柑橘溃疡病相关基因CsPGIP的克隆与表达

doi:10.3864/j.issn.0578-1752.2019.04.006

Abstract

Abstract:

【Objective】The objective of this study is to clone CsPGIP and analyze its expression characteristics, construct CsPGIP transgenic citrus and evaluate the resistance to citrus bacterial canker (CBC), and to provide a theoretical basis for molecular breeding of citrus bacterial canker.【Method】CsPGIP was annotated from the genomic databases and cloned from Wanjincheng and Calamondin. MEGA6 was used for multiple sequence alignment and phylogenetic tree was constructed. Two online softwares BaCelLo and SignalP 4.0 were used for the prediction of subcellular localization and signal peptide. The predicted result was then demonstrated by GFP transient expression. The expression profile of CsPGIP induced by Xanthomonas citri subsp. citri (Xcc) was also analyzed in Wanjincheng and Calamondin by using qRT-PCR method. The correlation between Xcc infection and CsPGIP expression was analyzed. Genetic transformation of Wanjincheng was conducted by Agrobacterium-mediated method. The over-expressed lines were identified by GUS staining, PCR and qRT-PCR. The phenotypic changes of transgenic and wild-type lines were observed, plant height and leaf phenotype were analyzed. In vitro acupuncture was used to evaluate the resistance of transgenic lines and wild-type lines to citrus bacterial canker. The effect of CsPGIP expression on resistance and susceptibility to citrus bacterial canker was analyzed by statistical analysis of lesion area (LA) and disease index (DI). 【Result】The PGIP cloned from Wanjincheng and Calamondin encodes 328 amino acids, which is 99.39% homology with the reported CsPGIP from Clementina, and contains two typical LRR domains (LRR_1 and LRR_2). In the phylogenetic tree, the genetic distance between CsPGIP and grape PGIP (GSVIVT01033370001) was the closest, and the similarity was 62.97%. It is inferred that CsPGIP and grape PGIP have similar resistance to disease. The prediction of subcellular localization and signal peptide indicated that CsPGIP was a secretory protein, and GFP transient expression proved that CsPGIP located on cell membrane and cell wall, which was consistent with the predicted results. The expression of CsPGIP in canker sensitive plant Wanjincheng and canker resistant plant Calamondin was different after inoculated with Xcc. The expression of CsPGIP was significantly down-regulated in Wanjincheng, but significantly up-regulated and maintained at a high level in Calamondin. It is speculated that CsPGIP was related to resistance to citrus bacterial canker. CsPGIP over-expression vector was constructed and transformed into Wanjincheng, and nine CsPGIP over-expression lines (OE1, OE3, OE4, OE5, OE6, OE9, OE10, OE12 and OE14) were identified as CsPGIP over-expression positive lines by PCR identification and qRT-PCR. Through the phenotypic observation of transgenic lines, it was found that the phenotypes of OE3 and OE14 lines were significantly different from those of wild-type lines. The plant was short, in which OE14 was also abnormal with curly property and greater thickness. The in vitro canker resistance of eight CsPGIP over-expression lines was evaluated. The results showed that the lesion area on the eight CsPGIP over-expression lines was smaller compared to that on the wild-type (24.11%-83.88%), and the lesion area of OE1 was the smallest. In terms of disease index, the disease index of CsPGIP over-expression lines (except OE3) was significantly lower than that of wild-type (23.12%-75.49%), and the decrease of OE1 was the most significant. The above results showed that over-expression of CsPGIP could effectively inhibit the growth of citrus bacterial canker.【Conclusion】CsPGIP is an important gene which can inhibit or reduce the incidence of citrus bacterial canker, and has a great application value in the mechanism study of citrus resistance to bacterial canker. In the same time, it can be used as a candidate gene for molecular breeding of citrus bacterial canker resistance.

Key words: citrus bacterial canker (CBC), polygalacturonase inhibitor protein, CsPGIP, over-expression, CBC resistance

HU AnHua,QI JingJing,ZHANG QingWen,CHEN ShanChun,ZOU XiuPing,XU LanZhen,PENG AiHong,LEI TianGang,YAO LiXiao,LONG Qin,HE YongRui,LI Qiang. Cloning and Expression Analysis of the Citrus Bacterial Canker-Related Gene CsPGIP in Citrus[J].Scientia Agricultura Sinica, 2019, 52(4): 639-650.

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References 37

[1]	PITINO M, ARMSTRONG C M, DUAN Y P . Rapid screening for citrus canker resistance employing pathogen-associated molecular pattern-triggered immunity responses. Horticulture Research, 2015,2:15042. doi: 10.1038/hortres.2015.42 pmid: 4595992
[2]	贾瑞瑞, 周鹏飞, 白晓晶, 陈善春, 许兰珍, 彭爱红, 雷天刚, 姚利晓, 陈敏, 何永睿, 李强 . 柑橘响应溃疡病菌转录因子CsBZIP40 的克隆及功能分析. 中国农业科学, 2017,50(13):2488-2497. doi: 10.3864/j.issn.0578-1752.2017.13.008
	JIA R R, ZHOU P F, BAI X J, CHEN S C, XU L Z, PENG A H, LEI T G, YAO L X, CHEN M, HE Y R, LI Q . Gene cloning and expression analysis of canker-related transcription factor CsBZIP40 in citrus. Scientia Agricultura Sinica, 2017,50(13):2488-2497. (in Chinese) doi: 10.3864/j.issn.0578-1752.2017.13.008
[3]	杨枫, 陈传武, 范七君, 石春梅, 谢宗周, 郭大勇, 刘继红 . 温度和多胺对柑橘溃疡病发生的影响及作用机制. 中国农业科学, 2018,51(10):1899-1907.
	YANG F, CHEN C W, FAN Q J, SHI C M, XIE Z Z, GUO D Y, LIU J H . Influence of temperature and polyamines on occurrence of citrus canker disease and underlying mechanisms. Scientia Agricultura Sinica, 2018,51(10):1899-1907. (in Chinese)
[4]	陈力, 王中康, 黄冠军, 曹月青, 夏玉先, 殷幼平 . 柑橘溃疡病生防菌株CQBS03的鉴定及其培养特性研究. 中国农业科学, 2008,41(8):2537-2545.
	CHEN L, WANG Z K, HUANG G J, CAO Y Q, XIA Y X, YIN Y P . Evaluation of Bacillus subtilis strain CQBS03 against Xanthomonas axonopodis pv. citri. Scientia Agricultura Sinica, 2008,41(8):2537-2545. (in Chinese)
[5]	陈波, 罗庆华, 谭雅芹, 闫慧清 . 柑橘PGIP的B细胞抗原表位分析和原核表达. 现代食品科技, 2018,34(4):18-22.
	CHEN B, LUO Q H, TAN Y Q, YAN H Q . B cell epitopes analysis and prokaryotic expression of PGIP in citrus. Modern Food Science and Technology, 2018,34(4):18-22. (in Chinese)
[6]	FREIBERG A, MACHNER M P, PFELI W, SCHUBERT W D, HEINZ D W, SECKLER R . Folding and stability of the leucine-rich repeat domain of internal in B from Listeri monocytogenes. Journal of Molecular Biology, 2004,337(2):453-461.
[7]	LEHMANN P . Structure and evolution of plant disease resistance genes. Journal of Applied Genetics, 2002,43(4):403-414. pmid: 12441626
[8]	FERRARI S, GALLETTI R, VAIRO D, GERVONE F, DE LORENZO G . Antisense expression of the Arabidopsis thaliana AtPGIP1 gene reduces polygalacturonase-inhibiting protein accumulation and enhances susceptibility to Botrytis cinerea. Molecular Plant-Microbe Interactions, 2006,19(8):931-936.
[9]	JOUBERT D A, KARS I, WAGEMAKERS L, BERGMANN C, KEMP G, VIVIER M A, VAN KAN J A . A polygalacturonase- inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction. Molecular Plant-Microbe Interactions, 2007,20(4):392-402.
[10]	CHENG Q, CAO Y Z, PAN H X, WANG M X, HUANG M R . Isolation and characterization of two genes encoding polygalacturonase- inhibiting protein from Populus deltoides. Journal of Genetics and Genomics, 2008,35(10):631-638. doi: 10.1016/S1673-8527(08)60084-3 pmid: 18937920
[11]	HEGEDUS D D, LI R, BUCHWALDT L, PARKIN I, WHITWILL S, COUTU C, BEKKAOUI D, RIMMER S R . Brassica napus possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to Sclerotinia sclerotiorum infection, wounding and defense hormone treatment. Planta, 2008,228(2):241-253.
[12]	JANNI M, SELLA L, FAVARON F, BLECHL A E, DE LORENZO G, D’OVIDO R . The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogenBipolaris sorokiniana. Molecular Plant-Microbe Interactions, 2008,21(2):171-177. doi: 10.1094/MPMI-21-2-0171 pmid: 18184061
[13]	DI C X, LI M, LONG F, BAI M P, LIU Y J, ZHENG X L, XU S J, XIANG Y, SUN Z L, AN L Z . Molecular cloning, functional analysis and localization of a novel gene encoding polygalacturonase- inhibiting protein in Chorispora bungeana. Planta, 2009,231(1):169-178. doi: 10.1007/s00425-009-1039-7 pmid: 19885675
[14]	HWANG B H, BAE H, LIM H S, KIM K B, KIM S J, IM M H, PARK B S, KIM D S, KIM J . Overexpression of polygalacturonase- inhibiting protein 2 (PGIP2) of Chinese cabbage(Brassica rapa ssp. pekinensis) increased resistance to the bacterial pathogen Pectobacterium carotovorum ssp. carotovorum. Plant Cell, Tissue Organ Culture, 2010,103(3):293-305.
[15]	D’OVIDIO R, RAIOLA A, CAPODICASA C, DEVOTO A, PONTIGGIA D, ROBERTI S, GALLETTI R, CONTI E, O’SULLIVAN D, DE LORENZO G . Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects. Plant Physiology, 2004,135(4):2424-2435. doi: 10.1104/pp.104.044644
[16]	LIU N N, ZHANG X Y, SUN Y, WANG P, LI X C, PEI Y K, LI F G, HOU Y X . Molecular evidence for the involvement of a polygalacturonase-inhibiting protein, GhPGIP1, in enhanced resistance to Verticillium and Fusarium wilts in cotton. Scientific Reports, 2017,7:39840. doi: 10.1038/srep39840 pmid: 5228132
[17]	JOUBERT D A, SLAUGHTER A R, KEMP C, BECKER J V, KROOSHOF C H, BERGMANN C, BENEN C, PRETORIUS I S, WIER M A . The polygalacturonase-inhibiting protein (VvPGIPl) reduces Botrytis cinerea susce in transgenic tobacco and differentially inhibits fungal polygalacturonases. Transgenic Research, 2006,15(6):687-702.
[18]	BORRAS-HIDALGO O, CAPRARI C, HERNANDEZ-ESTEVEZ I, DE LORENZO G, CERVONE F . A gene for plant protection: expression of a bean polygalacturonase inhibitor in tobacco confers a strong resistance against Rhizoctonia solani and two oomycetes. Frontiers in Plant Science, 2012,3:268. doi: 10.3389/fpls.2012.00268 pmid: 23264779
[19]	WANG A Y, WEI X N, RONG W, DANG L, DU L P, QI L, XU H J, SHAOY J, ZHANG Z Y . GmPGIP3 enhanced resistance to both take-all and common root rot diseases in transgenic wheat. Functional and Integrative Genomics, 2015,15(3):375-381. doi: 10.1007/s10142-014-0428-6 pmid: 25487419
[20]	MANFREDINI C, SICILIA F, FERRARI S, PONTIGGIA D, SALVI G, CAPRARI C, LORITO M, DE LORENZO G . Polygalacturonase- inhibiting protein 2 of Phaseolus vulgaris inhibits BcPGl, a polygalacturonase of Botrytis cinerea important for pathogenicity, and protects transgenic plants from infection. Physiological and Molecular Plant Pathology, 2005,67(2):108-115.
[21]	PRABHU S A, WAGENKNECHT M, MELVIN P, KUMAR B S G, VEENA M, SHAILASREE S, MOERSCHBACHER B M, KINI K R . Immuno-affinity purification of PglPGIP₁, a polygalacturonase inhibitor protein from pearl millet: studies on its inhibition of fungal polygalacturonases and role in resistance against the downy mildew pathogen. Molecular Biology Reports, 2015,42(6):1123-1138. doi: 10.1007/s11033-015-3850-5 pmid: 255967221
[22]	PRABHU S A, KINI K R, RAJ S N, MOERSCHBACHER B M, SHETTY H S . Polygalacturonase-inhibitor proteins in pearl millet: possible involvement in resistance against downy mildew. Acta Biochimic et Biophysica Sinica, 2012,44(5):415-423. doi: 10.1093/abbs/gms015 pmid: 22411686
[23]	AGÜERO C B, URATSU S L, GREVE C, POWELL A T, LABAVITCH J M, MEREDITH C P, DANDEKAR A M . Evaluation of tolerance to Pierce’s disease and Botrytis in transgenic plants of Vitis vinifera L. expressing the pear PGIP gene. Molecular Plant Pathology, 2005,6(1):43-51.
[24]	SCHACHT T, UNGER C, PICH A, WYDRA K . Endo- and exopolygalactuonases of Rslstonia solanacearum are inhibited by polygalactuonase-inhibiting protein (PGIP) activity in tomato stem extracts. Plant Physiology and Biochemistry, 2011,49(4):377-387. doi: 10.1016/j.plaphy.2011.02.001 pmid: 21367611
[25]	WANG R, LU L, PAN X, HU Z, LING F, YAN Y, LIU Y, LIN Y . Functional analysis of OsPGIP1 in rice sheath blight resistance. Plant Molecular Biology, 2015,87(1/2):181-191. doi: 10.1007/s11103-014-0269-7 pmid: 25488398
[26]	FENG C S, ZHANG X, WU T, YUAN B, DING X H, YAO F Y, CHU Z H . The polygalacturonase-inhibiting protein 4 (OsPGIP4), a potential component of the qBlsr5a locus, confers resistance to bacterial leaf streak in rice. Planta, 2016,243(5):1297-1308. doi: 10.1007/s00425-016-2480-z pmid: 26945855
[27]	GOODSTEIN D M, SHU S, HOWSON R, NEUPANE R, HAYES R D, FAZO J, MITROS T, DIRKS W, HELLSTEN U, PUTNAM N, ROKHSAR D S . Phytozome: a comparative platform for green plant genomics. Nucleic Acids Research, 2012,40(Database issue):D1178-D1186. doi: 10.1093/nar/gkr944 pmid: 22110026
[28]	TAMURA K, STECHER G, PETERSON D, FILIPSKI A, KUMAR S . MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 2013,30(12):2725-2729. doi: 10.1093/molbev/mst197 pmid: 24132122
[29]	PIERLEONI A, MARTELLI P L, FARISELLI P, CASADIO R . BaCelLo: a balanced subcellular localization predictor. Bioinformatics, 2006,22(14):e408-e416. doi: 10.1093/bioinformatics/btl222
[30]	PETERSEN T N, BRUNAK S, VON HEIJNE G, NIELSEN H . SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods, 2011,8(10):785-786. doi: 10.1038/nmeth.1701 pmid: 21959131
[31]	PENG A H, XU L Z, HE Y R, LEI T G, YAO L X, CHEN S C, ZOU X P . Efficient production of marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system. Plant Cell, Tissue and Organ Culture, 2015,123(1):1-13.
[32]	PENG A H, CHEN S C, LEI T G, XU L Z, HE Y R, WU L, ZOU X P . Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnology Journal, 2017,15(12):1509-1519. doi: 10.1111/pbi.12733 pmid: 5698050
[33]	POWELL A L, VAN KAN J, TEN HAVE A, VISSER J, GREVE L C, BENNETT A B, LABAVITCH J M . Transgenic expression of pear PGIP in tomato limits fungal colonization. Molecular Plant-Microbe Interactions, 2000,13(9):942-950. doi: 10.1094/MPMI.2000.13.9.942 pmid: 10975651
[34]	DE LORENZO G, D’OVIDIO R, CERVONE F . The role of polygalacturonase-inhibiting proteins (PGIPs) in defense against pathogenic fungi. Annual Review of Phytopathology, 2001,39(1):313-335. doi: 10.1146/annurev.phyto.39.1.313
[35]	KOBE B, KAJAVA A V . The leucine-rich repeat as a protein recognition motif. Current Opinion in Structural Biology, 2001,11(6):725-732. doi: 10.1016/S0959-440X(01)00266-4 pmid: 11751054
[36]	WANG X J, ZHU X P, TOOLEY P, ZHANG X G . Cloning and functional analysis of three genes encoding polygalacturonase- inhibiting proteins from Capsicum annuum and transgenic CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens. Plant Molecular Biology, 2013,81(4/5):379-400. doi: 10.1007/s11103-013-0007-6 pmid: 23334855
[37]	HU Y, ZHANG J L, JIA H G, SOSSO D, LI T, FROMMER W B, YANG B, WHITE F F, WANG N, JONES J B . Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease. Proceedings of the National Academy of Sciences of the United States of America, 2014,111(4):E521-E529.

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引物名称Primer name	引物序列Primer sequence (5′-3′)	酶切位点Enzyme site
OE-CsPGIP-f	CGGGATCCATGAGCAACACGTCACTGTTGTCT	BamHI
OE-CsPGIP-r	CGGAATTCTCACTTGCAGCTTTCGAGGGGCGC	EcoRI
SCL-CsPGIP-f	GGGGTACCATGAGCAACACGT CACTGTTGT	KpnI
SCL-CsPGIP-r	TCCCCCGGGCTTGCAGCTTTCG AGGGGCGCG	SmaI
qPCR-CsPGIP-f	AGAAGCTTGGCGCTCTTCAT	N/A
qPCR-CsPGIP-r	TCGCCTTCAAGCTTGTTCCT	N/A
qPCR-Actin-f	CATCCCTCAGCACCTTCC	N/A
qPCR-Actin-r	CCAACCTTAGCACTTCTCC	N/A
OE-f (35S)	AGTAAGGATCGAT CCCACAAAGT	N/A
OE-r (CsPGIP)	TTTTTGAAGAGTAGTGAAGCTGCA	N/A

Cloning and Expression Analysis of the Citrus Bacterial Canker-Related Gene CsPGIP in Citrus

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