摘要
Ⅲ型分泌系统(type Ⅲ secretion system,T3SS)是影响西瓜噬酸菌Acidovorax citrulli致病性的重要因素,hrcQ作为T3SS的保守基因,在组成和维持该系统功能方面扮演着重要角色,但hrcQ在西瓜噬酸菌中的具体生物学功能尚不明确。本试验以西瓜噬酸菌Aac5菌株为研究对象,通过基因敲除、致病力及相关表型测定等,解析了hrcQ基因的功能。结果表明:hrcQ缺失导致Aac5菌株对寄主的致病能力丧失,运动性及生物膜形成能力降低,并丧失了引起烟草过敏性反应能力;hrcJ和hrcR基因在hrcQ缺失突变株中的表达量下调,hrcQ对hrcJ和hrcR基因均为正调控。表明hrcQ在维持西瓜噬酸菌致病能力中起着不可或缺的作用。
Acidovorax citrulli is the causal agent of bacterial fruit blotch, which causes serious losses in watermelon and melon industry. Type Ⅲ secretion system(T3 SS) plays an important role in the pathogenicity of A.citrulli. The hrcQ is an important and conserved gene in forming and maintaining the function of T3 SS. However, the biological function of this gene in A.citrulli is unclear. Therefore, in this study, we analyzed the functions of hrcQ by gene knockout, virulence and phenotypic determinations based on the wild type strain Aac5. The results showed that the hrcQ gene deletion mutant strain caused significant reduction in pathogenicity, swimming motility and biofilm formation, and it lost the ability to induce hypersensitive reaction in non-host Nicotiana tabacum. Besides, the expressions of hrcJ and hrcR genes were decreased in the mutant strain, indicating a positive regulation of hrcQ to both genes. More importantly, we confirmed that the deletion of hrcQ resulted in the loss of virulence of Aac5 on its host. Taken together, these results indicated that hrcQ was the key gene for maintaining the pathogenicity of A.citrulli.
Acidovorax citrulli is the causal agent of bacterial fruit blotch, which causes serious losses in watermelon and melon industry. Type Ⅲ secretion system(T3 SS) plays an important role in the pathogenicity of A.citrulli. The hrcQ is an important and conserved gene in forming and maintaining the function of T3 SS. However, the biological function of this gene in A.citrulli is unclear. Therefore, in this study, we analyzed the functions of hrcQ by gene knockout, virulence and phenotypic determinations based on the wild type strain Aac5. The results showed that the hrcQ gene deletion mutant strain caused significant reduction in pathogenicity, swimming motility and biofilm formation, and it lost the ability to induce hypersensitive reaction in non-host Nicotiana tabacum. Besides, the expressions of hrcJ and hrcR genes were decreased in the mutant strain, indicating a positive regulation of hrcQ to both genes. More importantly, we confirmed that the deletion of hrcQ resulted in the loss of virulence of Aac5 on its host. Taken together, these results indicated that hrcQ was the key gene for maintaining the pathogenicity of A.citrulli.
引文
[1] BURDMAN S, WALCOTT R. Acidovorax citrulli: generating basic and applied knowledge to tackle a global threat to the cucurbit industry [J].Molecular Plant Pathology,2012,13(8):805-815.
[2] SCHAAD N W, SOWELL G J, GOTH R W, et al. Pseudomonas pseudoalcaligenes subsp. citrulli subsp. nov.[J].International Journal of Systematic Bacteriology,1978,28(1):117-125.
[3] SCHAAD N W, POSTNIKOVA E, SECHLER A, et al. Reclassification of subspecies of Acidovorax avenae as A.avenae (Manns 1905) emend. A.cattleyae (Pavarino, 1911) comb. nov. A.citrulli Schaad et al. 1978) comb. nov. and proposal of A.oryzae sp. nov.[J]. Systematic & Applied Microbiology, 2008, 31(6):434-446.
[4] DUTTA B, GITAITIS R, SMITH S, et al. Interactions of seedborne bacterial pathogens with host and non-host plants in relation to seed infestation and seedling transmission [J/OL]. PLoS ONE, 2014, 9(6): e99215.
[5] JIANG Chunhao, WU Fang, YU Zhenyun, et al. Study on screening and antagonistic mechanisms of Bacillus amyloliquefaciens 54 against bacterial fruit blotch (BFB) caused by Acidovorax avenae subsp. citrulli [J]. Microbiological Research, 2015, 170:95-104.
[6] BüTTNER D, HE Shengyang. Type Ⅲ protein secretion in plant pathogenic bacteria[J]. Plant Physiology, 2009, 150(4):1656-1664.
[7] GALáN J E, COLLMER A.Type Ⅲ secretion machines: bacterial devices for protein delivery into host cells [J]. Science, 1999, 284(5418):1322-1328.
[8] KVITKO B H, RAMOS A R, MORELLO J E, et al. Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are functionally similar to HrpK1 in promoting translocation of type Ⅲ secretion system effectors [J]. Journal of Bacteriology, 2007, 189(22):8059-8072.
[9] 李宾,李爱宁,魏强,等.欧美杨细菌性溃疡病菌hrcJ基因的功能分析[J].林业科学,2015,51(12):71-78.
[10] SUGIO A, YANG Bing, WHITE F F. Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae [J].Molecular Plant-Microbe Interactions,2005,18(6):546-554.
[11] 严婉荣, 王铁霖, 杨玉文,等. 瓜类细菌性果斑病菌hrcN基因的生物学功能分析[J]. 植物病理学报, 2015, 45(1):33-40.
[12] JOHNSON K L, MINSAVAGE G V, LE T, et al. Efficacy of a nonpathogenic Acidovorax citrulli strain as a biocontrol seed treatment for bacterial fruit blotch of cucurbits [J]. Plant Disease, 2014, 95(6):697-704.
[13] 任争光, 侯磊, 宋治国,等. 甜瓜细菌性果斑病菌致病性突变体筛选与hrcR基因的克隆[J]. 植物病理学报, 2009, 39(5):501-506.
[14] 汪新, 王卫, 钱国良,等. 瓜类细菌性果斑病菌过敏性反应和致病性(hrp)基因簇部分基因的克隆及功能分析[J]. 农业生物技术学报, 2011, 19(1):36-44.
[15] DENG Wanyin, MARSHALL N C, ROWLAND J L, et al. Assembly, structure, function and regulation of type Ⅲ secretion systems [J]. Nature Reviews Microbiology, 2017, 15(6):323-337.
[16] ZHANG Xiaoping, WANG Chunlian, ZHENG Chongke, et al. HrcQ is necessary for Xanthomonas oryzae, pv. oryzae, HR-induction in non-host tobacco and pathogenicity in host rice[J]. The Crop Journal, 2013, 1(2):143-150.
[17] LORENZ C, HAUSNER J, BüTTNER D. HrcQ provides a docking site for early and late type Ⅲ secretion substrates from Xanthomonas [J/OL]. PLoS ONE, 2012, 7(11):e51063.
[18] YAN Shasha, YANG Yuwen, WANG Tielin, et al. Genetic diversity analysis of Acidovorax citrulli in China [J]. European Journal of Plant Pathology, 2013, 136(1):171-181.
[19] KOVACH M E, ELZER P H, HILL D S, et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes[J]. Gene, 1995, 166(1):175-176.
[20] ZHANG Xiaoxiao, ZHAO Mei, YAN Jianpei, et al. Involvement of hrpX and hrpG in the virulence of Acidovorax citrulli strain Aac5, causal agent of bacterial fruit blotch in cucurbits[J]. Frontiers in Microbiology, 2018, 9:507.
[21] 陈亮. 西瓜食酸菌(Acidovorax citrulli)Ⅲ型分泌系统抑制剂的高通量筛选[D]. 北京: 中国农业大学, 2016.
[22] ZHANG Yanbao, WEI Chao, JIANG Wendi, et al. The HD-GYP domain protein RpfG of Xanthomonas oryzae pv. oryzicola regulates synthesis of extracellular polysaccharides that contribute to biofilm formation and virulence on rice [J/OL]. PLoS ONE, 2013, 8(3):e59428.
[23] PHILIPPE N, ALCARAZ J P, COURSANGE E, et al. Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria [J]. Plasmid, 2004, 51(3):246-255.
[24] WALCOTT R R, GITAITIS R D. Detection of Acidovorax avenae subsp. citrulli in watermelon seed using immunomagnetic separation and the polymerase chain reaction[J]. Plant Disease, 2000, 84(4):470-474.
[25] BAHAR O, KRITZMAN G, BURDMAN S. Bacterial fruit blotch of melon: screens for disease tolerance and role of seed transmission in pathogenicity[J]. European Journal of Plant Pathology, 2009, 123(1):71-83.
[26] BAHAR O, GOFFER T, BURDMAN S. Type Ⅳ Pili are required for virulence, twitching motility, and biofilm formation of Acidovorax avenae subsp. citrulli [J]. Molecular Plant-Microbe Interactions, 2009, 22(8):909.
[27] REN Zhengguang, JIANG Wenjun, NI Xingya, et al. Multiplication of Acidovorax citrulli in planta during infection of melon seedlings requires the ability to synthesize leucine[J]. Plant Pathology, 2014, 63(4):784-791.
[28] 张爱萍, 张晓晓, 吴林娜,等. 西瓜噬酸菌趋化性及鞭毛素基因ΔcheAΔfliC双突变体构建及功能[J]. 农业生物技术学报, 2017, 25(11):1838-1850.
[29] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method [J]. Methods, 2001, 25(4):402-408.
[30] 张鼎鼎, 邹丽芳, 赵梅勤,等. hrcQ基因决定水稻条斑病菌在非寄主烟草上的过敏性反应和在寄主水稻上的致病性[J]. 中国水稻科学, 2011, 25(1):11-18.
[31] WANG Tielin, GUAN Wei, HUANG Qi, et al. Quorum-sensing contributes to virulence, twitching motility, seed attachment and biofilm formation in the wild type strain Aac-5 of Acidovorax citrulli[J]. Microbial Pathogenesis, 2016, 100:133-140.
[32] TIAN Yanli, ZHAO Yuqiang, WU Xinrong, et al. The type Ⅵ protein secretion system contributes to biofilm formation and seed-to-seedling transmission of Acidovorax citrulli on melon [J]. Molecular Plant Pathology, 2015, 16(1):38-47.
[33] HOPKINS D L, THOMPSON C M. Seed transmission of Acidovorax avenae subsp. citrulli in cucurbits [J]. Hortscience A Publication of the American Society for Horticultural Science, 2002, 37(6):924-926.
[34] MOSS W, BYRNE J, CAMPBELL H, et al. Biological control of bacterial spot of tomato using hrp mutants of Xanthomonas campestris pv. vesicatoria [J]. Biological Control, 2007, 41(2):199-206.
[35] THARAUD M, LAURENT J, FAIZE M, et al. Fire blight protection with avirulent mutants of Erwinia amylovora [J]. Microbiology, 1997, 143(2):625-632.
[2] SCHAAD N W, SOWELL G J, GOTH R W, et al. Pseudomonas pseudoalcaligenes subsp. citrulli subsp. nov.[J].International Journal of Systematic Bacteriology,1978,28(1):117-125.
[3] SCHAAD N W, POSTNIKOVA E, SECHLER A, et al. Reclassification of subspecies of Acidovorax avenae as A.avenae (Manns 1905) emend. A.cattleyae (Pavarino, 1911) comb. nov. A.citrulli Schaad et al. 1978) comb. nov. and proposal of A.oryzae sp. nov.[J]. Systematic & Applied Microbiology, 2008, 31(6):434-446.
[4] DUTTA B, GITAITIS R, SMITH S, et al. Interactions of seedborne bacterial pathogens with host and non-host plants in relation to seed infestation and seedling transmission [J/OL]. PLoS ONE, 2014, 9(6): e99215.
[5] JIANG Chunhao, WU Fang, YU Zhenyun, et al. Study on screening and antagonistic mechanisms of Bacillus amyloliquefaciens 54 against bacterial fruit blotch (BFB) caused by Acidovorax avenae subsp. citrulli [J]. Microbiological Research, 2015, 170:95-104.
[6] BüTTNER D, HE Shengyang. Type Ⅲ protein secretion in plant pathogenic bacteria[J]. Plant Physiology, 2009, 150(4):1656-1664.
[7] GALáN J E, COLLMER A.Type Ⅲ secretion machines: bacterial devices for protein delivery into host cells [J]. Science, 1999, 284(5418):1322-1328.
[8] KVITKO B H, RAMOS A R, MORELLO J E, et al. Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are functionally similar to HrpK1 in promoting translocation of type Ⅲ secretion system effectors [J]. Journal of Bacteriology, 2007, 189(22):8059-8072.
[9] 李宾,李爱宁,魏强,等.欧美杨细菌性溃疡病菌hrcJ基因的功能分析[J].林业科学,2015,51(12):71-78.
[10] SUGIO A, YANG Bing, WHITE F F. Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae [J].Molecular Plant-Microbe Interactions,2005,18(6):546-554.
[11] 严婉荣, 王铁霖, 杨玉文,等. 瓜类细菌性果斑病菌hrcN基因的生物学功能分析[J]. 植物病理学报, 2015, 45(1):33-40.
[12] JOHNSON K L, MINSAVAGE G V, LE T, et al. Efficacy of a nonpathogenic Acidovorax citrulli strain as a biocontrol seed treatment for bacterial fruit blotch of cucurbits [J]. Plant Disease, 2014, 95(6):697-704.
[13] 任争光, 侯磊, 宋治国,等. 甜瓜细菌性果斑病菌致病性突变体筛选与hrcR基因的克隆[J]. 植物病理学报, 2009, 39(5):501-506.
[14] 汪新, 王卫, 钱国良,等. 瓜类细菌性果斑病菌过敏性反应和致病性(hrp)基因簇部分基因的克隆及功能分析[J]. 农业生物技术学报, 2011, 19(1):36-44.
[15] DENG Wanyin, MARSHALL N C, ROWLAND J L, et al. Assembly, structure, function and regulation of type Ⅲ secretion systems [J]. Nature Reviews Microbiology, 2017, 15(6):323-337.
[16] ZHANG Xiaoping, WANG Chunlian, ZHENG Chongke, et al. HrcQ is necessary for Xanthomonas oryzae, pv. oryzae, HR-induction in non-host tobacco and pathogenicity in host rice[J]. The Crop Journal, 2013, 1(2):143-150.
[17] LORENZ C, HAUSNER J, BüTTNER D. HrcQ provides a docking site for early and late type Ⅲ secretion substrates from Xanthomonas [J/OL]. PLoS ONE, 2012, 7(11):e51063.
[18] YAN Shasha, YANG Yuwen, WANG Tielin, et al. Genetic diversity analysis of Acidovorax citrulli in China [J]. European Journal of Plant Pathology, 2013, 136(1):171-181.
[19] KOVACH M E, ELZER P H, HILL D S, et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes[J]. Gene, 1995, 166(1):175-176.
[20] ZHANG Xiaoxiao, ZHAO Mei, YAN Jianpei, et al. Involvement of hrpX and hrpG in the virulence of Acidovorax citrulli strain Aac5, causal agent of bacterial fruit blotch in cucurbits[J]. Frontiers in Microbiology, 2018, 9:507.
[21] 陈亮. 西瓜食酸菌(Acidovorax citrulli)Ⅲ型分泌系统抑制剂的高通量筛选[D]. 北京: 中国农业大学, 2016.
[22] ZHANG Yanbao, WEI Chao, JIANG Wendi, et al. The HD-GYP domain protein RpfG of Xanthomonas oryzae pv. oryzicola regulates synthesis of extracellular polysaccharides that contribute to biofilm formation and virulence on rice [J/OL]. PLoS ONE, 2013, 8(3):e59428.
[23] PHILIPPE N, ALCARAZ J P, COURSANGE E, et al. Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria [J]. Plasmid, 2004, 51(3):246-255.
[24] WALCOTT R R, GITAITIS R D. Detection of Acidovorax avenae subsp. citrulli in watermelon seed using immunomagnetic separation and the polymerase chain reaction[J]. Plant Disease, 2000, 84(4):470-474.
[25] BAHAR O, KRITZMAN G, BURDMAN S. Bacterial fruit blotch of melon: screens for disease tolerance and role of seed transmission in pathogenicity[J]. European Journal of Plant Pathology, 2009, 123(1):71-83.
[26] BAHAR O, GOFFER T, BURDMAN S. Type Ⅳ Pili are required for virulence, twitching motility, and biofilm formation of Acidovorax avenae subsp. citrulli [J]. Molecular Plant-Microbe Interactions, 2009, 22(8):909.
[27] REN Zhengguang, JIANG Wenjun, NI Xingya, et al. Multiplication of Acidovorax citrulli in planta during infection of melon seedlings requires the ability to synthesize leucine[J]. Plant Pathology, 2014, 63(4):784-791.
[28] 张爱萍, 张晓晓, 吴林娜,等. 西瓜噬酸菌趋化性及鞭毛素基因ΔcheAΔfliC双突变体构建及功能[J]. 农业生物技术学报, 2017, 25(11):1838-1850.
[29] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method [J]. Methods, 2001, 25(4):402-408.
[30] 张鼎鼎, 邹丽芳, 赵梅勤,等. hrcQ基因决定水稻条斑病菌在非寄主烟草上的过敏性反应和在寄主水稻上的致病性[J]. 中国水稻科学, 2011, 25(1):11-18.
[31] WANG Tielin, GUAN Wei, HUANG Qi, et al. Quorum-sensing contributes to virulence, twitching motility, seed attachment and biofilm formation in the wild type strain Aac-5 of Acidovorax citrulli[J]. Microbial Pathogenesis, 2016, 100:133-140.
[32] TIAN Yanli, ZHAO Yuqiang, WU Xinrong, et al. The type Ⅵ protein secretion system contributes to biofilm formation and seed-to-seedling transmission of Acidovorax citrulli on melon [J]. Molecular Plant Pathology, 2015, 16(1):38-47.
[33] HOPKINS D L, THOMPSON C M. Seed transmission of Acidovorax avenae subsp. citrulli in cucurbits [J]. Hortscience A Publication of the American Society for Horticultural Science, 2002, 37(6):924-926.
[34] MOSS W, BYRNE J, CAMPBELL H, et al. Biological control of bacterial spot of tomato using hrp mutants of Xanthomonas campestris pv. vesicatoria [J]. Biological Control, 2007, 41(2):199-206.
[35] THARAUD M, LAURENT J, FAIZE M, et al. Fire blight protection with avirulent mutants of Erwinia amylovora [J]. Microbiology, 1997, 143(2):625-632.