Effects of Overexpression of CsEXPA8 on Growth and Canker Disease Resistance in‘Wanjincheng’Orange

doi:10.16420/j.issn.0513-353x.2023-0145

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (5): 971-981.doi: 10.16420/j.issn.0513-353x.2023-0145

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Effects of Overexpression of CsEXPA8 on Growth and Canker Disease Resistance in‘Wanjincheng’Orange

PENG Aihong¹^,^**(), ZHANG Jingyun², CHEN Zhiyi¹, SU Juan¹, HE Yongrui¹, YAO Lixiao¹

¹ Citrus Research Institute，Southwest University，Chongqing 400712，China
² Xi’an Jingkai No. 5 School，Xi’an 710018，China

Received:2023-05-17 Revised:2023-11-14 Online:2024-05-25 Published:2024-05-29

Abstract

Abstract:

To clarify the biological function of the expansion protein gene CsEXPA8，the vectors which excessed and inhibited the expression level of CsEXPA8 were constructed and then introduced in‘Wanjincheng’orange，respectively. The phenotype of transgenic plants was observed and their resistance to citrus canker was evaluated. The results showed that CsEXPA8 has tissue specific expression characteristics and its expression level in‘Wanjincheng’orange could be induced by Xanthomonas citri pv. citri，and that overexpression of CsEXPA8 could promote the growth of leaf length and width，and spring shoot，but also increase the sensitivity of transgenic plants to citrus canker，and that inhibiting the expression level of CsEXPA8 reduced the leaf length and width，and spring shoot，and enhanced the resistance of transgenic plants against citrus canker. These results indicated that CsEXPA8 positively and negatively regulates growth and resistance to citrus canker of‘Wanjincheng’orange，respectively.

Key words: citrus, expansins, CsEXPA8, plant growth, citrus canker, resistance

PENG Aihong, ZHANG Jingyun, CHEN Zhiyi, SU Juan, HE Yongrui, YAO Lixiao. Effects of Overexpression of CsEXPA8 on Growth and Canker Disease Resistance in‘Wanjincheng’Orange[J]. Acta Horticulturae Sinica, 2024, 51(5): 971-981.

Figures/Tables 8

Table 1 The sequences of primer used in this study

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
CsEXPA8克隆	CsEXPA8-F	ATGGCCAATATTGCAGCATCATCTA
Clone of CsEXPA8	CsEXPA8-R	TAGATGATGCTGCAATATTGGCCAT
CsEXPA8 CDS克隆	pCsEXPA8-F	ggtaccATGGCCAATATTGCAGCATCAT
Clone of CDS of CsEXPA8	pCsEXPA8-R	gaattcCTAAGCTTTGAGCAATTCCTT
检测柑橘Actin的表达量	Actin-F	CATCCCTCAGCACCTTCC
Expression analysis of citrus Actin	Actin-R	CCAACCTTAGCACTTCTCC
检测柑橘CsEXPA8的表达量	qCsEXPA8-F	TGCTTTCGTGGCACATTTGG
Expression analysis of citrus CsEXPA8	qCsEXPA8-R	ACCCCCGCCATAGAAAGTTG
克隆CsEXPA8干扰片段	rCsEXPA8-F	ggatccatttaaatCCTTATTCCTCCTTTCTTCGC
Clone of RNAi fragment of CsEXPA8	rCsEXPA8-R	gtcgacggcgcgccGGCGGGGGTGATGCTTCCGG
检测GUS基因	GUS-F	CATGAAGATGCGGACTTGCG
Detection of GUS gene	GUS-R	GCTAACGTATCCACGCCGTA

Fig. 1 Tissue specific（A）and induced by Xanthomonas citri pv. citri（B）expression analysis of CsEXPA8 Different lowercase letters indicate significant difference at 0.05 level. The same below.

Fig. 2 Evolutionary tree of CsEXPA8 and expansins in Arabidopsis thaliana（At）

Fig. 3 Structure（A）and CDS sequence（B）of CsEXPA8 gene and domain（C）of its encoded protein in citrus

Fig. 4 Detection of overexpression（A，C，E）and interference expression（B，D，F）of CsEXPA8 in transgenic citrus plants M：Marker；WT：Wild-type；P：Plasmid；p-：Overexpressed plants；RNAi-：Interfering expressed plants. The same below.

Fig. 5 Phenotypic analysis of CsEXPA8 overexpression（p-）and interference expression（RNAi-）plants in citrus

Fig. 6 Development of transgenic plants after inoculation with Xanthomonas citri pv. citri

Fig. 7 Resistant evaluation of transgenic plants to citrus canker

References 31

[1]	Abuqamar S, Ajeb S, Sham A, Enan M R, Iratni R. 2013. A mutation in the expansin-like A 2 gene enhances resistance to necrotrophic fungi and hypersensitivity to abiotic stress in Arabidopsis thaliana. Molecular Plant Pathology, 14 (8):813-827. doi: 10.1111/mpp.12049 pmid: 23782466
[2]	Chen L, Zou W, Wu G, Lin H, Xi D. 2018. Tobacco alpha-expansin EXPA4 plays a role in Nicotiana benthamiana defence against Tobacco mosaic virus. Planta, 247:355-368. doi: 10.1007/s00425-017-2785-6 pmid: 28993946
[3]	Cho H T, Cosgrove D J. 2002. Regulation of root hair initiation and expansin gene expression in Arabidopsis. The Plant Cell, 14:3237-3253.
[4]	Cosgrove D J. 1993. How do plant cell wall extend? Plant Physiology, 102:1-6. doi: 10.1104/pp.102.1.1 pmid: 11536544
[5]	Cosgrove D J. 2000. Loosening of plant cell walls by expansins. Nature, 407:321-326.
[6]	Dong B, Wang Q Q, Zhou D, Wang Y G, Miao Y F, Zhong S W, Fang Q, Yang L Y, Xiao Z, Zhao H B. 2024. Abiotic stress treatment reveals expansin like a gene OfEXLA1improving salt and drought tolerance of Osmanthus fragrans by responding to abscisic acid. Horticultural Plant Journal, 10 (2):573-585.
[7]	Gookin T E, Hunter D A, Reid M S. 2003. Temporal analysis of alpha and beta-expansin expression during floral opening and senescence. Plant Science, 164:769-781.
[8]	Han Y, Li A, Li F, Zhao M, Wang W. 2012. Characterization of a wheat(Triticum aestivum L.)expansin gene,TaEXPB23,involved in the abiotic stress response and phytohormone regulation. Plant Physiology and Biochemistry, 54:49-58.
[9]	Karlova R, Rosin F M, Busscherlange J, Parapunova V, Do P T, Fernie A R, Fraser P D, Baxter C, Angenent G C, Maagd R A D. 2011. Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening. The Plant Cell, 23:923-941.
[10]	Kuluev B R, Knyazev A B, Lebedev Y P, Chemeris A V. 2012. Morphological and physiological characteristics of transgenic tobacco plants expressing expansin genes:AtEXP10 from Arabidopsis and PnEXPA1 from poplar. Russian Journal of Plant Physiology, 59 (1):97-104.
[11]	Kumar S, Stecher G, Tamura K. 2016. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33:1870-1874.
[12]	Li F, Xing S C, Guo Q F, Zhao M R, Zhang J, Gao Q, Wang G P, Wang W. 2010. Drought tolerance through over-expression of expansin gene TaEXPB23 in transgenic tobacco. Journal of Plant Physiology, 168:960-966.
[13]	Liu X P, Dong S Y, Miao H, Bo K L, Li C X, Yang Y Y, Gu X F, Zhang S P. 2022. Genome-wide analysis of expansins and their role in fruit spine development in cucumber (Cucumis sativus L.). Horticultural Plant Journal, 8 (6):757-768.
[14]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method. Methods, 25:402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[15]	Ma Shuang. 2021. Genome-wide analysis of the expansin gene family in Phyllostachys edulis and functional analysis of PeEXPA8[M. D. Dissertation]. Mianyang: Southwest University of Science and Technology. (in Chinese)
	马霜. 2021. 毛竹扩展蛋白家族全基因组分析及PeEXPA8功能研究[硕士论文]. 绵阳: 西南科技大学.
[16]	Marowa P, Ding A, Xu Z, Kong Y. 2020. Overexpression of NtEXPA11 modulates plant growth and development and enhances stress tolerance in tobacco. Plant Physiology and Biochemistry, 151:477-485.
[17]	Mcqueenmason S. 2003. Expansins and cell growth. Current Opinion in Plant Biology, 6:603. doi: 10.1016/j.pbi.2003.09.003 pmid: 14611960
[18]	Muthusamy M, Kim J A, Jeong M J, Lee S I. 2020. Blue and red light upregulate α-expansin 1 (EXPA1) in transgenic Brassica rapa and its overexpression promotes leaf and root growth in Arabidopsis. Plant Growth Regulation, 91:75-87.
[19]	Peng A, Chen S, Lei T, Xu L, He Y, Wu L, Yao L, Zou X. 2017. Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnology Journal, 15:1509-1519.
[20]	Peng A, Xu L, He Y, Lei T, Yao L, Chen S, Zou X. 2015. 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, 123:1-13.
[21]	Peng A, Zou X, Xu L, He Y, Lei T, Yao L, Li Q, Chen S. 2019. Improved protocol for the transformation of adult Citrus sinensis Osbeck‘Tarocco’blood orange tissues. In Vitro Cellular & Developmental Biology-Plant, 55:659-667.
[22]	Sampedro J, Guttman M, Li L C, Cosgrove D J. 2015. Evolutionary divergence of β-expansin structure and function in grasses parallels emergence of distinctive primary cell wall traits. The Plant Journal, 81:108-120. doi: 10.1111/tpj.12715 pmid: 25353668
[23]	Sun Yongdong, Luo Weirong, Zhang Chuanlai. 2009. Protein sequence analysis for expansin family. China Journal of Bioinformatics, 7 (3):193-195. (in Chinese)
	孙涌栋, 罗未蓉, 张传来. 2009. 扩张蛋白家族蛋白序列分析. 生物信息学, 7 (3):193-195.
[24]	Tan J, Wang M, Shi Z, Miao X. 2018. OsEXPA10 mediates the balance between growth and resistance to biotic stress in rice. Plant Cell Reports, 37 (7):993-1002.
[25]	Trivedi P K, Nath P. 2004. MaExp1,an ethylene-induced expansin from ripening banana fruit. Plant Science, 167 (6):1351-1358.
[26]	Vreeburg R A M, Benschop J J, Peeters A J M, Colmer T D, Ammerlaan A H M, Staal M, Elzenga T M, Staals R H, Darley C P, McQueen-Mason S J, Voesenek L A C J. 2005. Ethylene regulates fast apoplastic acidification and expansin A transcription during submergence-induced petiole elongation in Rumex palustris. The Plant Journal, 43 (4):597-610.
[27]	Wang G F, Gao Y, Wang J J, Yang L W, Song R, Li X R, Shi J S. 2011. Overexpression of two cambium-abundant Chinese fir(Cunninghamia lanceolata)α-expansin genes ClEXPA1 and ClEXPA2 affect growth and development in transgenic tobacco and increase the amount of cellulose in stem cell walls. Plant Biotechnology Journal, 9 (4):486-502.
[28]	Xing S C, Li F, Guo Q F, Liu D R, Zhao X X, Wang W. 2009. The involvement of an expansin gene TaEXPB23 from wheat in regulating plant cell growth. Biology Plantarum, 53:429-434.
[29]	Xu Chaofeng. 2020. Functional identification of tomato expansin genes SlEXPA3 and SlEXLB1[M. D. Dissertation]. Wuhan: Huazhong Agricultural University. (in Chinese)
	徐超峰. 2020. 番茄扩张蛋白基因SlEXPA3和SlEXLB1的功能鉴定[硕士论文]. 武汉: 华中农业大学.
[30]	Yang Ruojiao. 2020. Functional analysis of expansin gene TaEXPA2 in wheat(Triticumaestivum L.)drought tolerance[M. D. Dissertation]. Tai’an: Shandong Agricultural University. (in Chinese)
	杨若娇. 2020. 扩展蛋白基因在小麦耐旱性中的功能分析[硕士论文]. 泰安: 山东农业大学.
[31]	Zust T, Grawal A A. 2017. Trade-offs between plant growth and defense against insect herbivory:an emerging mechanistic synthesis. Annual Review of Plant Biology, 68:513-534.

Effects of Overexpression of CsEXPA8 on Growth and Canker Disease Resistance in‘Wanjincheng’Orange

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 8

References 31

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LI Wenjing, SHI Shuo, ZHANG Hao, ZHANG Jiangyan, SONG Sihao, YANG Aizhen, SHEN Yuanyue, GUO Jiaxuan, and GAO Fan, . Effects of Exogenous Nitric Oxide and Putrescine on Drought Resistance of Strawberry Seedlings [J]. Acta Horticulturae Sinica, 2024, 51(9): 2131-2142.
[2]	DONG Liting, QU Rongrong, PANG Shuwei, MO Kaiqin, CHEN Shuang, SHANG Lanyue, and ZOU Xiuping. Expression Characteristics and Function Analysis of CsMEKK1-1 Gene in Response to Huanglongbing in Citrus [J]. Acta Horticulturae Sinica, 2024, 51(9): 2168-2182.
[3]	CHEN Ming, ZHANG Jieru, YANG Hangyun, WANG Yinbao, ZHENG Zhiyuan, ZENG Jiaoke, CHEN Jinyin, FU Yongqi, and XIANG Miaolian. Induction Mechanism of Methyl Jasmonate on Postharvest Navel Orange Fruit Against Blue Mold [J]. Acta Horticulturae Sinica, 2024, 51(9): 2183-2194.
[4]	ZHANG Wenlong, WAN Runchu, ZHENG Ni, CHEN Yan, LAI Hengxin, YU Xin, QIAN Chun, and CAO Li, . ‘Yangguang 1’–a New Monoembryonic Sterile Line of Citrus [J]. Acta Horticulturae Sinica, 2024, 51(9): 2221-2222.
[5]	ZHANG Yong, MA Jianxiang, WEI Chunhua, LI Hao, YANG Jianqiang, and ZHANG Xian. A New Muskmelon Cultivar‘Nongdatian 10’ [J]. Acta Horticulturae Sinica, 2024, 51(9): 2225-2226.
[6]	KUANG Meimei, LI Li, MA Jianwei, LIU Yuan, JIANG Hongfei, LEI Rui, MAN Yuping, WANG Yifan, HUANG Bo, WANG Yanchang, and LIU Shibiao, . Screening of Kiwifruit Canker Resistance-Related Genes Based on the Transcriptome Sequencing Analysis of Hybrids from Actinidia chinensis [J]. Acta Horticulturae Sinica, 2024, 51(8): 1743-1757.
[7]	YANG Jinghui, XU Yuan, XIAO Ting, CHU Shupin, LIU Jixiang, and YAO Kebing, . Resistance of Colletotrichum gloeosporioides Species Complexes from Grape to Azoxystrobin [J]. Acta Horticulturae Sinica, 2024, 51(8): 1906-1912.
[8]	GONG Xiaoya, LI Xian, ZHOU Xingang, and WU Fengzhi. Effect of Rhizosphere Microorganisms Induced by Potato-Onion on Tomato Root-Knot Nematode Disease [J]. Acta Horticulturae Sinica, 2024, 51(8): 1913-1926.
[9]	LI Yadi, WANG Hanxiang, HU Baigeng, YANG Hui, HU Xinxi, XIONG Xingyao, and WANG Wanxing, . Research Progress on Plant Growth Promoting Rhizobacteria to Alleviate Abiotic Stress Tolerance of Horticultural Crops [J]. Acta Horticulturae Sinica, 2024, 51(8): 1964-1976.
[10]	DU Meixia, PANG Shuwei, DONG Liting, MO Kaiqin, HOU Mengyuan, WANG Shuai, and ZOU Xiuping. Research Progress on the Molecular Mechanisms of Interactions Between Candidatus Liberibacter and Citrus [J]. Acta Horticulturae Sinica, 2024, 51(7): 1623-1638.
[11]	SHI Fengyan, WANG Zhidan, ZHANG Xi, WANG Xiuxue, and ZOU Chunlei, . Research Progress on the Mechanism of Pepper Resistance to Phytophthora Blight [J]. Acta Horticulturae Sinica, 2024, 51(7): 1665-1682.
[12]	CAO Huixiang, LUO Xin, LIU Wanrong, GUAN Shuping, WANG Tingting, WU Xiaomeng, GUO Wenwu, and XIE Kaidong, . Evaluation of Fruit Segmental Membrane Development and Mastication in Two Citrus Triploids [J]. Acta Horticulturae Sinica, 2024, 51(6): 1179-1188.
[13]	ZHOU Huizhen, ZHANG Jia, HU Junhua, LI Baixue, CAO Li, YU Xin, WANG Fusheng, ZOU Xiuping, ZHOU Yan. The Role of BAG1 in the Infection of Citrus with Alternaria Brown Spot [J]. Acta Horticulturae Sinica, 2024, 51(5): 956-970.
[14]	YANG Ting, XI Dehui, XIA Ming, LI Jianan. Studies on the Mechanism of α-Momordicin Gene Enhancing Tomato Resistance to Tobacco Mosaic Virus [J]. Acta Horticulturae Sinica, 2024, 51(5): 1126-1136.
[15]	WANG Qing, HU Yan, CHEN Shan, LU Jingwei, ZHENG Yang, TAO Weilin, SUN Xianchao, ZHOU Na, CHEN Guokang. Biocontrol Efficacy and Mechanisms of Streptomyces purpeofuscus CC2-6 Against Chinese Cabbage Clubroot Caused by Plasmodiophora brassicae [J]. Acta Horticulturae Sinica, 2024, 51(5): 1137-1150.