Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (16): 3182-3191.doi: 10.3864/j.issn.0578-1752.2024.16.007

• PLANT PROTECTION • Previous Articles     Next Articles

Optimization and Application of Rapid Evaluation System for Citrus Huanglongbing Resistance Mediated by Agrobacterium rhizogenes

CAO Peng1(), ZHOU JinHuan1, WANG XinLiang1, LI ChuXin1, LI JiaXIN1, JIANG Pei2, LIU JinXiang1, SONG Zhen1()   

  1. 1 Citrus Research Institute, Southwest University, Chongqing 400712
    2 National Agricultural Technology Extension and Service Center, Beijing 100125
  • Received:2024-04-20 Accepted:2024-06-13 Online:2024-08-16 Published:2024-08-27
  • Contact: SONG Zhen

RichHTML

17

PDF

140

Abstract:

【Objective】Citrus Huanglongbing (HLB) is a destructive citrus disease primarily caused by Candidatus Liberibacter asiaticus (CLas). The objective of this research is to investigate the growth pattern of CLas in citrus hairy roots at the initial stage of infection, optimize the rapid evaluation system of Agrobacterium rhizogenes-mediated HLB resistance and apply it to the resistance evaluation of antimicrobial peptides.【Method】A plant expression vector, pGNGM1300, was employed to transform different citrus varieties via the hairy root transformation system, which was mediated by A. rhizogenes K599. Subsequently, varieties with rapid induction and high transformation rate were then selected. Following inoculation with CLas, the presence of CLas in hairy roots was consistently demonstrated through the use of real-time quantitative PCR (qPCR), which allowed for the monitoring of its growth pattern. Further, the optimization of the HLB resistance evaluation system permitted the HLB resistance evaluation of antimicrobial peptides to be conducted via (RT-) qPCR analysis of CLas and related genes, quantification of callose deposition, microstructure observation, and symptom assessment.【Result】Among the 11 tested citrus varieties, citron (Citrus medica) exhibited the fastest induction of hairy roots (15 d), accompanied by the highest induction rate (73.75%) and a substantial transgene positivity rate (53.54%) in hairy roots. The results of regular qPCR monitoring indicated that CLas began to colonize roots on 20-30 days post-inoculation (dpi). At 30-50 dpi, there was a notable increase in the CLas content. The content of the CLas exhibited a slight fluctuation at a range of 60 to 120 dpi, though the discrepancy with 50 dpi was not statistically significant. The results of the antimicrobial peptide resistance evaluation, conducted on MaSAMP (stable antimicrobial peptide), HBD-4 (homo sapiens defensin beta 4), and CB (cecropin B), revealed that at 50-120 dpi, the content of CLas in both MaSAMP- and CB-expressing plants was significantly lower than that of the control. However, except that at 90 dpi, the CLas content was also significantly lower in HBD-4-expressing plants than the control. The callose content in all three plants expressing antimicrobial peptides was significantly lower than that of the control (60 dpi). There was no visible cell wall thickening in the phloem, and no significant deposition of starch grains or callus was observed. Furthermore, none of the plants exhibited signs of root death at 90 dpi.【Conclusion】This study elucidates the growth dynamics of CLas in citrus hairy roots during the early stages of infection and optimizes a rapid scoring system for A. rhizogenes-mediated HLB resistance. In addition, the results show that overexpression of MaSAMP, HBD-4 and CB effectively suppresses CLas proliferation, reduces callose deposition and alleviates HLB symptoms. These findings have the potential to be used in the prevention and control of HLB.

Key words: citrus Huanglongbing, antimicrobial peptide, Agrobacterium rhizogenes, hairy root transformation, resistance evaluation

Table 1

Information on the main primers used in this study"

引物名称Primer name 引物序列Primer sequence (5′-3′) 用途Usage
CLas16S-F TGAGTGCTAGCTGTTGGGTG CLas16S基因的qPCR分析
qPCR analysis for CLas16S gene
CLas16S-R CTGCGCGTTGCATCGAATTA
Cs18S-F AATTGTTGGTCTTCAACGAGGAA 柑橘内参基因
Citrus reference gene
Cs18S-R AAAGGGCAGGGACGTAGTCAA
CsCalS7-qF GACGCCTAACCGAGTACCTGC CsCalS7的qPCR分析
qPCR analysis for CsCalS7
CsCalS7-qR GTGCAGCTGGTGATCCATCA
CsPP2B15-qF GCTCTGAGATCTAGGGTTTCCG CsPP2B15的qPCR分析[24]
qPCR analysis for CsPP2B15
CsPP2B15-qR CCCCTTTCAGATGCTGACCC
2OG oxygenase-qF CGAAAGCACATGATGGAAAG 2OG oxygenase的qPCR分析[24]
qPCR analysis for 2OG oxygenase
2OG oxygenase-qR GCCACGAGTGAAGAAATGGT

Fig. 1

Induction hairy roots of citrus varieties mediated by A. rhizogenes K599"

Table 2

Hairy root induction and transformation of citrus varieties mediated by A. rhizogenes"

品种
Variety		 枝条总数
Total number of branches		 生根枝数
Number of branches with hairy roots		 生根时间
Hairy root
induction time (d)		 生根总数
Total number
of hairy roots		 阳性根数
Total number of
positive hairy
roots		 生根率
Hairy root induction rate (%)		 阳性根率
Hairy root
positive
rate (%)
锦橙C. sinensis 60 13 30 58 2 21.67 3.44
尤力克柠檬C. limon 262 179 20 722 313 68.32 43.35
香橼C. medica 160 118 15 480 257 73.75 53.54
枳壳P. trifoliata 40 6 60 19 9 15.00 47.36
卡里佐枳橙
C. sinensis×P. trifoliata		 40 14 30 53 8 35.00 15.09
粗柠檬C. limonia 40 29 20 116 46 72.50 39.65
墨西哥莱檬C. aurantiifolia 40 25 20 267 76 62.50 28.46
摩洛哥酸橙C. aurantium 40 20 25 88 48 50.00 54.54
甜橙C. sinensis 40 6 60 27 14 15.00 51.85
沙田柚* C. grandis 345 5 180 11 4 1.45 36.37
纽荷尔脐橙* C. sinensis 385 24 120 40 15 6.23 37.50
锦橙* C. sinensis 50 10 60 13 10 20.00 76.92

Table 3

Detection of CLas in hairy roots of C. medica by qPCR"

处理
Treatment		 植株编号
Plant number		 20 dpi 30 dpi 40 dpi
CLas Ct18S CLas Ct18S CLas Ct18S
pGNGM1300 cp2333-3 37.87 18.43 35.43 19.63 28.01 17.08
cp2333-5 38.87 17.15 36.61 19.06 31.72 18.87
cp2333-9 35.51 19.48 33.79 18.04 29.4 16.31
cp2333-12 36.96 18.77 31.68 17.64 29.41 17.62
cp2333-16 37.49 17.72 35.84 18.67 28.53 18.79
cp2333-17 37.71 18.57 33.31 17.86 27.21 18.94
CLas阳性对照
Positive control		 +CK 22.89 16.11 22.56 15.63 21.81 15.05
CLas阴性对照
Negative control		 -CK — 17.61 — 17.47 — 18.06

Fig. 2

(RT-) qPCR detection of CLas and related genes in transgenic hairy roots of C. medica"

Fig. 3

The effects of the antimicrobial peptide on CLas and related genes in transgenic hairy roots of C. medica"

Fig. 4

Callose content detection in transgenic hairy roots of C. medica"

Fig. 5

Observation of microscopic structure and phenotype of transgenic hairy roots of C. medica"

[1]
BOVÉ J M. Huanglongbing: A destructive, newly-emerging, century- old disease of citrus. Journal of Plant Pathology, 2006, 88(1): 7-37.
[2]
林孔湘. 柑桔黄梢(黄龙)病研究 Ⅱ. 关于病原的探讨. 植物病理学报, 1956, 2(1): 13-42.
LIN K X. Research on citrus Huanglongbing Ⅱ. Discussion on the pathogen. Acta Phytopathologica Sinica, 1956, 2(1): 13-42. (in Chinese)
[3]
ARITUA V, ACHOR D, GMITTER F G, ALBRIGO G, WANG N. Transcriptional and microscopic analyses of citrus stem and root responses to Candidatus Liberibacter asiaticus infection. PLoS ONE, 2013, 8(9): e73742.
[4]
郭文武, 邓秀新. 柑桔黄龙病及其抗性育种研究. 农业生物技术学报, 1998(1): 37-41.
GUO W W, DENG X X. Research on citrus Huanglongbing and its resistance breeding. Journal of Agricultural Biotechnology, 1998(1): 37-41. (in Chinese)
[5]
彭爱红. NPR和抗菌基因对柑橘黄龙病的抗性研究[D]. 重庆: 西南大学, 2021.
PENG A H. Development of transgenic HLB resistance in citrus with NPR and antimicrobial genes[D]. Chongqing: Southwest University, 2021. (in Chinese)
[6]
MAHLAPUU M, HÅKANSSON J, RINGSTAD L, BJÖRN C. Antimicrobial peptides: An emerging category of therapeutic agents. Frontiers in Cellular and Infection Microbiology, 2016, 6: 194.

doi: 10.3389/fcimb.2016.00194 pmid: 28083516
[7]
SOM A, VEMPARALA S, IVANOV I, TEW G N. Synthetic mimics of antimicrobial peptides. Biopolymers, 2008, 90(2): 83-93.

doi: 10.1002/bip.20970 pmid: 18314892
[8]
BAHAR A, REN D. Antimicrobial peptides. Pharmaceuticals, 2013, 6(12): 1543-1575.

doi: 10.3390/ph6121543 pmid: 24287494
[9]
AMSO Z, HAYOUKA Z. Antimicrobial random peptide cocktails: A new approach to fight pathogenic bacteria. Chemical Communications, 2019, 55(14): 2007-2014.

doi: 10.1039/c8cc09961h pmid: 30688322
[10]
DEHSORKHI A, CASTELLETTO V, HAMLEY I W. Self-assembling amphiphilic peptides. Journal of Peptide Science, 2014, 20(7): 453-467.

doi: 10.1002/psc.2633 pmid: 24729276
[11]
LEE D L, HODGES R S. Structure-activity relationships of de novo designed cyclic antimicrobial peptides based on gramicidin S. Biopolymers, 2003, 71(1): 28-48.
[12]
HUANG C Y, ARAUJO K, SÁNCHEZ J N, KUND G, TRUMBLE J, ROPER C, GODFREY K E, JIN H L. A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(6): e2019628118.
[13]
GARCÍA J R C, KRAUSE A, SCHULZ S, RODRÍGUEZ-JIMÉNEZ F J, KLÜVER E, ADERMANN K, FORSSMANN U, FRIMPONG- BOATENG A, BALS R, FORSSMANN W G. Human β-defensin 4: A novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity. The FASEB Journal, 2001, 15(10): 1819-1821.
[14]
YANAGI S, ASHITANI J, ISHIMOTO H, DATE Y, MUKAE H, CHINO N, NAKAZATO M. Isolation of human β-defensin-4 in lung tissue and its increase in lower respiratory tract infection. Respiratory Research, 2005, 6(1): 130.
[15]
张清杰, 张景宁, 黄自然, 谭石慈, 郭周仪. 柞蚕抗菌肽对柑桔黄龙病及溃疡病病原菌的杀菌作用. 蚕业科学, 1995, 21(2): 77-81.
ZHANG Q J, ZHANG J N, HUANG Z R, TAN S C, GUO Z Y. The bactericidal effect of Antherea pernyi antibacterial peptides on pathogenic bacteria of citrus Huanglongbing and citrus canker. Acta Sericologica Sinica, 1995, 21(2): 77-81. (in Chinese)
[16]
ZOU X P, JIANG X Y, XU L Z, LEI T G, PENG A H, HE Y R, YAO L X, CHEN S C. Transgenic citrus expressing synthesized cecropin B genes in the phloem exhibits decreased susceptibility to Huanglongbing. Plant Molecular Biology, 2017, 93(4): 341-353.
[17]
张琦, 段玉, 苏越, 蒋琪琪, 王春庆, 宾羽, 宋震. 基于柑橘叶斑驳病毒的表达载体构建及应用. 中国农业科学, 2022, 55(22): 4398-4407. doi: 10.3864/j.issn.0578-1752.2022.22.006.
ZHANG Q, DUAN Y, SU Y, JIANG Q Q, WANG C Q, BIN Y, SONG Z. Construction and application of expression vector based on citrus leaf blotch virus. Scientia Agricultura Sinica, 2022, 55(22): 4398-4407. doi: 10.3864/j.issn.0578-1752.2022.22.006. (in Chinese)
[18]
MA H, MENG X, XU K, LI M, GMITTER F G, LIU N G, GAI Y P, HUANG S Y, WANG M, WANG N, XU H R, LIU J H, SUN X P, DUAN S. Highly efficient hairy root genetic transformation and applications in citrus. Frontiers in Plant Science, 2022, 13: 1039094.
[19]
XIAO Y X, DUTT M, MA H, XIAO C, TONG Z, WANG Z Q, HE X J, SUN Z H, QIU W M. Establishment of an efficient root mediated genetic transformation method for gene function verification in citrus. Scientia Horticulturae, 2023, 321: 112298.
[20]
WANG M, QIN Y Y, WEI N N, XUE H Y, DAI W S. Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis. Frontiers in Plant Science, 2023, 14: 1293374.
[21]
IRIGOYEN S, RAMASAMY M, PANT S, NIRAULA P, BEDRE R, GURUNG M, ROSSI D, LAUGHLIN C, GORMAN Z, ACHOR D, et al. HPlant hairy roots enable high throughput identification of antimicrobials against Candidatus Liberibacter spp. Nature Communications, 2020, 11(1): 5802.
[22]
RAMASAMY M, DOMINGUEZ M M, IRIGOYEN S, PADILLA C S, MANDADI K K. Rhizobium rhizogenes-mediated hairy root induction and plant regeneration for bioengineering citrus. Plant Biotechnology Journal, 2023, 21(9): 1728-1730.

doi: 10.1111/pbi.14096 pmid: 37314751
[23]
CIFUENTES-ARENAS J C, BEATTIE G A C, PEÑA L, LOPES S A. Murraya paniculata and Swinglea glutinosa as short-term transient hosts of ‘Candidatus Liberibacter asiaticus’ and implications for the spread of Huanglongbing. Phytopathology, 2019, 109(12): 2064-2073.
[24]
文庆利, 谢竹, 吴柳, 何永睿, 陈善春, 邹修平. 柑橘响应黄龙病侵染的韧皮部蛋白2基因CsPP2B15的克隆与表达分析. 园艺学报, 2018, 45(12): 2347-2357.

doi: 10.16420/j.issn.0513-353x.2018-0377
WEN Q L, XIE Z, WU L, HE Y R, CHEN S C, ZOU X P. Clone and expression analysis of the citrus phloem protein 2 gene CsPP2B15 responding to Huanglongbing infection in citrus. Acta Horticulturae Sinica, 2018, 45(12): 2347-2357. (in Chinese)
[25]
钟晰. 马蜂柑响应黄龙病菌侵染前期与后期的转录组和蛋白组学研究[D]. 重庆: 西南大学, 2018.
ZHONG X. Transcriptomic and proteomic analysis of Citrus hystrix responses to ‘Candidatus Liberibacter asiaticus’ in early and late stage of infection[D]. Chongqing: Southwest University, 2018. (in Chinese)
[26]
ZHOU L L, WANG Y L, WANG P L, WANG C L, WANG J M, WANG X F, CHENG H M. Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene editing analysis in cotton. Frontiers in Plant Science, 2022, 13: 1059404.
[27]
CHENG Y Y, WANG X L, CAO L, JI J, LIU T F, DUAN K X. Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean. Plant Methods, 2021, 17(1): 73.
[28]
ISHIDA Y, HIEI Y, KOMARI T. Agrobacterium-mediated transformation of maize. Nature Protocols, 2007, 2(7): 1614-1621.

doi: 10.1038/nprot.2007.241 pmid: 17585302
[29]
PENG A H, ZOU X P, HE Y R, CHEN S C, LIU X F, ZHANG J Y, ZHANG Q W, XIE Z, LONG J H, ZHAO X C. Overexpressing a NPR1-like gene from Citrus paradisi enhanced Huanglongbing resistance in C. sinensis. Plant Cell Reports, 2021, 40(3): 529-541.
[30]
LEI T G, HE Y R, ZOU X P, WANG X F, FU S M, PENG A H, XU L Z, YAO L X, CHEN S C, ZHOU C Y. A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro. Journal of Integrative Agriculture, 2022, 21(6): 1683-1693.
[31]
谢竹. 柑橘黄龙病菌在根和叶中与寄主互作的比较分析[D]. 重庆: 西南大学, 2020.
XIE Z. Comparative analysis of the interaction of Candidatus Liberibacter asiaticus with root and leaf of host[D]. Chongqing: Southwest University, 2020. (in Chinese)
[1] LI ShengYou, WANG ChangLing, YAN ChunJuan, ZHANG LiJun, SUN XuGang, CAO YongQiang, WANG WenBin, SONG ShuHong. Evaluation of Drought Resistance in Soybean Germplasm and Identification of Candidate Drought-Resistant Genes [J]. Scientia Agricultura Sinica, 2024, 57(10): 1857-1869.
[2] FAN JunQiang, WU JunYan, LIU LiJun, MA Li, YANG Gang, PU YuanYuan, LI XueCai, SUN WanCang. Correlation Between Stomatal Characteristics and Cold Resistance of Brassica napus L. [J]. Scientia Agricultura Sinica, 2023, 56(4): 599-618.
[3] LI li, SUN ling, ZHANG JinHua, ZOU XiaoWei, SUN Hui, REN JinPing, JIANG ZhaoYuan, LIU XiaoMei. Evaluation of Resistance and Analysis of Utilization Value of the Major Japonica Rice Varieties in Jilin Province Based on the Physiological Race Variation of Magnaporthe oryzae [J]. Scientia Agricultura Sinica, 2023, 56(22): 4441-4452.
[4] ZHANG YaLing, GAO Qing, ZHAO Yuhan, LIU Rui, FU Zhongju, LI Xue, SUN Yujia, JIN XueHui. Evaluation of Rice Blast Resistance and Genetic Structure Analysis of Rice Germplasm in Heilongjiang Province [J]. Scientia Agricultura Sinica, 2022, 55(4): 625-640.
[5] HUANG JiaQuan,LI Li,WU FengNian,ZHENG Zheng,DENG XiaoLing. Proliferation of Two Types Prophage of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and their Pathogenicity [J]. Scientia Agricultura Sinica, 2022, 55(4): 719-728.
[6] ZHANG Qi,DUAN Yu,SU Yue,JIANG QiQi,WANG ChunQing,BIN Yu,SONG Zhen. Construction and Application of Expression Vector Based on Citrus Leaf Blotch Virus [J]. Scientia Agricultura Sinica, 2022, 55(22): 4398-4407.
[7] LI ZhenXi,LI WenTing,HUANG JiaQuan,ZHENG Zheng,XU MeiRong,DENG XiaoLing. Detection of ‘Candidatus Liberibacter asiaticus’ by Membrane Adsorption Method Combined with Visual Loop-Mediated Isothermal Amplification [J]. Scientia Agricultura Sinica, 2022, 55(1): 74-84.
[8] ZHAO Ke,ZHENG Lin,DU MeiXia,LONG JunHong,HE YongRui,CHEN ShanChun,ZOU XiuPing. Response Characteristics of Plant SAR and Its Signaling Gene CsSABP2 to Huanglongbing Infection in Citrus [J]. Scientia Agricultura Sinica, 2021, 54(8): 1638-1652.
[9] CUI YiPing,PENG AiTian,SONG XiaoBing,CHENG BaoPing,LING JinFeng,CHEN Xia. Investigation on Occurrence of Citrus Huanglongbing and Virus Diseases, and Prophage Genetic Diversity of Huanglongbing Pathogen in Meizhou, Guangdong [J]. Scientia Agricultura Sinica, 2020, 53(8): 1572-1582.
[10] CaiLing TENG,Xi ZHONG,HaoDi WU,Yan HU,ChangYong ZHOU,XueFeng WANG. Biologic and Transcriptomic Analysis of Citrus hystrix Responses to ‘Candidatus Liberibacter asiaticus’ at Different Infection Stages [J]. Scientia Agricultura Sinica, 2020, 53(7): 1368-1380.
[11] GUAN FangNian,LONG Li,YAO FangJie,WANG YuQi,JIANG QianTao,KANG HouYang,JIANG YunFeng,LI Wei,DENG Mei,LI Hao,CHEN GuoYue. Evaluation of Resistance to Stripe Rust and Molecular Detection of Important Known Yr Gene(s) of 152 Chinese Wheat Landraces from the Huang-huai-hai [J]. Scientia Agricultura Sinica, 2020, 53(18): 3629-3637.
[12] CHEN WenFeng,WANG HongFang,LIU ZhenGuo,ZHANG WeiXing,CHI XuePeng,XU BaoHua. Recombinant Expression and Antimicrobial Activity of Apidaecin in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2019, 52(4): 767-776.
[13] QU Qing,LI LiNa,LIU Jun,WANG ShaoXin,CAO ZhiYan,DONG JinGao. Resistance Evaluation of Some Commonly Used Maize Germplasm Resources to Fusarium Diseases in China [J]. Scientia Agricultura Sinica, 2019, 52(17): 2962-2971.
[14] HUANG Liang, LIU TaiGuo, XIAO XingZhi, QU ChunYan, LIU Bo, GAO Li, LUO PeiGao, CHEN WanQuan. Evaluation of Stripe Rust Resistance and Molecular Detection of Yr Genes of 79 Wheat Varieties (Lines) in China [J]. Scientia Agricultura Sinica, 2017, 50(16): 3122-3134.
[15] WU Yue, SU Hua-nan, HUANG Ai-jun, ZHOU Yan, LI Zhong-an, LIU Jin-xiang, ZHOU Chang-yong. Effect of Candidatus Liberibacter asiaticus Infection on Carbohydrate Metabolism in Citrus sinensis [J]. Scientia Agricultura Sinica, 2015, 48(1): 63-72.
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