西瓜食酸菌III型分泌效应物基因aop2功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1303

摘要/Abstract

摘要：

III型分泌效应物（type III secreted effectors, T3SEs）是细菌性果斑病（bacterial fruit blotch, BFB）的病原菌——西瓜食酸菌（Acidovorax citrulli）分泌的关键致病因子。鉴定西瓜食酸菌特异的、具有GNAT（Gcn5-related N-acetyltransferase）超家族结构域的T3SE基因aop2，分析其编码蛋白质影响植物免疫的方式，可为深入认识该基因在病菌致病机制中的作用奠定基础。利用生物信息学分析其序列特征；借助荧光定量PCR技术分析aop2的表达调控及其表达与抗病相关基因表达间的关系；利用基因突变及基因功能互补手段，通过分析致病性、寄主活性氧积累量等解析基因功能；使用瞬时表达技术了解Aop2抑制激发子诱导的细胞坏死能力及其亚细胞定位情况。aop2基因启动子区存在III型分泌系统（type III secretion system, T3SS）核心基因结合位点，其编码的蛋白不存在信号肽和跨膜螺旋区，含一个GNAT家族乙酰转移酶结构域但无同源蛋白；T3SS核心基因hrpG/hrpX突变体中aop2基因的表达量显著降低；缺失aop2基因的突变体对寄主黄瓜的致病力降低，但黄瓜子叶中活性氧积累量增加；Aop2可定位于烟草整个细胞，能够抑制由坏死因子NIP诱导的PCD（programmed cell death）；Aop2的表达增强了烟草叶片中病原相关分子模式触发的免疫（PAMP-triggered immunity, PTI）信号通路，以及SA和JA信号通路相关基因的表达。结果表明，Aop2为西瓜食酸菌一个含有GNAT结构域的特异T3SE，其在与寄主黄瓜互作中发挥毒性因子功能，在与烟草互作中参与调控植物细胞死亡及PTI和植物激素相关抗病防卫反应。

关键词: III型分泌效应物, aop2, 毒性因子, 细胞死亡, 乙酰转移酶, PTI

Abstract:

Type III secreted effectors are the key pathogenic factors secreted by Acidovorax citrulli, the pathogen of bacterial fruit blotch（BFB）. To identify the T3SE gene aop2 with GNAT（Gcn5-related N-acetyltransferase）superfamily domain, which is specific for Acidovorax citrulli, and to analyze the way that its encoded protein affects plant immunity, will lay a foundation for further understanding the role of this gene in pathogen pathogenesis. Bioinformatics was applied to analyze the sequence characteristics. Fluorescence quantitative PCR was used to analyze the expression regulation of aop2 and its relationship with the expression of disease-resistant genes. Gene mutation and gene function complementation was to analyze the gene function, including pathogenicity and host reactive oxygen species accumulation. Transient expression technique was used to investigate the ability of Aop2 inhibitory elicitor to induce necrosis and its subcellular localization. There was a core gene binding site of Type III secretion system（T3SS）in the promoter region of aop2 gene, and there was no signal peptide or transmembrane helical region in the encoded protein, and there was a GNAT family acetyltransferase domain but no homologous protein. The expression of aop2 gene in hrpG/hrpX mutant of T3SS significantly decreased. The mutant with aop2 gene deletion decreased the pathogenicity of host cucumber, but increased the accumulation of reactive oxygen species in cucumber cotyledon. Aop2 can be localized to whole tobacco cells and inhibited programmed cell death（PCD）induced by NIP. The expression of Aop2 enhanced the expression of genes involved in SA and JA signaling pathways triggered by pathogen-related molecular patterns in tobacco leaves. The results showed that Aop2 was a specific T3SE containing GNAT domain in A. citrulli. Aop2 played the function of virulence factor in the interaction with cucumber and was involved in the regulation of plant cell death and PTI and phytohormone-related disease resistance in the interaction with tobacco.

Key words: type III secreted effectors, aop2, virulence factor, cell death, acetyltransferase, PTI

陈宝强, 李莹莹, 马博雅, 肉扎古丽·马利克, 优丽图孜·乃比, 宋金迪, 刘君, 王希东. 西瓜食酸菌III型分泌效应物基因aop2功能分析[J]. 生物技术通报, 2023, 39(6): 286-297.

CHEN Bao-qiang, LI Ying-ying, MA Bo-ya, ROUZHAGULI Malike, YOULITUZI Naibi, SONG Jin-di, LIU Jun, WANG Xi-dong. Functional Analysis of the Type III Secreted Effector Gene aop2 in Acidovorax citrulli[J]. Biotechnology Bulletin, 2023, 39(6): 286-297.

图/表 10

表1 供试菌株、质粒

Table 1 Bacterial strains, plasmids used in this study

菌株和质粒 Strain and plasmid	名称 Name	特性 Characteristic	来源Source
菌株 Strain	FC440（WT）	Amp^R; wild type	Our laboratory
	FC440（∆aop2）	Amp^R; Kan^R; FC440 mutant defective in aop2	This study
	FC440（∆aop2-aop2）	Amp^R; Kan^R; Gm^R; FC440（∆aop2）complemented with the fragment of aop2 gene expressed by vector pBBR1MCS-5	This study
	FC440（∆hrpG）	AmpR; Kan^R; FC440 mutant defective in hrpG	[14]
	FC440（∆hrpX）	Amp^R; Kan^R; FC440 mutant defective in hrpX	[14]
	Trans T1（pBBR1MCS-5- aop2）	Gm^R; E. coli TransT1 strain containing vector pBBR1MCS-5-aop2	This study
	S17-1（pBBR1MCS-5- aop2）	Gm^R; E. coli S17-1 strain containing vector pBBR1MCS-5aop2	This study
	GV3101	Rif^R; wild type	Our laboratory
	GV3101（pAPK-aop2）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pAPK-aop2	This study
	GV3101（pAPK-GFP）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pAPK-GFP	[15]
	GV3101（pBINGFP2-aop2）	Kan^R; Rif^R; Gm^R; GV3101 strain containing vector pBINGFP2-aop2	This study
	GV3101（pBINGFP2）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pBINGFP2	[15]
	GV3101（BAX）	Kan^R; Rif^R; Gm^R; GV3101 strain containing BAX	[15]
	GV3101（NIP）	Kan^R; Rif^R; Gm^R; GV3101 strain containing NEP	[15]
	GV3101（NIP）	Kan^R; Rif^R; Gm^R; GV3101 strain containing NEP	[15]
	GV3101（Avh241）	Kan^R; Rif^R; Gm^R; GV3101 strain containing Avh241	[15]
	GV3101（INF1）	Kan^R; Rif^R; Gm^R; GV3101 strain containing INF1	[15]
质粒 Plasmid	pMD19-T	Amp^R; Cloning vector	TaKaRa
	pMD19-T- aop2	Amp^R; pMD19-T vector containing a 438-bp fragment with the aop2 gene	This study
	pK19mob2ΩHMB-aop2	Kan^R; pK19mob2ΩHMB vector containing a 301-bp fragment with the aop2 gene	This study
	pBBR1MCS-5-aop2	Gm^R; pBBR1MCS-5 vector containing aop2 gene; used to complement FC440（∆aop2）	This study
	pKannibal-aop2	Kan^R; pKannibal vector containing a 438-bp fragment with the aop2 gene	This study
	pAPK-aop2	Spec^R; pART27 vevtor containing pKannibal-aop2	This study
	pBINGFP2-aop2	Kan^R; pBINGFP2 vector containing a 438-bp fragment with the aop2 gene	This study

表1 供试菌株、质粒

Table 1 Bacterial strains, plasmids used in this study

菌株和质粒 Strain and plasmid	名称 Name	特性 Characteristic	来源Source
菌株 Strain	FC440（WT）	Amp^R; wild type	Our laboratory
	FC440（∆aop2）	Amp^R; Kan^R; FC440 mutant defective in aop2	This study
	FC440（∆aop2-aop2）	Amp^R; Kan^R; Gm^R; FC440（∆aop2）complemented with the fragment of aop2 gene expressed by vector pBBR1MCS-5	This study
	FC440（∆hrpG）	AmpR; Kan^R; FC440 mutant defective in hrpG	[14]
	FC440（∆hrpX）	Amp^R; Kan^R; FC440 mutant defective in hrpX	[14]
	Trans T1（pBBR1MCS-5- aop2）	Gm^R; E. coli TransT1 strain containing vector pBBR1MCS-5-aop2	This study
	S17-1（pBBR1MCS-5- aop2）	Gm^R; E. coli S17-1 strain containing vector pBBR1MCS-5aop2	This study
	GV3101	Rif^R; wild type	Our laboratory
	GV3101（pAPK-aop2）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pAPK-aop2	This study
	GV3101（pAPK-GFP）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pAPK-GFP	[15]
	GV3101（pBINGFP2-aop2）	Kan^R; Rif^R; Gm^R; GV3101 strain containing vector pBINGFP2-aop2	This study
	GV3101（pBINGFP2）	Spec^R; Rif^R; Gm^R; GV3101 strain containing vector pBINGFP2	[15]
	GV3101（BAX）	Kan^R; Rif^R; Gm^R; GV3101 strain containing BAX	[15]
	GV3101（NIP）	Kan^R; Rif^R; Gm^R; GV3101 strain containing NEP	[15]
	GV3101（NIP）	Kan^R; Rif^R; Gm^R; GV3101 strain containing NEP	[15]
	GV3101（Avh241）	Kan^R; Rif^R; Gm^R; GV3101 strain containing Avh241	[15]
	GV3101（INF1）	Kan^R; Rif^R; Gm^R; GV3101 strain containing INF1	[15]
质粒 Plasmid	pMD19-T	Amp^R; Cloning vector	TaKaRa
	pMD19-T- aop2	Amp^R; pMD19-T vector containing a 438-bp fragment with the aop2 gene	This study
	pK19mob2ΩHMB-aop2	Kan^R; pK19mob2ΩHMB vector containing a 301-bp fragment with the aop2 gene	This study
	pBBR1MCS-5-aop2	Gm^R; pBBR1MCS-5 vector containing aop2 gene; used to complement FC440（∆aop2）	This study
	pKannibal-aop2	Kan^R; pKannibal vector containing a 438-bp fragment with the aop2 gene	This study
	pAPK-aop2	Spec^R; pART27 vevtor containing pKannibal-aop2	This study
	pBINGFP2-aop2	Kan^R; pBINGFP2 vector containing a 438-bp fragment with the aop2 gene	This study

表2 供试引物

Table 2 Primers used in this study

Primer name	Sequnece（5'-3'）	Source
aop2-F	CGGAATTCTGCTGGAGGTCCTGCTGT（EcoR I）	This study
aop2-R	CCAAGCTTACTTCCATCGCATCGTCC（Hind III）	This study
aop2-QF	CCCTCGAGTGGCAACGATGTTTCCCC（Xho I）	This study
aop2-QR	GCTCTAGAGCGACAGCGACGGCTGAG（Xba I）	This study
AAC-1	GACCAGCCCACAACTGGGAC	[16]
AAC-2	CTGCCGCACTCCAGCGA	[16]
aop2-CF	TTTGGAGAGGACACGCTCGAGATGACAGACAGACTCAGCCGGC（Xho I）	This study
aop2-CR	TCATTAAAGCAGGACTCTAGATCAGTTCACCGTTGACGACGC（Xba I）	This study
aop2-DF	AGAGGATCCGTCGACCCCGGGATGAGCCTGCAGAACGCCA（Sma I）	This study
aop2-DR	CTGTACAAGGGTACCCCCGGGTCAGGCGTCCAGGGGCAG（Sma I）	This study
RT-aop2-F	CCTGGCTGGCGGACAAGT	This study
RT-aop2-R	CTGCCCGAAGAACTGCGA	This study
rpoB-F	GCGACAGCGTGCTCAAAGTG	[17]
rpoB-R	GGCCTTCGTTGGTGCGTTTCT	[17]
NbPti5-F	CCTCCAAGTTTGAGCTCGGATAGT	[18]
NbPti5-R	CCAAGAAATTCTCCATGCACTCTGTC	[18]
NbAcre31-F	AATTCGGCCATCGTGATCTTGGTC	[18]
NbAcre31-R	GAGAAACTGGGATTGCCTGAAGGA	[18]
NbGras2-F	TACCTAGCACCAAGCAGATGCAGA	[18]
NbGras2-R	TCATGAGGCGTTACTCGGAGCATT	[18]
NbEF1α-F	AAGGTCCAGTATGCCTGGGTGCTTGAC	[18]
NbEF1α-R	AAGAATTCACAGGGAC AGTTCCAATACCA	[18]
NbWRKY7-F	CACAAGGGTACAAACAACACAG	[19]
NbWRKY7-R	GGTTGCATTTGGTTCATGTAAG	[19]
NbWRKY8-F	AACAATGGTGCCAATAATGC	[19]
NbWRKY8-R	TGCATATCCTGAGAAACCATT	[19]
NbPR2b-F	TCCAACTTGGAATCAAAGGG	[20]
NbPR2b-R	GTGGACACTATACTCAGGTG	[20]
NbLOX-F	AAAACCTATGCCTCAAGAAC	[20]
NbLOX-R	ACTGCTGCATAGGCTTTGG	[20]
NbEFR1-F	GCTCTTAACGTCGGATGGTC	[20]
NbEFR1-R	AGCCAAACCCTAGCTCCATT	[20]
NbGAPDH-F	AGCTCAAGGGAATTCTCGATG	[21]
NbGAPDH-R	AACCTTAACCATGTCATCTCCC	[21]

图1 Aop2种内序列多态性分析

Fig. 1 Intraspecific sequence polymorphism analysis of Aop2

图2 Aop2序列的生物信息学分析 A：aop2基因转录起始位点上游PIP-box分析（第一行为革兰氏阴性菌T3SEs启动子区的保守基序的模体，第二行为aop2基因启动子区序列，A、T、C、G、B、N、Y、R为碱基单字母缩写）；B：Aop2蛋白的信号肽预测；C：Aop2蛋白的跨膜螺旋区预测；D：Aop2蛋白的结构域预测

Fig. 2 Bioinformatics analysis of Aop2 A: PIP-box analysis existed upstream of aop2 gene transcription start site（The first line is the motifs of the conservative sequences in the T3SEs promoter region of Gram-negative bacteria. The second line is the sequence of the aop2 gene promoter region. A, T, C, G, B, N, Y, and R are the single letter abbreviations of bases）. B: Signal peptide prediction of Aop2 protein. C: Transmembrane spiral region prediction of Aop2 protein. D: Domain prediction of Aop2 protein

图3 aop2基因在西瓜食酸菌hrpG/hrpX基因突变体中的表达分析 **表示在0.01水平上存在差异。下同

Fig. 3 Expression analysis of aop2 gene in hrpG or hrpX gene mutant in A. citrulli ** indicate significant difference at the 0.01 probability. The same below

图4 西瓜食酸菌aop2基因突变体及其衍生菌株的致病力表型 A：黄瓜子叶发病表型（图片采集于接菌1 d时）；B：病情指数分析；C：秦烟95的HR表型（图片采集于接菌1 d时）

Fig. 4 Virulence phenotype of aop2 mutants and their derived strains of A. citrulli A: The phenotype of cucumber cotyledon（Photos were taken at 1 d after inoculation）. B: Disease index analysis. C: HR phenotype of Qinyan 95（Photos were taken at 1 d after inoculation）

图5 西瓜食酸菌aop2基因突变体及其衍生菌株侵染下黄瓜ROS产量分析 A：黄瓜子叶ROS染色表型（图片采集于叶片接菌后1 d时）；B：相对染色强度分析。*表示在0.05平上存在差异

Fig. 5 Analysis of ROS production of cucumber infected by aop2 mutants and and their derived strains of A. citrulli A: ROS staining phenotype of cucumber cotyledon (Photos were taken at 1 d after inoculation). B: Relative staining intensity analysis. * indicates significant difference at the 0.05 probability

图6 Aop2在本氏烟的亚细胞定位图片采集于接菌2 d后

Fig. 6 Subcellular localization of Aop2 in N. benthamiana Photos were taken at 2 d after inoculation

图7 Aop2对5种激发子诱导PCD的抑制作用

Fig. 7 Inhibitory effect of Aop2 on the PCD induced by five elicitors A: Inhibitory effect of Aop2 on BAX, NIP and Avh241; 1: GV3101（pAPK-GFP）; 2: GV3101（pAPK-GFP）+GV3101（BAX）; 3: GV3101（pAPK-GFP）+GV3101（NIP）; 4: GV3101（pAPK-GFP）+GV3101（Avh241）; 5: GV3101（pAPK-aop2）; 6: GV3101（pAPK-aop2）+GV3101（BAX）; 7: GV3101（pAPK-aop2）+GV3101（NIP）; 8: GV3101（pAPK-aop2）+GV3101（Avh241）. B: Inhibitory effect of Aop2 on BAX, NIP and Avh241; 1: GV3101（pAPK-GFP）; 2: GV3101（pAPK-GFP）+GV3101（NEP）; 3: GV3101（pAPK-GFP）+GV3101（INF1）; 4: GV3101（pAPK-aop2）; 5: GV3101（pAPK-aop2）+GV3101（NEP）; 6: GV3101（pAPK-aop2）+GV3101（INF1）

图8 Aop2对本氏烟抗病信号通路的影响 A：Aop2对PTI信号通路的影响分析；B：Aop2对植物激素信号通路的影响分析（叶片于瞬时表达36 h时取样）

Fig. 8 Effects of Aop2 on the resistance signal pathway in N. benthamiana A: Analysis of the effect of Aop2 on PTI signal pathway. B: Analysis of the effect of Aop2 on plant hormone signal pathway（Leaf samples were taken 36 h after transient expression）

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