苹果黑腐皮壳菌CAP超家族蛋白基因鉴定及毒性功能分析

doi:10.3864/j.issn.0578-1752.2021.16.007

摘要/Abstract

摘要：

【目的】CAP（Cysteine-rich secretory protein,Antigen 5 and Pathogenesis related protein 1）超家族蛋白广泛存在于真菌、细菌、动植物等物种,并且参与病原菌的致病过程。本研究旨在鉴定苹果树腐烂病致病菌——苹果黑腐皮壳菌（Valsa mali）的CAP超家族基因并明确CAP超家族基因在病菌致病方面的作用。【方法】通过BLASTP在苹果黑腐皮壳菌全基因组中检索具有CAP保守结构域的基因;利用特异性引物对鉴定到的CAP基因进行PCR扩增和凝胶电泳检测;使用生物信息学软件和在线数据库进行蛋白序列特征和系统发育分析;利用RT-qPCR分析基因在病菌侵染过程中的表达模式;使用特异性引物扩增CAP基因上、下游片段并从PDL2质粒上扩增筛选标记基因;利用Double-joint PCR构建基因敲除盒并通过PEG介导的原生质转化技术进行基因敲除和基因回补;以遗传霉素（G418）抗性为筛选标记并利用4对引物PCR检测获得基因敲除突变体;以潮霉素（HPH）抗性为筛选标记获得基因回补菌株;通过对菌株进行培养皿内生长试验明确基因对病菌营养生长的影响;通过离体苹果枝条接种试验分析该病菌CAP超家族基因的毒性功能。【结果】在苹果黑腐皮壳菌中鉴定到3个具有CAP保守结构域的基因,分别命名为VmPR1a、VmPR1b和VmPR1c。序列特征分析发现,3个CAP蛋白均包含4个保守区：N端信号肽、N端延伸区（NTE）、CAP保守功能域和C端延伸区（CTE）。系统发育分析显示,3个CAP蛋白聚于不同的进化支,VmPR1a聚于clade2进化支并与粗糙链孢霉（Neurospora crassa）CAP蛋白进化关系较近;VmPR1b聚于clade3且与镰孢菌属（Fusarium spp.）CAP蛋白的进化关系更近;VmPR1c聚于clade1,并且也与粗糙链孢霉的CAP蛋白进化关系较近。RT-qPCR分析结果显示,VmPR1a、VmPR1b和VmPR1c在病菌侵染早期（6 h和12 h）均显著上调表达。利用PEG遗传转化技术获得VmPR1a、VmPR1b和VmPR1c敲除突变体（ΔVmPR1a-7/23、ΔVmPR1b-20/31和ΔVmPR1c-26/40）;营养生长观察发现,所有敲除突变体生长表型与野生型菌株03-8均无明显差异;致病力检测发现,ΔVmPR1b-20/31致病力较野生型无明显变化,而ΔVmPR1a-7/23和ΔVmPR1c-26/40致病力较野生型显著下降。将VmPR1a和VmPR1c分别回补至ΔVmPR1a和ΔVmPR1c,回补菌株（VmPR1a/C和VmPR1c/C）致病力恢复至野生型水平。【结论】苹果黑腐皮壳菌中存在3个CAP超家族基因（VmPR1a、VmPR1b和VmPR1c）,其中VmPR1a和VmPR1c是苹果黑腐皮壳菌重要的毒性因子。

关键词: 苹果黑腐皮壳菌, CAP 蛋白, 基因鉴定, 基因敲除, 毒性功能

Abstract:

【Objective】CAP (Cysteine-rich secretory protein, Antigen 5 and Pathogenesis related protein 1) superfamily proteins widely exist in fungi, bacteria, animal and plant. This kind of proteins participates in the pathogenic process of the pathogen. The purpose of this study was to identify the CAP superfamily genes in Valsa mali and clarify their virulence roles.【Method】BLASTP was used to retrieve genes with the conserved CAP domains in the whole genome of V. mali. PCR amplification and gel electrophoresis detection were carried out with specific primers. Bioinformatics software and online databases were used for protein sequence characterization and phylogenetic analysis. RT-qPCR was used to analyze the gene expression profiles. Double-joint PCR was used to construct gene knocking-out cassettes, PEG-mediated protoplast transformation was used to obtain knocking-out mutants and complementation strains. To obtain gene knockout mutants, geneticin (G418) was used as a selection marker, and transformants were validated by four pairs of primers. To obtain gene complementation transformants, hygromycin (HPH) was used as a selection marker. The vegetative growth of these strains was determined by cultivation on PDA medium, and the virulence of these strains was verified by inoculation on apple twigs.【Result】Three CAP superfamily genes were identified in V. mali, named VmPR1a, VmPR1b and VmPR1c, respectively. The three CAP proteins all contain four conserved regions, including a N-terminal signal peptide, a N-terminal extension region (NTE), a CAP domain and a C-terminal extension region (CTE). Phylogenetic analysis showed that the three proteins belonged to different clades. VmPR1a was clustered in clade2 and closely related to Neurospora crassa CAP protein. VmPR1b was clustered in clade3 and closely related to CAP proteins of Fusarium spp.. VmPR1c was clustered in clade1 and also closely related to N. crassa CAP protein. RT-qPCR analysis showed that VmPR1a, VmPR1b and VmPR1c were significantly up-regulated during early stages of infection (6 h and 12 h). VmPR1a, VmPR1b and VmPR1c knockout mutants (ΔVmPR1a-7/23, ΔVmPR1b-20/31 and ΔVmPR1c-26/40, respectively) were obtained, and all mutants showed no apparent alteration in filamentous growth compared with that of the wild-type strain. The virulence of ΔVmPR1b-20/31 was not obviously influenced. However, the virulence of ΔVmPR1a-7/23 and ΔVmPR1c-26/40 was significantly reduced compared with that of the wild-type strain. Moreover, the virulence of gene complementation strains (VmPR1a/C and VmPR1c/C) was restored to comparable level as that of the wild-type. 【Conclusion】Three CAP super-family genes were identified in V. mali (VmPR1a, VmPR1b and VmPR1c), and VmPR1a and VmPR1c are virulence factors of V. mali.

Key words: Valsa mali, CAP protein, gene identification, gene knockout, virulence

王程利,尹志远,聂嘉俊,林永辉,黄丽丽. 苹果黑腐皮壳菌CAP超家族蛋白基因鉴定及毒性功能分析[J]. 中国农业科学, 2021, 54(16): 3440-3450.

WANG ChengLi,YIN ZhiYuan,NIE JiaJun,LIN YongHui,HUANG LiLi. Identification and Virulence Analysis of CAP Superfamily Genes in Valsa mali[J]. Scientia Agricultura Sinica, 2021, 54(16): 3440-3450.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.16.007

https://www.chinaagrisci.com/CN/Y2021/V54/I16/3440

图/表 9

表1

图1

图2

图3

图4

图5

图6

图7

图8

参考文献 33

[1]	GIBBS G M, ROELANTS K, O’BRYAN M K. The CAP superfamily: Cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins—roles in reproduction, cancer, and immune defense. Endocrine Reviews, 2008, 29(7):865-897. doi: 10.1210/er.2008-0032
[2]	OLRICHS N K, HELMS J B. Novel insights into the function of the conserved domain of the CAP superfamily of proteins. AIMS Biophysics, 2016, 3(2):232-246. doi: 10.3934/biophy.2016.2.232
[3]	YAMAZAKI Y, MORITA T. Structure and function of snake venom cysteine-rich secretory proteins. Toxicon, 2004, 44(3):227-231. doi: 10.1016/j.toxicon.2004.05.023
[4]	KELLEHER A, DARWICHE R, REZENDE W C, FARIAS L P, LEITE L C C, SCHNEITER R, ASOJO O A. Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs. Acta Crystallographica, 2014, 71(8):2186-2196.
[5]	MILNE T J, ABBENANTE G, TYNDALL J D A, HALLIDAY J, LEWIS R J. Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily. Journal of Biological Chemistry, 2003, 278(33):31105-31110. doi: 10.1074/jbc.M304843200
[6]	KING T P, MORAN D, WANG D F, KOCHOUMIAN L, CHAIT B T. Structural studies of a hornet venom allergen antigen 5, Dol m V and its sequence similarity with other proteins. Protein Sequences and Data Analysis, 1990, 3(3):263-266.
[7]	VAN LOON L C, VAN KAMMEN A. Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. ‘Samsun’ and ‘Samsun NN’: II. Changes in protein constitution after infection with tobacco mosaic virus Virology, 1970, 40:190-211.
[8]	CHEN Y L, LEE C Y, CHENG K T, CHANG W H, HUANG R N, NAM H G, CHEN Y R. Quantitative peptidomics study reveals that a wound-induced peptide from PR-1 regulates immune signaling in tomato. The Plant Cell, 2014, 26:4135-4148. doi: 10.1105/tpc.114.131185
[9]	ROHM M, LINDEMANN E, HILLER E, ERMERT D, LEMUTH K, TRKULJA D, SOGUKPINAR O, BRUNNER H, RUPP S, URBAN C F, SOHN K. A family of secreted pathogenesis-related proteins in Candida albicans. Molecular Microbiology, 2013, 87(1):132-151. doi: 10.1111/mmi.2013.87.issue-1
[10]	PRADOS-ROSALES R C, ROLDAN-RODRIGUEZ R, SERENA C, LOPEZ-BERGES M S, GUARRO J, MARTÍNEZ-DEL-POZO Á, DI PIETRO A. A pr-1-like protein of Fusarium oxysporum functions in virulence on mammalian hosts. Journal of Biological Chemistry, 2012, 287(26):21970-21979. doi: 10.1074/jbc.M112.364034
[11]	TEIXEIRA P J P, THOMAZELLA D P T, VIDAL R O, DO PRADO P F V, REIS O, BARONI R M, FRANCO S F, MIECZKOWSKI P, PEREIRA G A G, MONDEGO J M C. The fungal pathogen Moniliophthora perniciosa has genes similar to plant PR-1 that are highly expressed during its interaction with cacao. PLoS ONE, 2012, 7(9):e45929. doi: 10.1371/journal.pone.0045929
[12]	LU S, EDWARDS M C. Molecular characterization and functional analysis of PR-1-like proteins identified from the wheat head blight fungus Fusarium graminearum. Phytopathology, 2018, 108(4):510-520. doi: 10.1094/PHYTO-08-17-0268-R
[13]	YIN Z Y, LIU H Q, LI Z P, KE X W, DOU D L, GAO X N, SONG N, DAI Q Q, WU Y X, XU J R, KANG Z S, HUANG L L. Genome sequence of Valsa canker pathogens uncovers a potential adaptation of colonization of woody bark. New Phytologist, 2015, 208(4):1202-1216. doi: 10.1111/nph.2015.208.issue-4
[14]	王旭丽. 中国苹果树腐烂病菌的种类: rDNA-ITS序列和表型比较研究[D]. 杨凌: 西北农林科技大学, 2007.
	WANG X L. Pathogen of apple tree valsa canker in China: A combined analysis of phenotypic characteristics and rDNA-ITS sequences[D]. Yangling: Northwest A&F University, 2007. (in Chinese)
[15]	KE X, HUANG L L, HAN Q M, GAO X N, KANG Z S. Histological and cytological investigations of the infection and colonization of apple bark by Valsa mali var. mali. Australasian Plant Pathology, 2013, 42(1):85-93. doi: 10.1007/s13313-012-0158-y
[16]	杜战涛, 李正鹏, 高小宁, 黄丽丽, 韩青梅. 陕西省苹果树腐烂病周年消长及分生孢子传播规律研究. 果树学报, 2013, 30(5):819-822.
	DU Z T, LI Z P, GAO X N, HUANG L L, HAN Q M. Study on the conidia dispersal and the disease dynamics of apple tree canker caused by Valsa mali var. mali in Shaanxi. Journal of Fruit Science, 2013, 30(5):819-822. (in Chinese)
[17]	林晓, 孙传茹, 王彩霞, 练森, 董向丽, 李保华. 影响苹果树腐烂病菌侵染致病的流行因子. 中国农业科学, 2021, 54(11):2333-2342.
	LIN X, SUN C R, WANG C X, LIAN S, DONG X L, LI B H. Epidemic factors affecting the infection and occurrence of Valsa mali. Scientia Agricultura Sinica, 2021, 54(11):2333-2342. (in Chinese)
[18]	BESSHO H, TSUCHIYA S, SOEJIMA J. Screening methods of apple trees for resistance to Valsa canker. Euphytica, 1994, 77(1/2):15-18. doi: 10.1007/BF02551454
[19]	ABE K, KOTODA N, KATO H, SOEJIMA J. Genetic studies on resistance to Valsa canker in apple: Genetic variance and breeding values estimated from intra- and inter-specific hybrid progeny populations. Tree Genetics and Genomes, 2011, 7(2):363-372. doi: 10.1007/s11295-010-0337-3
[20]	刘欣颖, 吕松, 王忆, 王昆, 李天红, 韩振海, 张新忠. 苹果种质资源对苹果树腐烂病抗性评价. 果树学报, 2011, 28(5):843-848.
	LIU X Y, LÜ S, WANG Y, WANG K, LI T H, HAN Z H, ZHANG X H. Evaluation of resistance of Malus germplasms to apple canker (Valsa ceratosperma). Journal of Fruit Science, 2011, 28(5):843-848. (in Chinese)
[21]	YIN Z Y, KE X W, HUANG D X, GAO X N, VOEGELE R T, KANG Z S, HUANG L L. Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR. World Journal of Microbiology and Biotechnology, 2013, 29(9):1563-1571. doi: 10.1007/s11274-013-1320-6
[22]	YU J, HAMARI Z, HAN K, SEO J, REYES-DOMÍNGUEZ Y, SCAZZOCCHIO C. Double-joint PCR: A PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genetics and Biology, 2004, 43:973-981.
[23]	高静, 李艳波, 柯希望, 康振生, 黄丽丽. PEG介导的苹果腐烂病菌原生质体转化. 微生物学报, 2011, 51(9):1194-1199.
	GAO J, LI Y B, KE X W, KANG Z S, HUANG L L. Development of genetic transformation system of Valsa mali of apple mediated by PEG. Acta Microbiologica Sinica, 2011, 51(9):1194-1199. (in Chinese)
[24]	臧睿, 黄丽丽, 康振生, 王旭丽. 陕西苹果树腐烂病菌(Cytospora spp.)不同分离株的生物学特性与致病性研究. 植物病理学报, 2007, 37(4):343-351.
	ZANG R, HUANG L L, KANG Z S, WANG X L. Biological characteristics and pathogenicity of different isolates of Cytospora spp. isolated from apple trees in Shaanxi Province. Acta Phytopathologica Sinica, 2007, 37(4):343-351. (in Chinese)
[25]	许春景. 苹果树腐烂病菌三个果胶酶基因的致病功能研究[D]. 杨凌: 西北农林科技大学, 2016.
	XU C J. Pathogenic function of three pectinase genes in Valsa mali[D]. Yangling: Northwest A&F University, 2016. (in Chinese)
[26]	WU Y X, XU L S, YIN Z Y, DAI Q Q, GAO X N, FENG H, VOEGELE R T, HUANG L L. Two members of the velvet family, VmVeA and VmVelB, affect conidiation, virulence and pectinase expression in Valsa mali. Molecular Plant Pathology, 2018, 19(7):1639-1651. doi: 10.1111/mpp.2018.19.issue-7
[27]	FENG H, XU M, ZHENG X, ZHU T Y, GAO X N, HUANG L L. MicroRNAs and their targets in apple (Malus domestica cv. “Fuji”) involved in response to infection of pathogen Valsa mali. Frontiers in Plant Science, 2017, 8:2081. doi: 10.3389/fpls.2017.02081
[28]	XU M, GUO Y, TIAN R Z, GAO C, GUO F R, VOEGELE R T, BAO J Y, LI C J, JIA C H, FENG H, HUANG L L. Adaptive regulation of virulence genes by microRNA-like RNAs in Valsa mali. New Phytologist, 2020, 227:899-913. doi: 10.1111/nph.v227.3
[29]	JONES J D, DANGL J L. The plant immune system. Nature, 2006, 444(7117):323-329. doi: 10.1038/nature05286
[30]	VLEESHOUWERS V G, OLIVER R P. Effectors as tools in disease resistance breeding against biotrophic, hemibiotrophic, and necrotrophic plant pathogens. Molecular Plant-Microbe Interactions, 2014, 27(3):196-206. doi: 10.1094/MPMI-10-13-0313-IA
[31]	LI Z, YIN Z Y, FAN Y, XU M, KANG Z S, HUANG L L. Candidate effector proteins of the necrotrophic apple canker pathogen Valsa mali can suppress BAX-induced PCD. Frontiers in Plant Science, 2015, 6:579.
[32]	ZHANG M, FENG H, ZHAO Y H, SONG L L, GAO C, XU X M, HUANG L L. Valsa mali pathogenic effector VmPxE1 contributes to full virulence and interacts with the host peroxidase MdAPX1 as a potential target. Frontiers in Microbiology, 2018, 9:821. doi: 10.3389/fmicb.2018.00821
[33]	ZHANG M, XIE S C, ZHAO Y H, MENG X, SONG L L, FENG H, HUANG L L. Hce2 domain-containing effectors contribute to the full virulence of Valsa mali in a redundant manner. Molecular Plant Pathology, 2019, 20(6):843-856. doi: 10.1111/mpp.2019.20.issue-6

引物 Primer	序列 Sequence (5′→3′)
VmPR1a-F	ggggactcttgaccatggtaATGAAGCCCGCTC
VmPR1a-R	ctcaccatcctaggactagtTAACGCCAGAAACAGTACCA
VmPR1b-F	ggggactcttgaccatggta ATGCACTTCTCAACTCGTCC
VmPR1b-R	ctcaccatcctaggactagtAGCAGAGGTTGGGGTCTC
VmPR1c-F	ggggactcttgaccatggta ATGAAGTCATCATCGCTCTT
VmPR1c-R	ctcaccatcctaggactagtTACGCTGACGTTCACGGA
pCA1302-F/R	TAAGGGATGACGCACAAT/CGGACACGCTGAACTTGT
qRT-VmPR1a-F/R	AGTCGCCAGACTCCGGATCCG/GACCAACTGCGTGAAGTGGC
qRT-VmPR1b-F/R	ACATCCTCGTCTGCTGCTGCTG/GGTCTCGCTAACTCCAGAACGA
qRT-VmPR1c-F/R	CATGACCTACGGTGACACGTA/GAGTCAGCGTCAGTAGAGGC
G6PDH-F/R	TCAGAACAAGTTCGAGGGCGACAA/TGAGGGCAATAGAGGGCTTGTTCA
VmPR1a-5F/6R	ACGCTTTCATCCAGACTTTT/CCTTCTCCTTGTGGCTTTGT
VmPR1b-5F/6R	CACTTCTCAACTCGTCCCG/ACCGTCCGAAGCAATACC
VmPR1c-5F/6R	GAGGTTACGACCGTTGAGAC/CAGTGAAAAGGGGATGGC
Neo-F/R	CACAGCACCGACCACAAA/CCAGCAGTAGACACTTGGAATC
PDL2-VmPR1a-F	cgactcactatagggcgaattgggtactcaaattggttataaACGCTTTCATCCAGACTTTT
PDL2-VmPR1a-R	ccaccccggtgaacagctcctcgcccttgctcacctcgagCCTTCTCCTTGTGGCTTTGT
PDL2-VmPR1c-F	cgactcactatagggcgaattgggtactcaaattggttataaCGATTTGGGCAACTTGAG
PDL2-VmPR1c-R	ccaccccggtgaacagctcctcgcccttgctcacctcgagGCGATAGCCAGAAAACATAG