Skip to main content
SpringerLink
Log in

GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3 ubiquitin ligase activity of GhPUB17

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Key message

A U-box E3 ubiquitin ligase GhPUB17 is inhibited by GhCyP3 with antifungal activity and acts as a negative regulator involved in cotton resistance to Verticillium dahliae.

Abstract

E3 ubiquitin ligases, the key component enzymes of the ubiquitin–proteasome system, which contains the most diverse structural and functional members involved in the determination of target specificity and the regulation of metabolism, have been well documented in previous studies. Here, we identify GhPUB17, a U-box E3 ligase in cotton that has ubiquitination activity and is involved in the cotton immune response to Verticillium dahliae. The expression level of GhPUB17 is downregulated in the ssn mutant with a constitutively activated immune response (Sun et al., Nat Commun 5:5372, 2014). Infection with V. dahliae or exogenous hormone treatment, including jasmonic acid and salicylic acid, significantly upregulated GhPUB17 in cotton roots, which suggested a possible role for this E3 ligase in the plant immune response to pathogens. Moreover, GhPUB17-knockdown cotton plants are more resistant to V. dahliae, whereas GhPUB17-overexpressing plants are more susceptible to the pathogen, which indicated that GhPUB17 is a negative regulator of cotton resistance to V. dahliae. A yeast two-hybrid (Y2H) assay identified GhCyP3 as a protein that interacts with GhPUB17, and this finding was confirmed by further protein interaction assays. The downregulation of GhCyP3 in cotton seedlings attenuated the plants’ resistance to V. dahliae. In addition, GhCyP3 showed antifungal activity against V. dahliae, and the E3 ligase activity of GhPUB17 was repressed by GhCyP3 in vitro. These results suggest that GhPUB17 negatively regulates cotton immunity to V. dahliae and that the antifungal protein GhCyP3 likely interacts with and inhibits the ligase activity of GhPUB17 and plays an important role in the cotton-Verticillium interaction.

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

Similar content being viewed by others

References

  • Andreas B, Gottfried W, Heinrich A, Antal R, Peter P (1997) Presence of cyclophilin A in synovial fluids of patients with rheumatoid arthritis. J Exp Med 185:975–980

    Article  Google Scholar 

  • Boller T, He SY (2009) Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science 324:742–744

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cao S, Yan L (2013) Construction of a high-quality yeast two-hybrid (Y2H) library and its application in identification of interacting proteins with key vernalization regulator TaVRN-A1 in wheat. BMC Res Notes 6:81

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dalio RJD, Magalhaes DM, Rodrigues CM, Arena GD, Oliveira TS, Souza-Neto RR, Picchi SC, Martins PMM, Santos PJC, Maximo HJ, Pacheco IS, De Souza AA, Machado MA (2017) PAMPs, PRRs, effectors and R-genes associated with citrus-pathogen interactions. Ann Bot 119:749–774

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dodds PN, Rathjen JP (2010) Plant immunity: towards an integrated view of plant-pathogen interactions. Nat Rev Genet 11:539–548

    Article  CAS  PubMed  Google Scholar 

  • Du Z, Zhou X, Li L, Su Z (2009) plantsUPS: a database of plants’ Ubiquitin Proteasome System. BMC Genom 10:227

    Article  CAS  Google Scholar 

  • Fernandes CJ, O’Sullivan MV, Cai Y, Kong F, Zeng X, Gilbert GL, Kotsiou G (2007) Agar dilution method for detection of inducible clindamycin resistance in Staphylococcus spp. J Clin Microbiol 45:4018–4020

    Article  PubMed  PubMed Central  Google Scholar 

  • Gao W, Long L, Zhu LF, Xu L, Gao WH, Sun LQ, Liu LL, Zhang XL (2013) Proteomic and virus-induced gene silencing (VIGS) analyses reveal that gossypol, brassinosteroids, and jasmonic acid contribute to the resistance of cotton to Verticillium dahliae. Mol Cell Proteomics 12:3690–3703

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Godoy AV, Lazzaro AS, Casalongué CA, Segundo BS (2000) Expression of a Solanum tuberosum cyclophilin gene is regulated by fungal infection and abiotic stress conditions. Plant Sci 152:123–134

    Article  CAS  Google Scholar 

  • Gonzalez-Lamothe R, Tsitsigiannis DI, Ludwig AA, Panicot M, Shirasu K, Jones JD (2006) The U-box protein CMPG1 is required for efficient activation of defense mechanisms triggered by multiple resistance genes in tobacco and tomato. Plant cell 18:1067–1083

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Göthel SF, Marahiel MA (1999) Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts. Cell Mol Life Sci 55:423

    Article  PubMed  Google Scholar 

  • He Q, McLellan H, Boevink PC, Sadanandom A, Xie C, Birch PR, Tian Z (2015) U-box E3 ubiquitin ligase PUB17 acts in the nucleus to promote specific immune pathways triggered by Phytophthora infestans. J Exp Bot 66:3189–3199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Horowitz DS, Lee EJ, Mabon SA, Misteli T (2002) A cyclophilin functions in pre-mRNA splicing. EMBO J 21:470–480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323–329

    Article  CAS  Google Scholar 

  • Kan YC, Liu SW, Guo ZJ, Li DB (2002) Characterization of a cyclophilin cDNA from soybean cells. Acta Bot Sin 44:173–176

    CAS  Google Scholar 

  • Kong HY, Lee SC, Hwang BK (2001) Expression of pepper cyclophilin gene is differentially regulated during the pathogen infection and abiotic stress conditions. Physiol Mol Plant Pathol 59:189–199

    Article  CAS  Google Scholar 

  • Kullertz G, Liebau A, Rucknagel P, Schierhorn A, Diettrich B, Fischer G, Luckner M (1999) Stress-induced expression of cyclophilins in proembryonic masses of Digitalis lanata does not protect against freezing/thawing stress. Planta 208:599–605

    Article  CAS  PubMed  Google Scholar 

  • Kwon SJ, Choi EY, Choi YJ, Ahn JH, Park OK (2006) Proteomics studies of post-translational modifications in plants. J Exp Bot 57:1547–1551

    Article  CAS  PubMed  Google Scholar 

  • Lee JR, Park SC, Kim JY, Lee SS, Park Y, Cheong GW, Hahm KS, Lee SY (2007) Molecular and functional characterization of a cyclophilin with antifungal activity from Chinese cabbage. Biochem Biophys Res Commun 353:672–678

    Article  CAS  PubMed  Google Scholar 

  • Lee JH, Jung JH, Park CM (2015) INDUCER OF CBF EXPRESSION 1 integrates cold signals into FLOWERING LOCUS C-mediated flowering pathways in Arabidopsis. Plant J 84:29–40

    Article  CAS  PubMed  Google Scholar 

  • Li C, He X, Luo X, Xu L, Liu L, Min L, Jin L, Zhu L, Zhang X (2014a) Cotton WRKY1 mediates the plant defense-to-development transition during infection of cotton by Verticillium dahliae by activating JASMONATE ZIM-DOMAIN1 expression. Plant Physiol 166:2179–2194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li M, Ma X, Chiang YH, Yadeta KA, Ding P, Dong L, Zhao Y, Li X, Yu Y, Zhang L, Shen QH, Xia B, Coaker G, Liu D, Zhou JM (2014b) Proline isomerization of the immune receptor-interacting protein RIN4 by a cyclophilin inhibits effector-triggered immunity in Arabidopsis. Cell Host Microbe 16:473–483

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liao D, Cao Y, Sun X, Espinoza C, Nguyen CT, Liang Y, Stacey G (2017) Arabidopsis E3 ubiquitin ligase PLANT U-BOX13 (PUB13) regulates chitin receptor LYSIN MOTIF RECEPTOR KINASE5 (LYK5) protein abundance. New Phytol 214:1646–1656

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP (2002) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30:415–429

    Article  CAS  PubMed  Google Scholar 

  • Ma Z, Zhu L, Song T, Wang Y, Zhang Q, Xia Y, Qiu M, Lin Y, Li H, Kong L, Fang Y, Ye W, Wang Y, Dong S, Zheng X, Tyler BM, Wang Y (2017) A paralogous decoy protects Phytophthora sojae apoplastic effector PsXEG1 from a host inhibitor. Science 355:710–714

    Article  CAS  PubMed  Google Scholar 

  • Mainali HR, Chapman P, Dhaubhadel S (2014) Genome-wide analysis of cyclophilin gene family in soybean (Glycine max). BMC Plant Biol 14:282

    Article  PubMed  PubMed Central  Google Scholar 

  • Navarro L, Zipfel C, Rowland O, Keller I, Robatzek S, Boller T, Jones JD (2004) The transcriptional innate immune response to flg22. Interplay and overlap with Avr gene-dependent defense responses and bacterial pathogenesis. Plant Physiol 135:1113–1128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oh K, Ivanchenko MG, White TJ, Lomax TL (2006) The diageotropica gene of tomato encodes a cyclophilin: a novel player in auxin signaling. Planta 224:133–144

    Article  CAS  PubMed  Google Scholar 

  • Orosa B, He Q, Mesmar J, Gilroy EM, McLellan H, Yang C, Craig A, Bailey M, Zhang C, Moore JD, Boevink PC, Tian Z, Birch PR, Sadanandom A (2017) BTB-BACK domain protein POB1 suppresses immune cell death by targeting ubiquitin E3 ligase PUB17 for degradation. PLoS Genet 13:e1006540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piechota-Polanczyk A, Demyanets S, Nykonenko O, Huk I, Mittlboeck M, Domenig CM, Neumayer C, Wojta J, Nanobachvili J, Klinger M (2013) Decreased tissue levels of cyclophilin A, a cyclosporine a target and phospho-ERK1/2 in simvastatin patients with abdominal aortic aneurysm. Eur J Vasc Endovasc Surg 45:682–688

    Article  CAS  PubMed  Google Scholar 

  • Sang Y, Wang Y, Ni H, Cazale AC, She YM, Peeters N, Macho AP (2018) The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses. Mol Plant Pathol 19:129–142

    Article  CAS  PubMed  Google Scholar 

  • Shi H, Liu Z, Zhu L, Zhang C, Chen Y, Zhou Y, Li F, Li X (2012) Overexpression of cotton (Gossypium hirsutum) dirigent1 gene enhances lignification that blocks the spread of Verticillium dahliae. Acta Biochim Biophys Sin 44:555–564

    Article  CAS  PubMed  Google Scholar 

  • Stone SL, Callis J (2007) Ubiquitin ligases mediate growth and development by promoting protein death. Curr Opin Plant Biol 10:624–632

    Article  CAS  PubMed  Google Scholar 

  • Sun L, Zhu L, Xu L, Yuan D, Min L, Zhang X (2014) Cotton cytochrome P450 CYP82D regulates systemic cell death by modulating the octadecanoid pathway. Nat Commun 5:5372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thomma BP, Nurnberger T, Joosten MH (2011) Of PAMPs and effectors: the blurred PTI-ETI dichotomy. Plant cell 23:4–15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Trujillo M (2018) News from the PUB: plant U-box type E3 ubiquitin ligases. J Exp Bot 69:371–384

    Article  CAS  PubMed  Google Scholar 

  • Trujillo M, Shirasu K (2010) Ubiquitination in plant immunity. Curr Opin Plant Biol 13:402–408

    Article  CAS  PubMed  Google Scholar 

  • Vierstra RD (2009) The ubiquitin-26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol 10:385–397

    Article  CAS  PubMed  Google Scholar 

  • Xu L, Zhang W, He X, Liu M, Zhang K, Shaban M, Sun L, Zhu J, Luo Y, Yuan D, Zhang X, Zhu L (2014) Functional characterization of cotton genes responsive to Verticillium dahliae through bioinformatics and reverse genetics strategies. J Exp Bot 65:6679–6692

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang CW, Gonzalez-Lamothe R, Ewan RA, Rowland O, Yoshioka H, Shenton M, Ye H, O’Donnell E, Jones JD, Sadanandom A (2006) The E3 ubiquitin ligase activity of arabidopsis PLANT U-BOX17 and its functional tobacco homolog ACRE276 are required for cell death and defense. Plant cell 18:1084–1098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yee D, Goring DR (2009) The diversity of plant U-box E3 ubiquitin ligases: from upstream activators to downstream target substrates. J Exp Bot 60:1109–1121

    Article  CAS  PubMed  Google Scholar 

  • You Q, Zhai K, Yang D, Yang W, Wu J, Liu J, Pan W, Wang J, Zhu X, Jian Y, Liu J, Zhang Y, Deng Y, Li Q, Lou Y, Xie Q, He Z (2016) An E3 ubiquitin ligase-BAG protein module controls plant innate immunity and broad-spectrum disease resistance. Cell Host Microbe 20:758–769

    Article  CAS  PubMed  Google Scholar 

  • Zarka DG, Vogel JT, Cook D, Thomashow MF (2003) Cold induction of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiol 133:910–918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhou B, Zeng L (2016) Conventional and unconventional ubiquitination in plant immunity. Mol Plant Pathol 18:1313–1330

    Article  Google Scholar 

  • Zhou B, Mural RV, Chen X. Oates ME, Connor RA, Martin GB, Gough J, Zeng L (2017) A subset of ubiquitin-conjugating enzymes is essential for plant immunity. Plant Physiol 173:1371–1390

    Article  CAS  PubMed  Google Scholar 

  • Zhu C, Wang Y, Li Y, Bhatti KH, Tian Y, Wu J (2011) Overexpression of a cotton cyclophilin gene (GhCyp1) in transgenic tobacco plants confers dual tolerance to salt stress and Pseudomonas syringae pv. tabaci infection. Plant Physiol Biochem 49:1264–1271

    Article  CAS  PubMed  Google Scholar 

  • Zipfel C, Oldroyd GE (2017) Plant signalling in symbiosis and immunity. Nature 543:328–336

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the Nature Science Foundation of Hubei Province (2016CFA054) and the International Science and Technology Cooperation Program of China (Grant No. 2015DFA30860).

Author information

Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, Hubei, China

    Tao Qin, Shiming Liu, Zhennan Zhang, Longqing Sun, Xin He, Longfu Zhu & Xianlong Zhang

  2. Department of Biosciences, Durham University, South Road, Durham, UK

    Keith Lindsey

Authors
  1. Tao Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shiming Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhennan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Longqing Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xin He
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Keith Lindsey
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Longfu Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xianlong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Professor LZ and Professor XZ designed the main thoughts of this study, TQ proceeded this study and finished the manuscript, SL and ZZ acted as the assistants in materials preparing and study proceeding, LS and XH provided significant suggestions in experiment designment, KL commented the research and revised the manuscript.

Corresponding author

Correspondence to Longfu Zhu.

Additional information

Publisher’s Note

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

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (XLSX 17 KB)

11103_2019_824_MOESM2_ESM.pptx

Supplementary material 2 GhPUB17 is a homologue of PUB17 and is involved in cotton disease resistance. a Alignment of amino acid sequences of homologues of PUB17 in Gossypium hirsutum (Gh), Arabidopsis thaliana (At), Nicotiana tabacum L. (Nt), Solanum tuberosum L. (St) and Solanum lycopsersicum Mill. (Sl); NtACRE74 (NtPUB21) was used as the outgroup. The coloured boxes indicate the conserved amino acids. The U-box domain is indicated by a black line, and the ARM domain is indicated by a green line. NtACRE74 contains a similar conversed U-box domain but no similar ARM domain. b RT-PCR analysis of GhPUB17 expression levels in different cotton tissues, including root, stem, cotyledon, growing point, leaf I (the first leaf from the top), leaf II (the second leaf from the top), ovule (on the day of anthesis), anther and petal. The transcript level of UB7 in cotton was used as a control. cGhPUB17 was induced by treatment with exogenous hormone salicylic acid (SA, 1 mM). dGhPUB17 was induced by treatment with exogenous hormone jasmonic acid (JA, 100 µM) (PPTX 362 KB)

11103_2019_824_MOESM3_ESM.pptx

Supplementary material 3 TRV-based virus induced gene silencing (VIGS) vectors. a TRV cDNA clones were inserted between the duplicated CaMV 35S promoter (2×35S) and the nopaline synthase terminator (NOST) in a T-DNA vector. LB and RB refer to the left and right borders of T-DNA. RdRp, RNA-dependent RNA polymerase. MP, movement protein. 16K, a 16-kDa cysteine-rich protein. Rz, self-cleaving ribozyme. CP, coat protein. MCS, multiple cloning sites. BamH I and Kpn I sites (marked with a red box) were used in this study. b The sequences of GhPUB17 (CDS 874-1098 bp) and GhCyP3 (CDS 107–474 bp) were used to generate the target vectors of both genes (PPTX 13108 KB)

11103_2019_824_MOESM4_ESM.pptx

Supplementary material 4 Identification and selection of GhPUB17 transformation lines used in this study. a & b Southern blot for detection of the copy numbers in stable GhPUB17 transformation lines. The lines marked with red boxes were used for the subsequent experiments. c & d Expression level of GhPUB17 in transformants and YZ1 plants (controls). Total RNA from leaves was extracted and used for RT-qPCR (PPTX 990 KB)

11103_2019_824_MOESM5_ESM.pptx

Supplementary material 5 Infection of GhPUB17 transgenic cotton lines with Botrytis cinerea. Fresh and healthy leaves were collected for B. cinerea infection. a Photographs of cotton leaves infected with B. cinerea. b Calculation of the mean lesion area in infected leaves using Digimizer software. The results showed no differences among the lines after B. cinerea infection (PPTX 480 KB)

11103_2019_824_MOESM6_ESM.pptx

Supplementary material 6 Recombinant proteins used in this study expressed with the prokaryotic expression system. Recombinant vectors were transformed into E. coli (BL21) and induced by cultivation with different levels of IPTG at 28℃ with shaking. a GhCyP3 was fused to GST and HIS tags at its N terminus and induced in an E. coli prokaryotic expression system through the addition of IPTG (0.1 mol/L) at 28℃. The purified proteins were assessed using an SDS-PAGE gel and stained with Coomassie blue. HIS-GST protein was used as the control, and the target proteins are marked in the image. b GhPUB17 was fused with an MBP tag at its N terminus, extracted using an MBP extraction kit, and checked with an SDS-PAGE gel stained with Coomassie blue. The extracted proteins contain full-length MBP-GhPUB17 and its two fragments. c The mutated version of GhPUB17 was expressed as the form MBP-GhPUB17 V311I (the conversed U-box domain with a Val-to-Ile substitution mutation at residue 311, MBP-GhPUB17 V311I) as a control for E3 ligase activity assay of GhPUB17. d Identification of fragments from the MBP-GhPUB17 extraction. The MBP-GhPUB17 structure is displayed with different coloured boxes, and the detected sites matching the reference sequence are marked with purple stripes (PPTX 98 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Qin, T., Liu, S., Zhang, Z. et al. GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3 ubiquitin ligase activity of GhPUB17. Plant Mol Biol 99, 379–393 (2019). https://doi.org/10.1007/s11103-019-00824-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-019-00824-y

Keywords

Search

Navigation