葡萄霜霉菌糖基水解酶基因的表达模式与功能分析

doi:10.3864/j.issn.0578-1752.2023.05.006

摘要/Abstract

摘要：

【目的】克隆葡萄霜霉菌（Plasmopara viticola）糖基水解酶（glycosyl hydrolase，GH）基因（PvGH），分析其特征以及在葡萄霜霉菌侵染葡萄叶片过程中的表达模式，研究其抑制/促进烟草叶片程序性细胞死亡（PCD）以及影响烟草疫霉（Phytophthora nicotianae）侵染的能力，为深入探究调控寄主植物免疫的机制提供理论依据。【方法】以葡萄霜霉菌Pv5-27菌株作为试材，采用RT-PCR方法扩增获得8个PvGH基因全长，并对其基因及编码蛋白进行生物信息学分析；利用酵母信号肽诱捕系统（SST）验证PvGH的分泌活性；利用实时荧光定量PCR技术检测葡萄霜霉菌侵染葡萄叶片过程中PvGH的表达模式；同时利用农杆菌介导的PVX病毒表达系统在本氏烟上瞬时表达PvGH效应蛋白，分析其对INF1和BAX触发的烟草PCD的抑制能力以及促进烟草疫霉侵染的能力。【结果】8个PvGH基因序列与通过全基因组预测的序列完全一致，长度为1 092—1 392 bp，分别编码364—464个氨基酸，与其他卵菌同源蛋白相似性高达60.62%—86.36%。8个PvGH均不含跨膜结构域，其二级结构差异较大，三级结构与其他蛋白三级结构相似性较低，呈现非常独特的三级结构。使用SingalP 5.0软件对编码的蛋白进行信号肽预测，发现它们均含有20—26 aa的信号肽，但通过SST验证结果显示PvG09279和PvG13517并不具有分泌特性。保留、缺失或者替换信号肽的8个PvGH均可抑制BAX触发的本氏烟PCD，并促进烟草疫霉的侵染，表明PvGH效应蛋白发挥其毒力不依赖于信号肽。PvGH在葡萄霜霉菌侵染初期表达上调，进一步表明其在病原菌与寄主互作中的重要作用。【结论】在侵染寄主过程中，葡萄霜霉菌分泌的PvGH通过抑制寄主PTI反应参与致病过程。

关键词: 葡萄霜霉菌, 糖基水解酶, 生物信息学, 功能分析

Abstract:

【Objective】The objective of this study is to clone glycosyl hydrolase genes from Plasmopara viticola (PvGHs), analyze their characteristics and expression patterns in infected grape leaves, and the abilities to inhibit or promote programmed cell death (PCD) and affect Phytophthora nicotianae infection in tobacco leaves, so as to provide a theoretical basis for further study on mechanism of regulating host plant immunity.【Method】Eight PvGHs with full-length were amplified by RT-PCR from P. viticola Pv5-27 strain. The sequences of these eight PvGHs and encoded proteins were analyzed by bioinformatics. Yeast signal peptide trap system (SST) was used to verify the secretory activity of the PvGH proteins. The expression pattern of PvGHs during infection grape leaves was detected by qRT-PCR. At the same time, eight PvGH effector proteins were transiently expressed in Nicotiana benthamiana by Agrobacterium-mediated PVX virus expression system. Moreover, their inhibitory abilities to inhibit INF1- and BAX-triggered PCD and to promote P. nicotianae infection were also analyzed.【Result】The sequences of eight PvGHs were completely consistent with the prediction in genome sequence, with the length of 1 092 to 1 392 bp, encoding 364-464 amino acids, respectively. The similarity with homologous proteins from other oomycetes was as high as 60.62%-86.36%. None of them had transmembrane domains. Their secondary structures were quite different from each other, and their tertiary structures were less similar to those of other proteins, which showed a very unique tertiary structure. SingalP 5.0 software was used to predict signal peptide of these proteins. It was found that all the PvGH proteins contained signal peptide sequences of 20-26 aa in length. However, the SST verification results showed that PvG09279 and PvG13517 do not have secretion activity. The retention, deletion or replacement of signal peptide sequences of the eight PvGH proteins could inhibit the BAX-triggered PCD and all these PvGH proteins could promote P. nicotianae infection in tobacco leaves. It suggests that the potential virulence of eight PvGH effectors does not dependent on the signal peptide. The up-regulated expression of PvGHs in the early stage of infection of P. viticola further indicated that PvGHs play an important role in the interaction between pathogen and host.【Conclusion】During downy mildew infection, PvGHs secreted by P. viticola are involved in pathogenic process by inhibiting the host PTI response.

Key words: Plasmopara viticola, glycosyl hydrolase, bioinformatics, function analysis

潘凤英, 曲俊杰, 刘露露, 孙大运, 郭泽西, 韦晓丽, 韦淑梅, 尹玲. 葡萄霜霉菌糖基水解酶基因的表达模式与功能分析[J]. 中国农业科学, 2023, 56(5): 879-891.

PAN FengYing, QU JunJie, LIU LuLu, SUN DaYun, GUO ZeXi, WEI XiaoLi, WEI ShuMei, YIN Ling. Expression and Functional Analysis of Glycosyl Hydrolase Genes from Plasmopara viticola[J]. Scientia Agricultura Sinica, 2023, 56(5): 879-891.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.05.006

https://www.chinaagrisci.com/CN/Y2023/V56/I5/879

图/表 9

表1

本研究所用引物信息"

引物Primer	序列Sequence (5′-3′)	用途Purpose
pSUC2-G11117F	GGATCTTCCAGAGATGAATTCATGACCAAGACATTGCATTCGG	扩增PvG11117信号肽序列 To amplify signal peptide sequence of PvG11117
pSUC2-G11117R	CTGCCGTTCGACGATCTCGAGCGCATCAACGCTGAGAGTAGAC
G11117ClaⅠF	TCAGCACCAGCTAGCATCGATATGACCAAGACATTGCATTCGG	扩增PvG11117保留或剔除信号肽和替换为PR1信号肽的片段 To amplify fragments of PvG11117 with or without signal peptide and the fragment that replaces PvG11117 signal peptide with PR1 signal peptide
G11117ClaⅠNSPF	TCAGCACCAGCTAGCATCGATATGGACAAGTTATGTTCCATTCCG
G11117ClaⅠPR1F	TCAGCACCAGCTAGCATCGATATGGGATTTGTTCTCTTTTCGCA
G11117SalⅠR	AACCGTTCATCGGCGGTCGACCTAAAACTGGGTGATAGGGGCC
pSUC2-G13516F	GGATCTTCCAGAGATGAATTCATGGTGAACGTCCTGTGTGCG	扩增PvG13516信号肽 To amplify signal peptide of PvG13516
pSUC2-G13516R	CTGCCGTTCGACGATCTCGAGGGCATCAGCAGAACCGACG	扩增PvG13516信号肽 To amplify signal peptide of PvG13516
G13516ClaⅠF	TCAGCACCAGCTAGCATCGATATGGTGAACGTCCTGTGTGCG	扩增PvG13516保留或剔除信号肽和替换为PR1信号肽的片段 To amplify fragments of PvG13516 with or without signal peptide and the fragment that replaces PvG13516 signal peptide with PR1 signal peptide
G13516ClaⅠNSPF	TCAGCACCAGCTAGCATCGATATGAGTCTGTTTAAGTGCTCGGG
G13516ClaⅠPR1F	TCAGCACCAGCTAGCATCGATATGGGATTTGTTCTCTTTTCGCA
G13516SalⅠR	AACCGTTCATCGGCGGTCGACTTACATGGCGCAGTTCTTGTGC
引物Primer	序列Sequence (5′-3′)	用途Purpose
pSUC2-G10113F	GGATCTTCCAGAGATGAATTCATGGTTGCCAACTCGTTGCG	扩增PvG10113信号肽 To amplify signal peptide of PvG10113
pSUC2-G10113R	CTGCCGTTCGACGATCTCGAGAACAATTGATGATAAAAAGAGTGCAAC	扩增PvG10113信号肽 To amplify signal peptide of PvG10113
G10113ClaⅠF	TCAGCACCAGCTAGCATCGATATGGTTGCCAACTCGTTGCG	扩增PvG10113保留或剔除信号肽和替换为PR1信号肽的片段 To amplify fragments of PvG10113 with or without signal peptide and the fragment that replaces PvG10113 signal peptide with PR1 signal peptide
G10113ClaⅠNSPF	TCAGCACCAGCTAGCATCGATATGGCCGAAGACCTGTGCT
G10113ClaⅠPR1F	TCAGCACCAGCTAGCATCGATATGGGATTTGTTCTCTTTTCGCA
G10113SalⅠR	AACCGTTCATCGGCGGTCGACTTACTGCAAGAATTGCTTTCTTACTCG
pSUC2-G01792F	GGATCTTCCAGAGATGAATTCATGTTTCGCGAAGTTGCTTTCT	扩增PvG01792信号肽 To amplify signal peptide of PvG01792
pSUC2-G01792R	CTGCCGTTCGACGATCTCGAGCGCTAAAGTCGAAGACCCTAACA	扩增PvG01792信号肽 To amplify signal peptide of PvG01792
G01792ClaⅠF	TCAGCACCAGCTAGCATCGATATGTTTCGCGAAGTTGCTTTCT	扩增PvG01792保留或剔除信号肽和替换为PR1信号肽的片段 To amplify fragments of PvG01792 with or without signal peptide and the fragment that replaces PvG01792 signal peptide with PR1 signal peptide
G01792ClaⅠNSPF	TCAGCACCAGCTAGCATCGATATGCTGGATCAGAAGCTGTATGG
G01792ClaⅠPR1F	TCAGCACCAGCTAGCATCGATATGGGATTTGTTCTCTTTTCGCA
G01792SalⅠR	AACCGTTCATCGGCGGTCGACTTACAAATACCAGAAAACGGTGGA
pSUC2-G09279F	GGATCTTCCAGAGATGAATTCATGCAGCTTCTTTTTACTGCAATC	扩增PvG09279信号肽 To amplify signal peptide of PvG09279
pSUC2-G09279R	CTGCCGTTCGACGATCTCGAGACTCTCAATCGTCATCATGAGCAG	扩增PvG09279信号肽 To amplify signal peptide of PvG09279
G09279ClaⅠF	TCAGCACCAGCTAGCATCGATATGCAGCTTCTTTTTACTGCAATC	扩增PvG09279保留或剔除信号肽和替换为PR1信号肽的片段 To amplify fragments of PvG09279 with or without signal peptide and the fragment that replaces PvG9279 signal peptide with PR1 signal peptide
G09279ClaⅠNSPF	TCAGCACCAGCTAGCATCGATATGTCCCAAACGCTTCACCA
G09279ClaⅠPR1F	TCAGCACCAGCTAGCATCGATATGGGATTTGTTCTCTTTTCGCA
G09279SalⅠR	AACCGTTCATCGGCGGTCGACCTAGAGAGATATGCCAGGGGGA
pSUC2-G02199F	GGATCTTCCAGAGATGAATTCATGAAGCTCTCTTCCTCTTTATCCC	扩增PvG02199信号肽 To amplify signal peptide of PvG02199
pSUC2-G02199R	CTGCCGTTCGACGATCTCGAGAGCTGATAAAAGGGACGACGAC	扩增PvG02199信号肽 To amplify signal peptide of PvG02199
G02199ClaⅠF	TCAGCACCAGCTAGCATCGATATGAAGCTCTCTTCCTCTTTATCCC	扩增PvG02199保留或剔除信号肽和替换为PR1信号肽的片段 To amplify fragments of PvG02199 with or without signal peptide and the fragment that replaces PvG02199 signal peptide with PR1 signal peptide
G02199ClaⅠNSPF	TCAGCACCAGCTAGCATCGATATGCAGCAGATTGGAACGAATA
G02199ClaⅠPR1F	TCAGCACCAGCTAGCATCGATATGGGATTTGTTCTCTTTTCGCA
G02199SalⅠR	AACCGTTCATCGGCGGTCGACTCAGTACGTGGTTCCGATGTCG
pSUC2-G13517F	GGATCTTCCAGAGATGAATTCATGCTATCCTCCAAACTCCTCG	扩增PvG13517信号肽 To amplify signal peptide of PvG13517
pSUC2-G13517R	CTGCCGTTCGACGATCTCGAGTCCCGCCGCTTTTTTGCC	扩增PvG13517信号肽 To amplify signal peptide of PvG13517
G13517ClaⅠF	TCAGCACCAGCTAGCATCGATATGCTATCCTCCAAACTCCTCG	扩增PvG13517保留或剔除信号肽和替换为PR1信号肽的片段 To amplify fragments of PvG13517 with or without signal peptide and the fragment that replaces PvG013517 signal peptide with PR1 signal peptide
G13517ClaⅠNSPF	TCAGCACCAGCTAGCATCGATATGCAGAATTTTTGTATCCATGGC
G13517ClaⅠPR1F	TCAGCACCAGCTAGCATCGATATGGGATTTGTTCTCTTTTCGCA
G13517SalⅠR	AACCGTTCATCGGCGGTCGACTTACATGCTTGTTTGGCACTCTTC
pSUC2-G13010F	GGATCTTCCAGAGATGAATTCATGTTGCCAGCTTTGCTGTTT	扩增PvG13010信号肽 To amplify signal peptide of PvG13010
pSUC2-G13010R	CTGCCGTTCGACGATCTCGAGAGCGCCAATTCCGTCAGTG	扩增PvG13010信号肽 To amplify signal peptide of PvG13010
G13010ClaⅠF	TCAGCACCAGCTAGCATCGATATGTTGCCAGCTTTGCTGTTT	扩增PvG13010保留或剔除信号肽和替换为PR1信号肽的片段 To amplify fragments of PvG13010 with or without signal peptide and the fragment that replaces PvG13010 signal peptide with PR1 signal peptide
G13010ClaⅠNSPF	TCAGCACCAGCTAGCATCGATATGGTCGCTGTCGATACGCT
G13010ClaⅠPR1F	TCAGCACCAGCTAGCATCGATATGGGATTTGTTCTCTTTTCGCA
G13010SalⅠR	AACCGTTCATCGGCGGTCGACCTAAACCTGAAAAAGCACTAATGCC

表1

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表2

表3

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图5

图6

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基因号 Gene ID	长度 Length (bp)	氨基酸 Amino acid (aa)	NCBI保守结构域 Conserved domain	区间 Interval (aa)	PFam保守结构域 Conserved domain	区间 Interval (aa)	同源蛋白 <BOLD>H</BOLD>omologous protein	相似度 <BOLD>S</BOLD>imilarity (%)	信号肽 Signal peptide (aa)
PvG11117	1131	377	Glyco_hydro_6 super family	44-297	Glycosyl hydrolases family 6	35-300	GH family 6 protein (P. infestans) (KAF4028604.1)	60.62	1-25
PvG13516	1221	407	Glyco_hydro super family	20-300	Glycosyl hydrolases family 17	196-311	Glucan 1,3-beta-D-glucosidase (P. cinamomi) (CAJ90910.1)	72.90	1-23
PvG10113	1179	393	Glyco_hydro_6 super family Cell division protein DedD	46-295 331-380	Glycosyl hydrolases family 6	33-301	1,4-cellobiohydrolase (P. cactorum) (KAF1773299.1)	73.83	1-26
PvG01792	1170	390	Glyco_hydro super family	6-289	Glycosyl hydrolases family 17	203-300	GH family 17 protein (P. infestans) (KAF4032687.1)	72.92	1-21
PvG09279	1158	386	Glyco_hydro_28 super family	72-373	Glycosyl hydrolases family 28	72-380	Polygalacturonase (P. cinamomi) (KAG6616063.1)	61.04	1-20
PvG02199	1392	464	GH7_CBH_EG super family	26-462	Glycosyl hydrolase family 7	26-463	GH family 7 protein (P. infestans) (KAF4031525.1)	86.36	1-23
PvG13517	1092	364	Glyco_hydro super family	1-320	Glycosyl hydrolases family 17	31-309	GH family 3 protein (P. pseudosyringae) (KAG7379682.1)	76.62	1-24
PvG13010	1092	364	Glyco_hydro_6 super family	55-300	Glycosyl hydrolases family 6	32-302	GH family 6 protein (P. halstedii) (XP_024583306.1)	81.82	1-20

蛋白 Protein	二级结构 Secondary structure				三级结构 Tertiary structure
蛋白 Protein	α-螺旋 α-helix (%)	β-转角 β-turn (%)	无规则卷曲Random coil (%)	延伸链 Extend (%)	与之最相似的蛋白 <BOLD>M</BOLD>ost similar protein	相似性 <BOLD>S</BOLD>imilarity (%)
PvG11117	32.98	5.59	48.67	12.77	Glycoside hydrolase family 6	26.24
PvG13516	36.45	6.40	41.63	15.52	β-1,3-glucanosyltr-ansferase	31.56
PvG10113	36.22	6.63	45.15	11.99	Cellobiohydrolase cel6A	24.49
PvG01792	43.70	3.86	39.07	13.37	β-1,3-glucanosyltr-ansferase	33.33
PvG09279	5.71	5.71	49.61	38.96	Endo-polygalacturonase	39.88
PvG02199	14.47	7.78	56.80	20.95	1,4-β-Cellulase	59.86
PvG13517	41.05	3.31	39.67	15.98	β-1,3-Glucosyltransf-erase	30.30
PvG13010	34.71	6.34	42.98	15.98	Chimeric cel6A	26.82