Skip to main content
SpringerLink
Log in

Overexpressing a NPR1-like gene from Citrus paradisi enhanced Huanglongbing resistance in C. sinensis

  • Original Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

Overexpression of CiNPR4 enhanced resistance of transgenic citrus plants to Huanglongbing by perceiving the salicylic acid and jasmonic acid signals and up-regulating the transcriptional activities of plant–pathogen interaction genes.

Abstract

Developing transgenic citrus plants with enhanced immunity is an efficient strategy to control citrus Huanglongbing (HLB). Here, a nonexpressor of pathogenesis-related gene 1 (NPR1) like gene from HLB-tolerant ‘Jackson’ grapefruit (Citrus paradisi Macf.), CiNPR4, was introduced into ‘Wanjincheng’ orange (Citrus sinensis Obseck). CiNPR4 expression was determined in transgenic citrus plants using quantitative real-time PCR analyses. The Candidatus Liberibacter asiaticus (CLas) pathogen of HLB was successfully transmitted to transgenic citrus plants by grafting infected buds. HLB symptoms developed in transgenic and wild-type (WT) plants by 9 months after inoculation. A CLas population analysis showed that 26.9% of transgenic lines exhibited significantly lower CLas titer levels compared with the CLas-infected WT plants at 21 months after inoculation. Lower starch contents and anatomical aberration levels in the phloem were observed in transgenic lines having enhanced resistance compared with CLas-infected WT plants. CiNPR4 overexpression changed the jasmonic acid, but not salicylic acid, level. Additionally, the jasmonic acid and salicylic acid levels increased after CLas infection. Transcriptome analyses revealed that the enhanced resistance of transgenic plants to HLB resulted from the up-regulated transcriptional activities of plant–pathogen interaction-related genes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • An C, Mou Z (2011) Salicylic acid and its function in plant immunity. J Integr Plant Biol 53:412–428

    Article  CAS  PubMed  Google Scholar 

  • Belasque J, Bassanezi RB, Yamamoto PT, Ayres AJ, Tachibana A, Violante AR, Tank A, Di Giorgi F, Tersi FEA, Menezes GM, Dragone J, Jank RH, Bové JM (2010) Lessons from Huanglongbing management in São Paulo State, Brazil. J Plant Pathol 92:285–302

    Google Scholar 

  • Bové JM (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J Plant Pathol 88:7–37

    Google Scholar 

  • Chen X, Barnaby JY, Sreedharan A, Huang X, Orbović V, Grosser JW, Wang N, Dong X, Song WY (2013) Over-expression of the citrus gene CtNH1 confers resistance to bacterial canker disease. Physiol Mol Plant 84:115–122

    Article  CAS  Google Scholar 

  • Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R (2020) TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant 13(8):1194–1202

    Article  CAS  PubMed  Google Scholar 

  • da Graça JV, Douhan GW, Halbert SE, Keremane ML, Lee RF, Vidalakis G, Zhao H (2016) Huanglongbing: an overview of a complex pathosystem ravaging the world’s citrus. J Integr Plant Biol 58(4):373–387

    Article  PubMed  Google Scholar 

  • Ding Y, Sun T, Ao K, Peng Y, Zhang Y, Li X, Zhang Y (2018) Opposite roles of salicylic acid receptors NPR1 and NPR3/NPR4 in transcriptional regulation of plant immunity. Cell 173:1454–1467

    Article  CAS  PubMed  Google Scholar 

  • Dutt M, Barthe G, Irey M, Grosser J (2015) Transgenic citrus expressing an Arabidopsis NPR1 gene exhibit enhanced resistance against Huanglongbing (HLB; Citrus Greening). PLoS ONE 10(9):e0137134

    Article  PubMed  PubMed Central  Google Scholar 

  • Fu ZQ, Yan S, Saleh A, Wang W, Ruble J, Oka N, Mohan R, Spoel SH, Tada Y, Zheng N, Dong X (2012) NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature 486:228–232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gómez-muñoz N, Velázquez K, Vives MC, Ruiz-ruiz S, Pina JA, Flores R, Moreno P, Guerri J (2017) The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways. Mol Plant Pathol 18(9):1253–1266

    Article  PubMed  Google Scholar 

  • He YR, Chen SC, Peng AH, Zou XP, Xu LZ, Lei TG, Liu XF, Yao LX (2011) Production and evaluation of transgenic sweet orange (Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary shoots. Sci Hort 128:99–107

    Article  CAS  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Le Henanff G, Heitz T, Mestre P, Mutterer J, Walter B, Chong J (2009) Characterization of Vitis vinifera NPR1 homologs involved in the regulation of pathogenesis-related gene expression. BMC Plant Biol 9:54

    Article  PubMed  PubMed Central  Google Scholar 

  • Liu X, Liu Z, Niu X, Xu Q, Yang L (2019) Genome-wide identification and analysis of the NPR1-like gene family in bread wheat and its relatives. Int J Mol Sci 20(23):5974

    Article  CAS  PubMed Central  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆Ct method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Makandar R, Essig JS, Schapaug MA, Trick HN, Shah J (2006) Genetically engineered resistance to Fusarium head blight in wheat by expression of Arabidopsis NPR1. Mol Plant Microbe Interact 19:123–129

    Article  CAS  PubMed  Google Scholar 

  • Mao X, Cai T, Olyarchuk JG, Wei L (2005) Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 21:3787–3793

    Article  CAS  PubMed  Google Scholar 

  • Meur G, Budatha M, Srinivasan T, Kumar KRR, Gupta AD, Kirti PB (2008) Constitutive expression of Arabidopsis NPR1 confers enhanced resistance to the early instars of Spodoptera litura in transgenic tobacco. Physiol Plant 133:765–775

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Ohta M, Matsui K, Hiratsu K, Shinshi H, Ohme-Takagi M (2001) Repression domains of class II ERF transcriptional repressors share an essential motif for active repression. Plant Cell 13:1959–1968

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 Tiss Organ Cult 123:1–13

    Article  CAS  Google Scholar 

  • 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. Vitro Cell Dev-Pl 55:659–667

    Article  Google Scholar 

  • Peraza-Echeverria S, Santamaría JM, Fuentes G, Menéndez-Cerón MDLÁ, Vallejo-Reyna MÁ, Herrera-Valencia VA (2012) The NPR1 family of transcription cofactors in papaya: insights into its structure, phylogeny and expression. Genes Genom 34:379–390

    Article  CAS  Google Scholar 

  • Pilotti M, Brunetti A, Gallelli A, Loreti S (2008) NPR1-like genes from cDNA of rosaceous trees: cloning strategy and genetic variation. Tree Genet Genomes 4:49–63

    Article  Google Scholar 

  • Qiu W, Soares J, Pang Z, Huang Y, Sun Z, Wang N, Grosser J, Dutt M (2020) Potential mechanisms of AtNPR1 mediated resistance against Huanglongbing (HLB) in citrus. Int J Mol Sci 21(6):2009

    Article  CAS  PubMed Central  Google Scholar 

  • Shao Y, Zhang H, He H, Cheng B, Xiang Y (2013) Molecular cloning and characterization of orthologues of NPR1 gene from poplar. J Phytopathol 161:35–42

    Article  CAS  Google Scholar 

  • Shi Z, Zhang Y, Maximova SN, Guiltinan MJ (2013) TcNPR3 from Theobroma cacao functions as a repressor of the pathogen defense response. BMC Plant Biol 13:204

    Article  PubMed  PubMed Central  Google Scholar 

  • Shu L, Liao J, Lin N, Chung C (2018) Identification of a strawberry NPR-like gene involved in negative regulation of the salicylic acid-mediated defense pathway. PLoS ONE 13(10):e0205790

    Article  PubMed  PubMed Central  Google Scholar 

  • Spoel SH, Koornneef A, Claessens SMC, Korzelius JP, Pelt JAV, Mueller MJ, Buchala AJ, Métraux JP, Brown R, Kazan K, Loon LCV, Dong X, Pieterse CMJ (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 15:760–770

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sticher L, MauchMani B, Metraux JP (1997) Systemic acquired resistance. Annu Rev Phytopathol 35:235–270

    Article  CAS  PubMed  Google Scholar 

  • Wally O, Jayaraj J, Punja ZK (2009) Broad-spectrum disease resistance to necrotrophic and biotrophic pathogens in transgenic carrots (Daucus carota L.) expressing an Arabidopsis NPR1 gene. Planta 231:131–141

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Zhou L, Yu X, Stover E, Luo F, Duan Y (2016) Transcriptome profiling of Huanglongbing (HLB) tolerant and susceptible citrus plants reveals the role of basal resistance in HLB tolerance. Front Plant Sci 7:1–13

    PubMed  PubMed Central  Google Scholar 

  • Wang L, Guo Z, Zhang Y, Wang Y, Yang G, Yang L, Wang L, Wang R, Xie Z (2017) Overexpression of LhSorNPR1, a NPR1-like gene from the oriental hybrid lily ‘Sorbonne’, conferred enhanced resistance to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis. Physiol Mol Biol Plants 23(4):793–808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009) Jalview version 2–A multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu Y, Zhang D, Chu JY, Boyle P, Wang Y, Brindle ID, De Luca V, Després C (2012) The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid. Cell Rep 1:639–647

    Article  CAS  PubMed  Google Scholar 

  • Yamada S, Kano A, Tamaoki D, Miyamoto A, Shishido H, Miyoshi S, Taniguchi S, Akimitsu K, Gomi K (2012) Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice. Plant Cell Physiol 53(12):2060–2072

    Article  CAS  PubMed  Google Scholar 

  • Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11:R14

    Article  PubMed  PubMed Central  Google Scholar 

  • Yuan Y, Zhong S, Li Q, Zhu Z, Lou Y, Wang L, Wang J, Wang M, Li Q, Yang D, He Z (2007) Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility. Plant Biotechnol J 5:313–324

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Cheng YT, Qu N, Zhao Q, Bi D, Li X (2006) Negative regulation of defense responses in Arabidopsis by two NPR1 paralogs. Plant J 48:647–656

    Article  CAS  PubMed  Google Scholar 

  • Zhang X, Francis MI, Dawson WO, Graham JH, Orbović V, Triplett EW, Mou Z (2010) Over-expression of the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker. Eur J Plant Pathol 128:91–100

    Article  CAS  Google Scholar 

  • Zou X, Jiang X, Xu L, Lei T, Peng A, He Y, Yao L, Chen S (2017) Transgenic citrus expressing synthesized cecropin B genes in the phloem exhibits decreased susceptibility to Huanglongbing. Plant Mol Biol 93:341–353

    Article  CAS  PubMed  Google Scholar 

  • Zou X, Bai X, Wen Q, Xie Z, Wu L, Peng A, He Y, Xu L, Chen S (2019) Comparative analysis of tolerant and susceptible citrus reveals the role of methyl salicylate signaling in the response to Huanglongbing. J Plant Growth Regul 38:1516–1528

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the grants from the National Key R&D Program of China (2018YFD0201500), the Key-Area Research and Development Program of Guangdong Province (2018B020202009), Science and Technology Major Project of Guangxi (Gui Ke AA18118046-6) and the Earmarked Fund for China Agriculture Research System (CARS-26). We thank Lesley Benyon, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

Author information

Authors and Affiliations

  1. Citrus Research Institute, Southwest University, National Citrus Engineering Research Center, Chongqing, 400712, People’s Republic of China

    Aihong Peng, Xiuping Zou, Yongrui He, Shanchun Chen, Xiaofeng Liu, Jingyun Zhang, Qingwen Zhang, Zhu Xie, Junhong Long & Xiaochun Zhao

Authors
  1. Aihong Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiuping Zou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yongrui He
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shanchun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaofeng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jingyun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Qingwen Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zhu Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Junhong Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xiaochun Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

AP constructed the plasmid, performed the resistance evaluation of transgenic plants and wrote the manuscript. XZ performed the RNA-seq analysis. YH and SC performed citrus transformations and analyzed the GUS activities of regenerants. XL performed the phylogenetic analysis and amino acid sequence alignment. JZ performed the starch content analysis. QZ and ZX observed the anatomical phloem structures. JL performed the PCR and expression analyses. XZ designed the experiments and revised the manuscript. All the authors have read and approved the manuscript.

Corresponding author

Correspondence to Xiuping Zou.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Communicated by Leena Tripathi.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Peng, A., Zou, X., He, Y. et al. Overexpressing a NPR1-like gene from Citrus paradisi enhanced Huanglongbing resistance in C. sinensis. Plant Cell Rep 40, 529–541 (2021). https://doi.org/10.1007/s00299-020-02648-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-020-02648-3

Keywords

Search

Navigation