Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (15): 2986-2996.doi: 10.3864/j.issn.0578-1752.2024.15.006

• PLANT PROTECTION • Previous Articles     Next Articles

Screening and Verification of Pepper Host Factors Interacting with the 126 kDa Protein of Pepper Mild Mottle Virus by Yeast Two-Hybrid System

HE Yong(), FAN XiaoZhu, CHEN XinYue, DUAN ShuJing, HU TingTing, XIE RuXue, WANG YuQing, CHEN Jing()   

  1. School of Plant Protection, Anhui Agricultural University, Hefei 230036
  • Received:2024-04-05 Accepted:2024-05-24 Online:2024-08-05 Published:2024-08-05
  • Contact: CHEN Jing

RichHTML

11

PDF

94

Abstract:

【Objective】Pepper mild mottle virus (PMMoV) is one of the major viruses that harm peppers worldwide. The 126 kDa protein is an important pathogenic factor encoded by PMMoV, but its pathogenic mechanism remains unclear. This study aims to screen for pepper host factors that interact with the 126 kDa protein, and to provide a theoretical basis for elucidating the pathogenic mechanism of PMMoV.【Method】Firstly, the bait vector pGBK-126 kDa was constructed using homologous recombination. Using pepper leaves as experimental materials, total RNA was extracted from pepper leaves using the Trizol method, and a pepper yeast cDNA library was prepared. Subsequently, the cDNA library was screened using pGBK-126 kDa, and the screening results were subjected to sequence alignment and bioinformatics functional analysis using NCBI and Uniprot. Based on the alignment and analysis results, host factors that may participate in plant disease resistance pathways were selected, and their full-length CDSs were cloned and constructed into the pGADT7 vector. Yeast two-hybrid (Y2H), BiFC, and LCI were used to further verify the interaction between 126 kDa and host factors. Finally, the role of transiently overexpressed host proteins during PMMoV infection was analyzed.【Result】High-quality pepper RNA was extracted without degradation. A high-quality yeast cDNA library was obtained, and the bait plasmid pGBK-126 kDa was successfully constructed. A total of 18 pepper host factors that interact with the 126 kDa protein were screened. Bioinformatics analysis revealed that these 18 host factors are widely involved in multiple pathways such as plant enzyme systems, regulation of material and energy metabolism, DNA-binding transcription, hormone synthesis, and defense responses. Among them, three host factors (LA2, PDHE1, BXL1) showed interactions with 126 kDa in one-to-one Y2H interaction verification, indicating the reliability of the initial screening results. The interaction between 126 kDa and BXL1 was further verified in vitro and in vivo using BiFC and LCI. Transient overexpression of BXL1 significantly inhibited PMMoV infection.【Conclusion】The pGBK-126 kDa bait plasmid was successfully constructed. Based on this plasmid, 18 interacting host factors were obtained when screening the yeast cDNA library, which are widely involved in multiple pathways of plant life activities. The screening results were verified to be reliable. Among them, BXL1 interacts with 126 kDa both in vitro and in vivo, and can inhibit PMMoV infection. The results can provide a good theoretical and material basis for further exploring of the infection mechanism of PMMoV.

Key words: pepper mild mottle virus (PMMoV), pepper (Capsicum annuum), yeast two-hybrid (Y2H), 126 kDa protein, pathogenic mechanism

Table 1

PCR amplification primers"

引物Primer 序列Sequence (5′-3′)
pGAD-LA2-F CGCTCATATGGCCATGGAGGCCAGTGAATTCatgggtggttgggctatagcg
pGAD-LA2-R GATTCATCTGCAGCTCGAGCTCGATGGATCCctattcccaaataccaacttcc
pGAD-BXL1-F CGCTCATATGGCCATGGAGGCCAGTGAATTCatgagtaaaaaatattccagt
pGAD-BXL1-R GATTCATCTGCAGCTCGAGCTCGATGGATCCctaaaaggaaaagtaggttagt
pGAD-PDHE1-F CGCTCATATGGCCATGGAGGCCAGTGAATTCatggctgctattatacaaggaa
pGAD-PDHE1-R GATTCATCTGCAGCTCGAGCTCGATGGATCCtcactggcaaagctgttcaaca
pGBK-126 kDa-F GAGGAGGACCTGCATATGGCCATGGAGGCCGAATTCatggcttacacacaacaagctacc
pGBK-126 kDa-R ATGCTAGTTATGCGGCCGCTGCAGGTCGACGGATCCctattgagtcgacacatcaactttg

Fig. 1

Symptoms of pepper infected with PMMoV"

Fig. 2

Electrophoresis patterns of total RNA isolated from pepper"

Fig. 3

Electrophoresis patterns of PCR amplification of 126 kDa gene"

Fig. 4

Growth status of yeast containing pGBK-126 kDa and pGBKT7 plasmids on SD/-Trp medium"

Fig. 5

Growth status of yeast co-transforming pGBK-126 kDa and pGADT7 plasmids on different SD dropout media"

Table 2

Eighteen host factors of pepper interacting with PMMoV 126 kDa protein"

GenBank登录号
GenBank accession		 基因注释
Gene annotation		 克隆数
Number of clone
PHT30078.1 AGO 1A蛋白Protein argonaute 1A 2
AVP43435.1 叶绿体诱导的β-淀粉酶1 Chloroplast induced beta amylase 1 1
KAF3682718.1 乙烯反应转录因子9 Ethylene-responsive transcription factor 9 1
XP_016562445.1 乙烯反应转录因子RAP2-12 Ethylene-responsive transcription factor RAP2-12 isoform X2 1
PHT62104.1 p-1, 3-葡聚糖苷酶Glucan endo-1, 3-beta-D-glucosidase 2
XP_006359610.1 预测的酰胺基磷酸核糖基转移酶，叶绿体Predicted amidophosphoribosyl transferase, chloroplastic-like 1
XP_009794582.1 预测的延长因子类似子-2 Predicted elongation factor 2-like 1
XP_016568807.1 可能的异天冬氨酰肽酶L-天冬酰胺酶2 Probable isoaspartyl peptidase L-asparaginase 2 1
XP_016569480.1 类ROH1蛋白Protein ROH1-like 2
PHU30596.1 推定的β-木糖苷酶2 Putative beta-D-xylosidase 2 1
KAF3627957.1 推定的木葡聚糖半乳糖基转移酶类KATAMARI 1 Putative xyloglucan galactosyltransferase KATAMARI 1-like 1
MCE0481271.1 丙酮酸脱氢酶E1 β亚基，叶绿体Pyruvate dehydrogenase E1 component subunit beta-3, chloroplastic 1
NP_001385273.1 RuBisCO小亚基，叶绿体Ribulose bisphosphate carboxylase small subunit, chloroplastic 1
KAF3647305.1 RuBisCO大亚基结合蛋白β亚基，叶绿体RuBisCO large subunit-binding protein subunit beta, chloroplastic 1
AFR41329.1 S-腺苷甲硫氨酸合成酶S-adenosylmethionine synthetase 3
PHT42653.1 mec-8和unc-52蛋白样蛋白2抑制因子Suppressor of mec-8 and unc-52 protein-like protein 2 1
PHU13958.1 伤诱导蛋白1 Wound-induced protein 1 3
TKY48728.1 WRKY转录因子42 WRKY transcription factor 42 1

Fig. 6

Annotation by gene ontology"

Fig. 7

Interaction between 126 kDa and LA2, PDHE1 and BXL1 in the yeast two-hybrid system"

Fig. 8

Interaction between 126 kDa and BXL1 in LCI experiment"

Fig. 9

Interaction between 126 kDa and BXL1 in BiFC experiment"

Fig. 10

The function of transient overexpression of BXL1 in PMMoV infection"

[1]
ZOU Z Y, ZOU X X. Geographical and ecological differences in pepper cultivation and consumption in China. Frontiers in Nutrition, 2021, 8: 718517.
[2]
ZHU X Q, LIU D M, HONG Q C, LU Y F, PEI D L. First report of chili pepper fruit rot caused by Fusarium incarnatum in China. Plant Disease, 2021, 105(10): 3304.
[3]
HAN K L, ZHENG H Y, JI M F, CUI W J, HU S Z, PENG J J, ZHAO J P, LU Y W, LIN L, LIU Y, CHEN J P, YAN F. A single amino acid in coat protein of pepper mild mottle virus determines its subcellular localization and the chlorosis symptom on leaves of pepper. Journal of General Virology, 2020, 101(5): 565-570.

doi: 10.1099/jgv.0.001398 pmid: 32149597
[4]
JI M F, ZHAO J P, HAN K L, CUI W J, WU X Y, CHEN B H, LU Y W, PENG J J, ZHENG H Y, RAO S F, WU G W, CHEN J P, YAN F. Turnip mosaic virus P1 suppresses JA biosynthesis by degrading cpSRP54 that delivers AOCs onto the thylakoid membrane to facilitate viral infection. PLoS Pathogens, 2021, 17(12): e1010108.
[5]
JIAO Y B, ZHAO X X, HAO K Q, GAO X R, XING D, WANG Z P, AN M N, XIA Z H, WU Y H. Characterization of small interfering RNAs derived from pepper mild mottle virus in infected pepper plants by high-throughput sequencing. Virus Research, 2022, 307: 198607.
[6]
ZHANG Z M, CHANG X F, LUO S X, WANG Y H, XUAN S X, ZHAO J J, SHEN S X, MA W, CHEN X P. Transcriptome analysis of two pepper genotypes infected with pepper mild mottle virus. Frontiers in Genetics, 2023, 14: 1164730.
[7]
向本春, 谢浩, 崔星明, 李春, 刘素萍, 席德慧, 尹玉琦. 新疆辣椒轻微斑驳病毒的分离鉴定. 病毒学报, 1994, 10(3): 240-245.
XIANG B C, XIE H, CUI X M, LI C, LIU S P, XI D H, YIN Y Q. Isolation and identification of pepper mid mottle tobamo virus in Xinjiang. Chinese Journal of Virology, 1994, 10(3): 240-245. (in Chinese)
[8]
ZHOU W P, LI Y Y, LI F, TAN G L. First report of natural infection of tomato by pepper mild mottle virus in China. Journal of Plant Pathology, 2021, 103(1): 363.
[9]
OH C, XUN G H, LANE S T, PETROV V A, ZHAO H M, NGUYEN T H. Portable, single nucleotide polymorphism-specific duplex assay for virus surveillance in wastewater. Science of the Total Environment, 2024, 912: 168701.
[10]
邵煜尊, 张万红, 王惠, 王玉洁, 苗圃, 申莉莉, 李莹, 焦裕冰, 杨金广. 辣椒轻斑驳病毒RT-LAMP快速检测方法的建立. 中国蔬菜, 2023(12): 42-48.
SHAO Y Z, ZHANG W H, WANG H, WANG Y J, MIAO P, SHEN L L, LI Y, JIAO Y B, YANG J G. Establishment of a rapid RT-LAMP detection method for pepper mild mottle virus. China Vegetables, 2023(12): 42-48. (in Chinese)
[11]
JIAO Y B, AN M N, LI X D, YU M, ZHAO X X, XIA Z H, WU Y H. Transcriptomic and functional analyses reveal an antiviral role of autophagy during pepper mild mottle virus infection. BMC Plant Biology, 2020, 20(1): 495.
[12]
HAN K L, ZHENG H Y, YAN D K, ZHOU H J, JIA Z X, ZHAI Y S, WU J, LU Y W, WU G W, RAO S F, CHEN J P, PENG J J, QI R D, YAN F. Pepper mild mottle virus coat protein interacts with pepper chloroplast outer envelope membrane protein OMP24 to inhibit antiviral immunity in plants. Horticulture Research, 2023, 10(5): uhad046.
[13]
TSUDA S, KUBOTA K, KANDA A, OHKI T, MESHI T. Pathogenicity of pepper mild mottle virus is controlled by the RNA silencing suppression activity of its replication protein but not the viral accumulation. Phytopathology, 2007, 97(4): 412-420.

doi: 10.1094/PHYTO-97-4-0412 pmid: 18943281
[14]
DAGVADORJ B, OUTRAM M A, WILLIAMS S J, SOLOMON P S. The necrotrophic effector ToxA from Parastagonospora nodorum interacts with wheat NHL proteins to facilitate Tsn1-mediated necrosis. The Plant Journal, 2022, 110(2): 407-418.
[15]
YANG C L, LIU Q Y, PENG M, CHEN X L, ZHU W L, CHEN X H, LI Q Y, ZENG D G, ZHAO Y Z. Penaeus stylirostris densovirus proteins CP and NS1 interact with peritrophin of Litopenaeus vannamei. Fish & Shellfish Immunology, 2020, 106: 357-364.
[16]
宾羽, 张琦, 王春庆, 赵晓春, 宋震, 周常勇. 利用酵母双杂交系统筛选与柑橘黄化脉明病毒CP互作的寄主因子. 中国农业科学, 2023, 56(10): 1881-1892. doi: 10.3864/j.issn.0578-1752.2023.10.006.
BIN Y, ZHANG Q, WANG C Q, ZHAO X C, SONG Z, ZHOU C Y. Screening of the host factors interacting with CP of citrus yellow vein clearing virus by yeast two-hybrid system. Scientia Agricultura Sinica, 2023, 56(10): 1881-1892. doi: 10.3864/j.issn.0578-1752.2023.10.006. (in Chinese)
[17]
HU T, GUO D Q, LI B W, WANG L Q, LIU H, YIN J L, JIN T T, LUAN H X, SUN L, LIU M Z, ZHI H J, LI K. Soybean 40S ribosomal protein S8 (GmRPS8) interacts with 6K1 protein and contributes to soybean susceptibility to soybean mosaic virus. Viruses, 2023, 15(12): 2362.
[18]
MÉNDEZ-LÓPEZ E, DONAIRE L, GOSÁLVEZ B, DÍAZ-VIVANCOS P, SÁNCHEZ-PINA M A, TILSNER J, ARANDA M A. Tomato SlGSTU38 interacts with the PepMV coat protein and promotes viral infection. New Phytologist, 2023, 238(1): 332-348.
[19]
ZONG T X, YIN J L, JIN T T, WANG L Q, LUO M X, LI K, ZHI H J. A DnaJ protein that interacts with soybean mosaic virus coat protein serves as a key susceptibility factor for viral infection. Virus Research, 2020, 281: 197870.
[20]
HAN S H, PARK J S, HAN J Y, GONG J S, PARK C H, KIM J K, SEO E Y, DOMIER L L, HAMMOND J, LIM H S. New Korean isolates of pepper mild mottle virus (PMMoV) differ in symptom severity and subcellular localization of the 126 kDa protein. Virus Genes, 2017, 53(3): 434-445.
[21]
ZHENG K Y, ZHANG R H, WAN Q L, ZHANG G, LU Y W, ZHENG H Y, YAN F, PENG J J, WU J. Pepper mild mottle virus can infect and traffick within Nicotiana benthamiana plants in non-virion forms. Virology, 2023, 587: 109881.
[22]
LEE G E, KIM H J, CHO I S, JEONG R D. First report on natural infection of Nepenthes alata by pepper mild mottle virus in Korea. Journal of Plant Pathology, 2023, 105(4): 1725.
[23]
YU M, LIU H, ZHENG H Y, YAN F, ZHAO X X, XIA Z H, AN M N, WU Y H. Viral sequences required for efficient viral infection differ between two Chinese pepper mild mottle virus isolates. Virus Research, 2019, 267: 9-15.

doi: S0168-1702(19)30175-3 pmid: 31039366
[24]
LI N, ZHANG R, ZHOU J P, HUANG Z X. Structures, biochemical characteristics, and functions of β-xylosidases. Journal of Agricultural and Food Chemistry, 2023, 71(21): 7961-7976.
[25]
FARIAS M D P, ALBUQUERQUE P B S, SOARES P A G, DE SÁ D M A T, VICENTE A A, CARNEIRO-DA-CUNHA M G. Xyloglucan from Hymenaea courbaril var. courbaril seeds as encapsulating agent of L-ascorbic acid. International Journal of Biological Macromolecules, 2018, 107: 1559-1566.
[26]
JEWARIA P K, YU M, LI X J. Cell wall and hormone interplay controls growth asymmetry. Trends in Plant Science, 2021, 26(7): 665-667.

doi: 10.1016/j.tplants.2021.04.003 pmid: 33958277
[27]
YU Z P, ZHANG F, FRIML J, DING Z J. Auxin signaling: Research advances over the past 30 years. Journal of Integrative Plant Biology, 2022, 64(2): 371-392.

doi: 10.1111/jipb.13225
[28]
ZHAO S S, LI Y. Current understanding of the interplays between host hormones and plant viral infections. PLoS Pathogens, 2021, 17(2): e1009242.
[29]
PADMANABHAN M S, GOREGAOKER S P, GOLEM S, SHIFERAW H, CULVER J N. Interaction of the tobacco mosaic virus replicase protein with the Aux/IAA protein PAP1/IAA26 is associated with disease development. Journal of Virology, 2005, 79(4): 2549-2558.

doi: 10.1128/JVI.79.4.2549-2558.2005 pmid: 15681455
[30]
ZHANG H H, LI L L, HE Y Q, QIN Q Q, CHEN C H, WEI Z Y, TAN X X, XIE K L, ZHANG R F, HONG G J, et al. Distinct modes of manipulation of rice auxin response factor OsARF 17 by different plant RNA viruses for infection. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(16): 9112-9121.
[31]
LE X H, LEE C P, MILLAR A H. The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism. The Plant Cell, 2021, 33(8): 2776-2793.
[32]
SGRIGNANI J, CHEN J J, ALIMONTI A, CAVALLI A. How phosphorylation influences E1 subunit pyruvate dehydrogenase: A computational study. Scientific Reports, 2018, 8: 14683.

doi: 10.1038/s41598-018-33048-z pmid: 30279533
[33]
ZHOU W J, NIU Y J, NIE Z W, KIM J Y, XU Y N, YAN C G, CUI X S. Nuclear accumulation of pyruvate dehydrogenase alpha 1 promotes histone acetylation and is essential for zygotic genome activation in porcine embryos. Biochimica et Biophysica Acta. Molecular Cell Research, 2020, 1867(4): 118648.
[34]
LIU Z B, SONG J S, MIAO W, YANG B Z, ZHANG Z Q, CHEN W C, TAN F J, SUO H, DAI X Z, ZOU X X, OU L J. Comprehensive proteome and lysine acetylome analysis reveals the widespread involvement of acetylation in cold resistance of pepper (Capsicum annuum L.). Frontiers in Plant Science, 2021, 12(8): 730489.
[35]
YANG Z, DU J, TAN X, ZHANG H, LI L, LI Y, WEI Z, XU Z, LU Y, CHEN J, SUN Z. Histone deacetylase OsHDA706 orchestrates rice broad-spectrum antiviral immunity and is impeded by a viral effector. Cell Reports, 2024, 43(3): 113838.
[36]
DING B, BELLIZZI M D, NING Y S, MEYERS B C, WANG G L. HDT701, a histone H4 deacetylase, negatively regulates plant innate immunity by modulating histone H4 acetylation of defense-related genes in rice. The Plant Cell, 2012, 24(9): 3783-3794.

doi: 10.1105/tpc.112.101972 pmid: 22968716
[37]
CHEN B, WANG Z C, JIAO M J, ZHANG J, LIU J, ZHANG D M, LI Y B, WANG G N, KE H F, CUI Q X, et al. Lysine 2- hydroxyisobutyrylation- and succinylation-based pathways act inside chloroplasts to modulate plant photosynthesis and immunity. Advanced Science, 2023, 10(27): e2301803.
[38]
BATOOL T, MAKKY E A, JALAL M, YUSOFF M M. A comprehensive review on L-asparaginase and its applications. Applied Biochemistry and Biotechnology, 2016, 178(5): 900-923.

doi: 10.1007/s12010-015-1917-3 pmid: 26547852
[39]
HWANG I S, AN S H, HWANG B K. Pepper asparagine synthetase 1 (CaAS1) is required for plant nitrogen assimilation and defense responses to microbial pathogens. The Plant Journal, 2011, 67(5): 749-762.

doi: 10.1111/j.1365-313X.2011.04622.x pmid: 21535260
[40]
SEIFI H S, DE VLEESSCHAUWER D, AZIZ A, HÖFTE M. Modulating plant primary amino acid metabolism as a necrotrophic virulence strategy. The immune-regulatory role of asparagine synthetasein Botrytis cinerea-tomato interaction. Plant Signaling & Behavior, 2014, 9(2): e27995.
[41]
LIU C Y, TIAN S R, LV X, PU Y D, PENG H R, FAN G J, MA X Z, MA L S, SUN X C. Nicotiana benthamiana asparagine synthetase associates with IP-L and confers resistance against tobacco mosaic virus via the asparagine-induced salicylic acid signalling pathway. Molecular Plant Pathology, 2022, 23(1): 60-77.
[1] JIANG XingLin, YU LianWei, FU Han, AI Niu, CUI YingJun, LI HaoHai, XIA ZiHao, YUAN HongXia, LI HongLian, YANG Xue, SHI Yan. The Transcription Factor NbMYB1R1 Inhibits Viral Infection by Promoting ROS Accumulation [J]. Scientia Agricultura Sinica, 2024, 57(8): 1490-1505.
[2] YU LianWei, JIANG XingLin, YANG LingLing, WANG He, ZHANG YuYang, XIE LiNa, XIA ZiHao, LI HongLian, YANG Xue, SHI Yan. Function of Transcription Factor NbERF RAP2-1 in Cucumber Green Mottle Mosaic Virus Infection [J]. Scientia Agricultura Sinica, 2023, 56(15): 2919-2928.
[3] WANG ShaoLi, TAN WeiPing, YANG YuanYuan, DAI HuiJie, SUN XiaoHui, QIAO Ning, ZHU XiaoPing. Molecular Detection and Identification of Main Viruses on Pepper in Shandong Province [J]. Scientia Agricultura Sinica, 2017, 50(14): 2728-2738.
[4] LUO Wei-yu, ZHU Peng-yang, ZHANG Jie, HU Yong-hao, KONG Hui-hui, LIANG Li-bin, ZHOU Yuan, LI Cheng-jun, JIANG Li, CHEN Hua-lan. Construction of cDNA Library Derived from Human Lung Epithelial Cell Lines and Screening for Host Cellular Proteins Interacting with Influenza Virus Nucleoprotein [J]. Scientia Agricultura Sinica, 2016, 49(22): 4451-4459.
[5] Shi-Qiangg GAO Chenyang He. Transcriptional profile of plant pathogenic bacteria revealed by DNA microarray analysis [J]. Scientia Agricultura Sinica, 2008, 41(5): 1341-1346 .
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd