Overexpressing GH3.1 and GH3.1L reduces susceptibility to Xanthomonas citri subsp. citri by repressing auxin signaling in citrus (Citrus sinensis Osbeck)

Autoři: Xiuping Zou aff001;  Junhong Long aff001;  Ke Zhao aff001;  Aihong Peng aff001;  Min Chen aff001;  Qin Long aff001;  Yongrui He aff001;  Shanchun Chen aff001
Působiště autorů: Citrus Research Institute, Chinese Academy of Agricultural Sciences and National Center for Citrus Variety Improvement, Chongqing, P. R. China aff001;  Citrus Research Institute, Southwest University, Chongqing, P. R. China aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0220017


The auxin early response gene Gretchen Hagen3 (GH3) plays dual roles in plant development and responses to biotic or abiotic stress. It functions in regulating hormone homeostasis through the conjugation of free auxin to amino acids. In citrus, GH3.1 and GH3.1L play important roles in responding to Xanthomonas citri subsp. citri (Xcc). Here, in Wanjingcheng orange (Citrus sinensis Osbeck), the overexpression of CsGH3.1 and CsGH3.1L caused increased branching and drooping dwarfism, as well as smaller, thinner and upward curling leaves compared with wild-type. Hormone determinations showed that overexpressing CsGH3.1 and CsGH3.1L decreased the free auxin contents and accelerated the Xcc-induced decline of free auxin levels in transgenic plants. A resistance analysis showed that transgenic plants had reduced susceptibility to citrus canker, and a transcriptomic analysis revealed that hormone signal transduction-related pathways were significantly affected by the overexpression of CsGH3.1 and CsGH3.1L. A MapMan analysis further showed that overexpressing either of these two genes significantly downregulated the expression levels of the annotated auxin/indole-3-acetic acid family genes and significantly upregulated biotic stress-related functions and pathways. Salicylic acid, jasmonic acid, abscisic acid, ethylene and zeatin levels in transgenic plants displayed obvious changes compared with wild-type. In particular, the salicylic acid and ethylene levels involved in plant resistance responses markedly increased in transgenic plants. Thus, the overexpression of CsGH3.1 and CsGH3.1L reduces plant susceptibility to citrus canker by repressing auxin signaling and enhancing defense responses. Our study demonstrates auxin homeostasis’ potential in engineering disease resistance in citrus.

Klíčová slova:

Auxins – Citrus – Gene expression – Genetically modified plants – Hyperexpression techniques – Leaves – Plant cell walls – Transcription factors


1. Brunings AM, Gabriel DW. Xanthomonas citri: breaking the surface. Molecular Plant Pathology. 2003;4(3):141–57. doi: 10.1046/j.1364-3703.2003.00163.x 20569374.

2. Mendonça LBD, Zambolim L, Badel JL. Bacterial citrus diseases: Major threats and recent progress. Journal of Bacteriology & Mycology. 2017;5:340–50. doi: 10.15406/jbmoa.2017.05.00143

3. Hu Y, Zhang JL, Jia HG, Sosso D, Li T, Frommer WB, et al. 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–E9. doi: 10.1073/pnas.1313271111 24474801

4. Li Z, Zou L, Ye G, Xiong L, Ji Z, Zakria M, et al. A potential disease susceptibility gene CsLOB of citrus is targeted by a major virulence effector PthA of Xanthomonas citri subsp. citri. Molecular plant. 2014;7(5):912–5. doi: 10.1093/mp/sst176 24398629

5. Cernadas RA, Benedetti CE. Role of auxin and gibberellin in citrus canker development and in the transcriptional control of cell-wall remodeling genes modulated by Xanthomonas axonopodis pv. citri. Plant Science. 2009;177(3):190–5. doi: 10.1016/j.plantsci.2009.05.006

6. Peng AH, Chen SC, Lei TG, Xu LZ, He YR, Wu L, et al. Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant biotechnology journal. 2017;15:1509–19. doi: 10.1111/pbi.12733 28371200

7. Jia H, Zhang Y, Orbovic V, Xu J, White F, Jones J, et al. Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant biotechnology journal. 2017;15:817–23. doi: 10.1111/pbi.12677 27936512.

8. Glickmann E, Gardan L, Jacquet S, Hussain S, Elasri M, Petit A, et al. Auxin production is a common feature of most pathovars of Pseudomonas syringae. Molecular Plant-Microbe Interactions. 1998;11(2):156–62. doi: 10.1094/MPMI.1998.11.2.156 9450337

9. Kazan K, Manners JM. Linking development to defense: auxin in plant–pathogen interactions. Trends in Plant Science. 2009;14(7):373–82. doi: 10.1016/j.tplants.2009.04.005 19559643

10. Chen ZY, Agnew JL, Cohen JD, He P, Shan LB, Sheen J, et al. Pseudomonas syringae type III effector AvrRpt2 alters Arabidopsis thaliana auxin physiology. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(50):20131–6. doi: 10.1073/pnas.0704901104 18056646

11. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, et al. A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science. 2006;312(5772):436–9. doi: 10.1126/science.aae0382 16627744.

12. Truman WM, Bennett MH, Turnbull CGN, Grant MR. Arabidopsis auxin mutants are compromised in systemic acquired resistance and exhibit aberrant accumulation of various indolic compounds. Plant physiology. 2010;152(3):1562–73. doi: 10.1104/pp.109.152173 20081042

13. Zhang Z, Li Q, Li Z, Staswick PE, Wang M, Zhu Y, et al. Dual regulation role of GH3.5 in salicylic acid and auxin signaling during Arabidopsis-Pseudomonas syringae interaction. Plant physiology. 2007;145(2):450–64. doi: 10.1104/pp.107.106021 17704230

14. Wang D, Pajerowska-Mukhtar K, Culler AH, Dong XN. Salicylic acid inhibits pathogen growth in plants through repression of the auxin signaling pathway. Current Biology. 2007;17(20):1784–90. doi: 10.1016/j.cub.2007.09.025 17919906

15. Fu J, Liu HB, Li Y, Yu HH, Li XH, Xiao JH, et al. Manipulating broad-spectrum disease resistance by suppressing pathogen-induced auxin accumulation in rice. Plant physiology. 2011;155(1):589–602. doi: 10.1104/pp.110.163774 21071600

16. Costacurta A, Mazzafera P, Rosato YB. Indole-3-acetic acid biosynthesis by Xanthomonas axonopodis pv. citri is increased in the presence of plant leaf extracts. FEMS Microbiology Letters. 2010;159(2):215–20. doi: 10.1111/j.1574-6968.1998.tb12863.x

17. Hagen G, Guilfoyle T. Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant molecular biology. 2002;49(3):373–85. doi: 10.1023/a:1015207114117

18. Staswick PE, Serban B, Rowe M, Tiryaki I, Maldonado MT, Maldonado MC, et al. Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. The Plant Cell. 2005;17(2):616–27. doi: 10.1105/tpc.104.026690 15659623

19. Yuan H, Zhao K, Lei H, Shen X, Liu Y, Liao X, et al. Genome-wide analysis of the GH3 family in apple (Malus × domestica). BMC Genomics. 2013;14(1):297. doi: 10.1186/1471-2164-14-297 23638690

20. Feng S, Yue R, Tao S, Yang Y, Zhang L, Xu M, et al. Genome-wide identification, expression analysis of auxin-responsive GH3 family genes in maize (Zea mays L.) under abiotic stresses. Journal of Integrative Plant Biology. 2015;57(9):783–95. doi: 10.1111/jipb.12327 25557253

21. Yang Y, Yue R, Sun T, Zhang L, Chen W, Zeng H, et al. Genome-wide identification, expression analysis of GH3 family genes in Medicago truncatula under stress-related hormones and Sinorhizobium meliloti infection. Applied Microbiology and Biotechnology. 2015;99(2):841–54. doi: 10.1007/s00253-014-6311-5 25529315

22. Nobuta K, Okrent RA, Stoutemyer M, Rodibaugh N, Kempema L, Wildermuth MC, et al. The GH3 acyl adenylase family member PBS3 regulates salicylic acid-dependent defense responses in Arabidopsis. Plant physiology. 2007;144(2):1144–56. doi: 10.1104/pp.107.097691 17468220.

23. Westfall CS, Sherp AM, Zubieta C, Alvarez S, Schraft E, Marcellin R, et al. Arabidopsis thaliana GH3.5 acyl acid amido synthetase mediates metabolic crosstalk in auxin and salicylic acid homeostasis. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(48):13917–22. Epub 11/14. doi: 10.1073/pnas.1612635113 27849615.

24. Ding X, Cao Y, Huang L, Zhao J, Xu C, Li X, et al. Activation of the indole-3-acetic acid-amido synthetase GH3-8 suppresses expansin expression and promotes salicylate- and jasmonate-independent basal immunity in rice. The Plant Cell. 2008;20(1):228–40. doi: 10.1105/tpc.107.055657 18192436.

25. Domingo C, Andres F, Tharreau D, Iglesias DJ, Talon M. Constitutive expression of OsGH3.1 reduces auxin content and enhances defense response and resistance to a fungal pathogen in rice. Molecular Plant-Microbe Interactions. 2009;22(2):201–10. doi: 10.1094/MPMI-22-2-0201 19132872.

26. Chen M, Yongrui HE, Lanzhen XU, Peng A, Lei T, Yao L, et al. Cloning and Expression Analysis of Citrus Genes CsGH3.1 and CsGH3.6 Responding to Xanthomonas axonopodis pv. citri Infection. Horticultural Plant Journal. 2016;2(04):18–27. doi: 10.1016/j.hpj.2016.10.001

27. Zou X, Jiang X, Xu L, Lei T, Peng A, He Y, et al. Transgenic citrus expressing synthesized cecropin B genes in the phloem exhibits decreased susceptibility to Huanglongbing. Plant molecular biology. 2017;93:341–53. doi: 10.1007/s11103-016-0565-5 27866312.

28. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods. 2001;25(4):402–8. doi: 10.1006/meth.2001.1262 11846609

29. Wu Y, Hu B. Simultaneous determination of several phytohormones in natural coconut juice by hollow fiber-based liquid–liquid–liquid microextraction-high performance liquid chromatography. Journal of Chromatography A. 2009;1216(45):7657–63. doi: 10.1016/j.chroma.2009.09.008 19783255

30. Górka B, Wieczorek PP. Simultaneous determination of nine phytohormones in seaweed and algae extracts by HPLC-PDA. Journal of Chromatography B. 2017;1057:32–9. doi: 10.1016/j.jchromb.2017.04.048 28499204

31. Meigh DF, Norris KH, Craft CC, Lieberman M. Ethylene Production by Tomato and Apple Fruits. Nature. 1960;186(4728):902–3. doi: 10.1038/186902a0

32. Peng A, Xu L, He Y, Lei T, Yao L, Chen S, et al. 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 (PCTOC). 2015;123(1):1–13. doi: 10.1007/s11240-015-0799-y

33. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biology. 2013;14(4):R36. doi: 10.1186/gb-2013-14-4-r36 23618408

34. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods. 2008;5:621–8. doi: 10.1038/nmeth.1226 18516045

35. Wang L, Wang X, Wang X, Zhang X, Feng Z. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics. 2009;26(1):136–8. doi: 10.1093/bioinformatics/btp612 19855105

36. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biology. 2010;11(2):R14. doi: 10.1186/gb-2010-11-2-r14 20132535

37. Mao X, Tao CJGO, Wei L. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics. 2005;21(19):3787–93. doi: 10.1093/bioinformatics/bti430 15817693

38. Thimm O, Blasing O, Gibon Y, Nagel A, Meyer S, Kruger P, et al. MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. The Plant Journal. 2010;37(6):914–39. doi: 10.1111/j.1365-313X.2004.02016.x 14996223

39. Ludwigmüller J. Auxin conjugates: their role for plant development and in the evolution of land plants. Journal of Experimental Botany. 2011;62(6):1757–73. doi: 10.1093/jxb/erq412 21307383

40. Sherry L, Rosie T, RebekahA R, T MS, Bonnie B. Characterization of a family of IAA-amino acid conjugate hydrolases from Arabidopsis. Journal of Biological Chemistry. 2002;277(23):20446–52. doi: 10.1074/jbc.M111955200 11923288

41. Pereira ALA, Carazzolle MF, Abe VY, de Oliveira MLP, Domingues MN, Silva JC, et al. Identification of putative TAL effector targets of the citrus canker pathogens shows functional convergence underlying disease development and defense response. BMC Genomics. 2014;15:157. doi: 10.1186/1471-2164-15-157 24564253

42. Soprano AS, Abe VY, Smetana JH, Benedetti CE. Citrus MAF1, a repressor of RNA polymerase III, binds the Xanthomonas citri canker elicitor PthA4 and suppresses citrus canker development. Plant physiology. 2013;163(1):232–42. doi: 10.1104/pp.113.224642 23898043.

43. Soprano AS, Giuseppe PO, Shimo HM, Lima TB, Batista FAH, Righetto GL, et al. Crystal Structure and Regulation of the Citrus Pol III Repressor MAF1 by Auxin and Phosphorylation. Structure. 2017;25(9):1360–70. doi: 10.1016/j.str.2017.07.004 28781084.

44. Rashid A. Defense responses of plant cell wall non-catalytic proteins against pathogens. Physiological and Molecular Plant Pathology. 2016;94:38–46. doi: 10.1016/j.pmpp.2016.03.009

45. Bellincampi D, Cervone F, Lionetti V. Plant cell wall dynamics and wall-related susceptibility in plant-pathogen interactions. Frontiers in plant science. 2014;5:228. doi: 10.3389/fpls.2014.00228 24904623.

46. Fendrych M, Leung J, Friml J. TIR1/AFB-Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls. eLife. 2016;5:e19048. doi: 10.7554/eLife.19048 27627746

47. Kunkel BN, Brooks DM. Cross talk between signaling pathways in pathogen defense. Current Opinion in Plant Biology. 2002;5(4):325–31. doi: 10.1016/s1369-5266(02)00275-3 12179966

48. Di X, Gomila J, Takken FLW. Involvement of salicylic acid, ethylene and jasmonic acid signalling pathways in the susceptibility of tomato to Fusarium oxysporum. Molecular Plant Pathology. 2017;18(7):1024–35. doi: 10.1111/mpp.12559 28390170

49. Francis MI, Redondo A, Burns JK, Graham JH. Soil application of imidacloprid and related SAR-inducing compounds produces effective and persistent control of citrus canker. European Journal of Plant Pathology. 2009;124(2):283–92. doi: 10.1007/s10658-008-9415-x

50. Zhongqin Z, Muyang W, Zhimiao L, Qun L, Zuhua H. Arabidopsis GH3.5 regulates salicylic acid-dependent and both NPR1-dependent and independent defense responses. Plant Signaling & Behavior. 2008;3(8):537–42. doi: 10.4161/psb.3.8.5748 19513247

51. Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, et al. Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(21):11655–60. doi: 10.1073/pnas.97.21.11655 11027363.

52. O'Donnell PJ, Jones JB, Antoine FR, Ciardi J, Klee HJ. Ethylene-dependent salicylic acid regulates an expanded cell death response to a plant pathogen. The Plant Journal. 2001;25(3):315–23. doi: 10.1046/j.1365-313x.2001.00968.x 11208023

53. Lawton KA, Potter SL, Uknes S, Ryals J. Acquired resistance signal transduction in Arabidopsis is ethylene independent. The Plant Cell. 1994;6(5):581–8. doi: 10.1105/tpc.6.5.581 12244251

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden