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Interaction between Brassica napus polygalacturonase inhibition proteins and Sclerotinia sclerotiorum polygalacturonase: implications for rapeseed resistance to fungal infection

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Abstract

Main conclusion

BnPGIPs interacted with Sclerotinia sclerotiorum PGs to improve rapeseed SSR resistance at different levels; the BnPGIP-overexpression lines did not affect plant morphology or seed quality traits.

Abstract

Plant polygalacturonase-inhibiting proteins (PGIPs) play a crucial role in plant defence against phytopathogenic fungi by inhibiting fungal polygalacturonase (PG) activity. We overexpressed BnPGIP2, BnPGIP5, and BnPGIP10 genes in an inbred line 7492 of rapeseed (Brassica napus). Compared with 7492WT, the overexpression of BnPGIP2 lines significantly increased Sclerotinia sclerotiorum resistance in both seedlings and adult plants. BnPGIP5 overexpression lines exhibited decreased S. sclerotiorum disease symptoms in seedlings only, whereas BnPGIP10 overexpression lines did not improve Sclerotinia resistance for seedlings or adult plants. Quantitative real-time PCR analysis of S. sclerotiorum PG1, SsPG3, SsPG5, and SsPG6 genes in overexpressing BnPGIP lines showed that these pathogenic genes in the Sclerotinia resistance transgenic lines exhibited low expression in stem tissues. Split-luciferase complementation experiments confirmed the following: BnPGIP2 interacts with SsPG1 and SsPG6 but not with SsPG3 or SsPG5; BnPGIP5 interacts with SsPG3 and SsPG6 but not with SsPG1 or SsPG5; and BnPGIP10 interacts with SsPG1 but not SsPG3, SsPG5, or SsPG6. Leaf crude protein extracts from BnPGIP2 and BnPGIP5 transgenic lines displayed high inhibitory activity against the SsPG crude protein. BnPGIP-overexpression lines with Sclerotinia resistance displayed a lower accumulation of H2O2 and higher expression of the H2O2-removing gene BnAPX (ascorbate peroxidase) than 7492WT, as well as elevated expression of defence response genes including jasmonic acid/ethylene and salicylic acid pathways after S. sclerotiorum infection. The plants overexpressing BnPGIP exhibited no difference in either agronomic traits or grain yield from 7492WT. This study provides potential target genes for developing S. sclerotiorum resistance in rapeseed.

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Abbreviations

dpi:

Days post inoculation

hpi:

Hours post inoculation

JA:

Jasmonic acid

PCW:

Plant cell wall

PG:

Polygalacturonase

PGIP:

Polygalacturonase inhibitor

PR:

Pathogenesis-related gene

SA:

Salicylic acid

SSR:

Sclerotinia stem rot

References

  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22(2):195–201

    Article  CAS  PubMed  Google Scholar 

  • Bashi ZD, Rimmer SR, Khachatourians GG, Hegedus DD (2012) Factors governing the regulation of Sclerotinia sclerotiorum cutinase A and polygalacturonase 1 during different stages of infection. Can J Microbiol 58(5):605–616

    Article  CAS  Google Scholar 

  • Bashi ZD, Rimmer SR, Khachatourians GG, Hegedus DD (2013) Brassica napus polygalacturonase inhibitor proteins inhibit Sclerotinia sclerotiorum polygalacturonase enzymatic and necrotizing activities and delay symptoms in transgenic plants. Can J Microbiol 59(2):79–86

    Article  CAS  PubMed  Google Scholar 

  • Benedetti M, Leggio C, Federici L, De Lorenzo G, Pavel NV, Cervone F (2011) Structural resolution of the complex between a fungal polygalacturonase and a plant polygalacturonase-inhibiting protein by small-angle X-ray scattering. Plant Physiol 157(2):599–607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Benedetti M, Andreani F, Leggio C, Galantini L, Di Matteo A, Pavel NV, De Lorenzo G, Cervone F, Federici L, Sicilia F (2013) A single amino-acid substitution allows endo-polygalacturonase of Fusarium verticillioides to acquire recognition by PGIP2 from Phaseolus vulgaris. PLoS ONE 8(11):e80610

    Article  PubMed  PubMed Central  Google Scholar 

  • Berendzen KW, Böhmer M, Wallmeroth N, Peter S, Vesić M, Zhou Y, Tiesler FK, Schleifenbaum F, Harter K (2012) Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry. Plant Methods 8(1):1–17

    Article  Google Scholar 

  • Boland GJ, Hall R (1994) Index of plant hosts of Sclerotinia sclerotiorum. Can J Plant Pathol 16(2):93–108

    Article  Google Scholar 

  • Bolton MD, Thomma BPHJ, Nelson BD (2006) Sclerotiniasclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. Mol Plant Pathol 7(1):1–16

    Article  CAS  PubMed  Google Scholar 

  • Borras-Hidalgo O, Caprari C, Hernandezestevez I, Lorenzo GD, Cervone F (2012) A gene for plant protection: expression of a bean polygalacturonase inhibitor in tobacco confers a strong resistance against Rhizoctonia solani and two oomycetes. Front Plant Sci 3:268

    Article  PubMed  PubMed Central  Google Scholar 

  • Chen X, Chen Y, Zhang L, He Z, Huang B, Chen C, Zhang Q, Zuo S (2019) Amino acid substitutions in a polygalacturonase inhibiting protein (OsPGIP2) increases sheath blight resistance in rice. Rice 12(1):56

    Article  PubMed  PubMed Central  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. Molecular Plant 13(8):1194–1202

    Article  CAS  PubMed  Google Scholar 

  • De Lorenzo G, D’Ovidio R, Cervone F (2001) The role of polygalacturonase-inhibiting proteins (PGIPs) in defense against pathogenic fungi. Annu Rev Phytopathol 39(1):313–335

    Article  PubMed  Google Scholar 

  • Derbyshire M, Denton-Giles M (2016) The control of sclerotinia stem rot on oilseed rape (Brassica napus): current practices and future opportunities. Mol Plant Pathol 65(6):859–877

    Article  CAS  Google Scholar 

  • Di Matteo A, Federici L, Mattei B, Salvi G, Johnson K, Savino C, De Lorenzo G, Tsernoglou D, Cervone F (2003) The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense. Proc Natl Acad Sci USA 100(17):10124

    Article  PubMed  PubMed Central  Google Scholar 

  • Federici L, Di Matteo A, Fernandez-Recio J, Tsernoglou D, Cervone F (2006) Polygalacturonase inhibiting proteins: players in plant innate immunity? Trends Plant Sci 11(2):65–70

    Article  CAS  PubMed  Google Scholar 

  • Ferrari S, Sella L, Janni M, De Lorenzo G, Favaron F, D’Ovidio R (2012) Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum. Plant Biol 14(1):31–38

    Article  CAS  PubMed  Google Scholar 

  • Ferrari S, Savatin DV, Sicilia F, Gramegna G, Cervone F, Lorenzo GD (2013) Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development. Front Plant Sci 4:49

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hegedus DD, Li R, Buchwaldt L, Parkin I, Whitwill S, Coutu C, Bekkaoui D, Rimmer SR (2008) Brassica napus possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to Sclerotinia sclerotiorum infection, wounding and defense hormone treatment. Planta 228(2):241–253

    Article  CAS  PubMed  Google Scholar 

  • Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 8(3):275–282

    CAS  PubMed  Google Scholar 

  • Jonoubi P, Mousavi A, Majd A, Salmanian AH, Javaran MJ, Daneshian J (2005) Efficient regeneration of Brassicanapus L. hypocotyls and genetic transformation by Agrobacteriumtumefaciens. Biol Plant 49(2):175–180

    Article  CAS  Google Scholar 

  • Joubert DA, Slaughter AR, Kemp G, Becker JV, Krooshof GH, Bergmann C, Benen J, Pretorius IS, Vivier MA (2006) The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases. Transgenic Res 15(6):687–702

    Article  CAS  PubMed  Google Scholar 

  • Joubert DA, Kars I, Wagemakers L, Bergmann C, Kemp G, Vivier MA, van Kan JA (2007) A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction. Mol Plant-Microbe Interact 20(4):392–402

    Article  CAS  PubMed  Google Scholar 

  • Kaewwongwal A, Chen J, Somta P, Kongjaimun A, Yimram T, Chen X, Srinives P (2017) Novel alleles of two tightly linked genes encoding polygalacturonase-inhibiting proteins (VrPGIP1 and VrPGIP2) associated with the Br locus that confer bruchid (Callosobruchus spp.) resistance to mungbean (Vignaradiata) accession V2709. Front Plant Sci 8:1692

    Article  PubMed  PubMed Central  Google Scholar 

  • Kalunke RM, Tundo S, Benedetti M, Cervone F, De Lorenzo G, D’Ovidio R (2015) An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens. Front Plant Sci 6:146

    Article  PubMed  PubMed Central  Google Scholar 

  • Kumar S, Chauhan JS, Kumar A (2010) Screening for erucic acid and GSLs content in rapeseed-mustard seeds using near infrared reflectance spectroscopy. J Food Sci Tech 47(6):690–692

    Article  CAS  Google Scholar 

  • Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35(6):1547–1549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lehmann S, Serrano M, L’Haridon F, Tjamos SE, Metraux J-P (2015) Reactive oxygen species and plant resistance to fungal pathogens. Phytochemistry 112:54–62

    Article  CAS  PubMed  Google Scholar 

  • Li H, Smigocki AC (2018) Sugar beet polygalacturonase-inhibiting proteins with 11 LRRs confer Rhizoctonia, Fusarium and Botrytis resistance in Nicotiana plants. Physiol Mol Plant Pathol 102:200–208

    Article  CAS  Google Scholar 

  • Li R, Rimmer R, Yu M, Sharpe AG, Seguin-Swartz G, Lydiate D, Hegedus DD (2003) Two Brassica napus polygalacturonase inhibitory protein genes are expressed at different levels in response to biotic and abiotic stresses. Planta 217(2):299–308

    Article  CAS  PubMed  Google Scholar 

  • Li R, Rimmer R, Buchwaldt L, Sharpe AG, Séguin-Swartz G, Hegedus DD (2004) Interaction of Sclerotinia sclerotiorum with Brassica napus: cloning and characterization of endo- and exo-polygalacturonases expressed during saprophytic and parasitic modes. Fungal Genet Biol 41(8):754–765

    Article  CAS  PubMed  Google Scholar 

  • Lin Z-JD, Liebrand TW, Yadeta KA, Coaker G (2015) PBL13 is a serine/threonine protein kinase that negatively regulates Arabidopsis immune responses. Plant Physiol 169(4):2950–2962

    CAS  PubMed  PubMed Central  Google Scholar 

  • Liu N, Zhang X, Sun Y, Wang P, Li X, Pei Y, Li F, Hou Y (2017) Molecular evidence for the involvement of a polygalacturonase-inhibiting protein, GhPGIP1, in enhanced resistance to Verticillium and Fusarium wilts in cotton. Sci Rep 7(1):1–18

    Google Scholar 

  • Ma QH, Tian B, Li YL (2010) Overexpression of a wheat jasmonate-regulated lectin increases pathogen resistance. Biochimie 92(2):187–193

    Article  CAS  PubMed  Google Scholar 

  • Magliery TJ, Wilson CG, Pan W, Mishler D, Ghosh I, Hamilton AD, Regan L (2005) Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. J Am Chem Soc 127(1):146–157

    Article  CAS  PubMed  Google Scholar 

  • Marone D, Russo MA, Laidò G, De Leonardis AM, Mastrangelo AM (2013) Plant nucleotide binding site–leucine-rich repeat (NBS-LRR) genes: active guardians in host defense responses. Int J Mol Sci 14(4):7302–7326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Misasvillamil JC, Ra VDH (2008) Enzyme-inhibitor interactions at the plant-pathogen interface. Curr Opin Plant Biol 11(4):380–388

    Article  CAS  Google Scholar 

  • Nafisi M, Goregaoker S, Botanga CJ, Glawischnig E, Olsen CE, Halkier BA, Glazebrook J (2007) Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-3-acetaldoxime in camalexin synthesis. Plant Cell 19(6):2039–2052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oelofse D, Dubery IA, Meyer R, Arendse MS, Gazendam I, Berger DK (2006) Apple polygalacturonase inhibiting protein expressed in transgenic tobacco inhibits polygalacturonases from fungal pathogens of apple and the anthracnose pathogen of lupins. Phytochemistry 67(3):255–263

    Article  CAS  PubMed  Google Scholar 

  • Ohashi K, Kiuchi T, Shoji K, Sampei K, Mizuno K (2012) Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments. Biotechniques 52(1):45–50

    Article  CAS  PubMed  Google Scholar 

  • Oliveira MB, Nascimento LB, Junior ML, Petrofeza S (2010) Characterization of the dry bean polygalacturonase-inhibiting protein (PGIP) gene family during Sclerotinia sclerotiorum (Sclerotiniaceae) infection. Genet Mol Res 9(2):994–1004

    Article  CAS  PubMed  Google Scholar 

  • Peumans WJ, Van Damme EJ (1995) The role of lectins in plant defence. Histochem J 27(4):253–271

    Article  CAS  PubMed  Google Scholar 

  • Prabhu SA, Wagenknecht M, Melvin P, Gnanesh Kumar BS, Veena M, Shailasree S, Moerschbacher BM, Kini KR (2015) Immuno-affinity purification of PglPGIP1, a polygalacturonase-inhibitor protein from pearl millet: studies on its inhibition of fungal polygalacturonases and role in resistance against the downy mildew pathogen. Mol Biol Rep 42(6):1123–1138

    Article  CAS  PubMed  Google Scholar 

  • Rasmussen U, Bojsen K, Collinge DB (1992) Cloning and characterization of a pathogen-induced chitinase in Brassica napus. Plant Mol Biol 20(2):277–287

    Article  CAS  PubMed  Google Scholar 

  • Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42(W1):W320–W324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sharma RV, Somidi AK, Dalai AK (2015) Preparation and properties evaluation of biolubricants derived from canola oil and canola biodiesel. J Agric Food Chem 63(12):3235–3242

    Article  CAS  PubMed  Google Scholar 

  • Sicilia F, Fernandez-Recio J, Caprari C, De LG, Tsernoglou D, Cervone F, Federici L (2005) The polygalacturonase-inhibiting protein PGIP2 of Phaseolus vulgaris has evolved a mixed mode of inhibition of endopolygalacturonase PG1 of Botrytis cinerea. Plant Physiol 139(3):1380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1(4):2019

    Article  CAS  PubMed  Google Scholar 

  • Taylor RJ, Secor GA (1988) An improved diffusion assay for quantifying polygalacturonase content of Erwinia culture filtrates. Phytopathology 78:1101–1103

    Article  CAS  Google Scholar 

  • Torres MA, Jones JD, Dangl JL (2006) Reactive oxygen species signaling in response to pathogens. Plant Physiol 141(2):373–378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Volpi C, Raiola A, Janni M, Gordon A, O’Sullivan DM, Favaron F, D’Ovidio R (2013) Claviceps purpurea expressing polygalacturonases escaping PGIP inhibition fully infects PvPGIP2 wheat transgenic plants but its infection is delayed in wheat transgenic plants with increased level of pectin methyl esterification. Plant Physiol Biochem 73:294–301

    Article  CAS  PubMed  Google Scholar 

  • Voragen AG, Coenen G-J, Verhoef RP, Schols HA (2009) Pectin, a versatile polysaccharide present in plant cell walls. Struct Chem 20(2):263

    Article  CAS  Google Scholar 

  • Wang R, Lu L, Pan X, Hu Z, Ling F, Yan Y, Liu Y, Lin Y (2015) Functional analysis of OsPGIP1 in rice sheath blight resistance. Plant Mol Biol 87(1–2):181–191

    Article  CAS  PubMed  Google Scholar 

  • Wang Z, Wan L, Xin Q, Chen Y, Zhang X, Dong F, Hong D, Yang G (2018) Overexpression of OsPGIP2 confers Sclerotinia sclerotiorum resistance in Brassica napus through increased activation of defense mechanisms. J Exp Bot 69(12):3141–3155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Z, Ma LY, Cao J, Li YL, Ding LN, Zhu KM, Yang YH, Tan XL (2019) Recent advances in mechanisms of plant defense to Sclerotinia sclerotiorum. Front Plant Sci 10:1314

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang Z, Zhao FY, Tang MQ, Chen T, Bao LL, Cao J, Li YL, Yang YH, Zhu KM, Liu S, Tan XL (2020) BnaMPK6 is a determinant of quantitative disease resistance against Sclerotinia sclerotiorum in oilseed rape. Plant Sci 291:110362

    Article  CAS  PubMed  Google Scholar 

  • Wu J, Cai G, Tu J, Li L, Liu S, Luo X, Zhou L, Fan C, Zhou Y (2013) Identification of QTLs for resistance to sclerotinia stem rot and BnaC.IGMT5.a as a candidate gene of the major resistant QTL SRC6 in Brassicanapus. PLoS ONE 8(7):217–227

    Article  Google Scholar 

  • Wu J, Zhao Q, Yang Q, Liu H, Li Q, Yi X, Cheng Y, Guo L, Fan C, Zhou Y (2016) Comparative transcriptomic analysis uncovers the complex genetic network for resistance to Sclerotinia sclerotiorum in Brassica napus. Sci Rep 6:19007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu T, Peng C, Li B, Wu W, Kong L, Li F, Chu Z, Liu F, Ding X (2019) OsPGIP1-mediated resistance to bacterial leaf streak in rice is beyond responsive to the polygalacturonase of Xanthomonasoryzaepv. oryzicola. Rice 12(1):90

    Article  PubMed  PubMed Central  Google Scholar 

  • Xing S, Wallmeroth N, Berendzen KW, Grefen C (2016) Techniques for the analysis of protein-protein interactions in vivo. Plant Physiol 171(2):727–758

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yang G, Tang L, Gong Y, Xie J, Fu Y, Jiang D, Li G, Collinge DB, Chen W, Cheng J (2018) A cerato-platanin protein SsCP1 targets plant PR1 and contributes to virulence of Sclerotinia sclerotiorum. New Phytol 217(2):739–755

    Article  CAS  PubMed  Google Scholar 

  • Zhu G, Liang E, Lan X, Li Q, Qian J, Tao H, Zhang M, Xiao N, Zuo S, Chen J, Gao Y (2019) ZmPGIP3 gene encodes a polygalacturonase-inhibiting protein that enhances resistance to sheath blight in rice. Phytopathology 109(10):1732–1740

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This research was supported by the China Postdoctoral Science Foundation (2019M652726) and the Postdoctoral Program of Wuhan Academy of Agricultural Sciences (bshxm201801). We would like to thank Editage (http://www.editage.cn) for English language editing.

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Authors and Affiliations

  1. Institute of Crops, Wuhan Academy of Agricultural Sciences, Wuhan, 430065, Hubei, China

    Zhuanrong Wang, Lili Wan & Yuhong Sun

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

    Xiaohui Zhang, Qiang Xin, Yixian Song, Dengfeng Hong & Guangsheng Yang

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  1. Zhuanrong Wang
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  2. Lili Wan
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Wang, Z., Wan, L., Zhang, X. et al. Interaction between Brassica napus polygalacturonase inhibition proteins and Sclerotinia sclerotiorum polygalacturonase: implications for rapeseed resistance to fungal infection. Planta 253, 34 (2021). https://doi.org/10.1007/s00425-020-03556-2

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