Temporal proteomic profiling via 4D-DIA reveals early defense mechanisms and core resistance determinants in soybean against Phakopsora pachyrhizi

Abstract

Asian soybean rust, caused by Phakopsora pachyrhizi, is a devastating fungal disease threatening global soybean production, particularly in tropical regions where chemical control is increasingly unsustainable. This study employed cutting-edge 4D-DIA proteomics to investigate molecular defense mechanisms in resistant (SX6907) and susceptible (Tianlong 1) soybean cultivars during early infection (12 hpi and 3 dpi). We identified 12,852 proteins, with 1,510 differentially expressed proteins (DEPs) revealing genotype-specific responses. Resistant plants exhibited sustained upregulation of immune receptors (CRKs, LRR-RLKs), MAPK signaling components, and cell wall reinforcement proteins (peroxidases, XTHs), alongside dynamic modulation of calcium signaling and ROS homeostasis. These patterns suggest key pathways enriched in resistance may include phenylpropanoid biosynthesis, isoflavonoid production, and ER stress responses, while susceptible plants showed suppression of photosynthesis and defense pathways. Weighted Protein Co-expression Network Analysis(WPCNA) highlighted co-expression modules linked to resistance, potentially including NLR-mediated effector-triggered immunity. Crucially, DIR proteins and organelle-specific defense hubs (e.g., chloroplasts, nuclei) were implicated in rust resistance. Validation by qPCR confirmed concordance for 84% of tested DEPs. Our findings provide a protein-level blueprint of soybean rust resistance, identifying candidate targets for marker-assisted breeding and genetic engineering to develop durable resistant varieties, reducing reliance on fungicides.

Keywords

Soybean / Phakopsora pachyrhizi / 4D-DIA proteomics / Plant immunity / Resistance mechanisms / Molecular breeding

References

[1] Ahuja I, Kissen R, Bones AM. Phytoalexins in defense against pathogens. Trends Plant Sci, 2012, 17: 73-90

[2] Bacete L, Mélida H, Miedes E, Molina A. Plant cell wall-mediated immunity: cell wall changes trigger disease resistance responses. Plant J, 2018, 93: 614-636

[3] Birkenbihl RP, Kracher B, Ross A, Kramer K, Finkemeier I, Somssich IE. Principles and characteristics of the Arabidopsis WRKY regulatory network during early MAMP-triggered immunity. Plant J, 2018, 96: 487-502

[4] Chen HF, Zhao S, Yang ZL, Sha AH, Wan Q, Zhang CJ, Chen LM, Yuan SL, Qiu DZ, Chen SL, Shan ZH, Zhou XA. Genetic analysis and molecular mapping of resistance gene to Phakopsora pachyrhizi in soybean germplasm SX6907. Theor Appl Genet, 2015, 128: 733-743

[5] Chicowski AS, Bredow M, Utiyama AS, Marcelino-Guimarães FC, Whitham SA. Soybean-Phakopsora pachyrhizi interactions: towards the development of next-generation disease-resistant plants. Plant Biotechnol J, 2024, 22: 296-315

[6] Childs SP, King ZR, Walker DR, Harris DK, Pedley KF, Buck JW, Boerma HR, Li Z. Discovery of a seventh Rpp soybean rust resistance locus in soybean accession PI 605823. Theor Appl Genet, 2018, 131: 27-41

[7] Dixon RA, Achnine L, Kota P, Liu CJ, Reddy MS, Wang L. The phenylpropanoid pathway and plant defence-a genomics perspective. Mol Plant Pathol, 2002, 3: 371-390

[8] Favoretto VR, Murithi HM, Leles EP, da Santos FM, Chigeza G, Goldsmith P, Coyne D, Clough SJ. Soybean rust-resistant and tolerant varieties identified through the pan-African trial network. Pest Manag Sci, 2025, 81: 2769-2775

[9] Gillet LC, Navarro P, Tate S, Röst H, Selevsek N, Reiter L, Bonner R, Aebersold R. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics, 2012, 11(6 O111.016717

[10] Gimenez-Ibanez S, Boter M, Solano R. Novel players fine-tune plant trade-offs. Essays Biochem, 2015, 58: 83-100

[11] Godoy C (2011) Risk and management of fungicide resistance in the Asian soybean rust fungus Phakopsora pachyrhizi. In: Thind TS (eds) Fungicide resistance in crop protection: risk and management. https://doi.org/10.1079/9781845939052.0087

[12] Godoy CV, Bueno ADF, Gazziero DLP. Brazilian soybean pest management and threats to its sustainability. Outlooks on Pest Management, 2015

[13] Godoy CV, Seixas CDS, Soares RMG, Franscismar C, Maurício C, Leila M. Asian soybean rust in Brazil: past, present, and future. Pesq Agropec Bras, 2016, 51: 407-421

[14] Goellner K, Loehrer M, Langenbach C, Conrath U, Koch E, Schaffrath U. Phakopsora pachyrhizi, the causal agent of Asian soybean rust. Mol Plant Pathol, 2010, 11: 169-177

[15] Görg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics. Proteomics, 2004, 4: 3665-3685

[16] Grant MR, Jones JD. Hormone (dis)harmony moulds plant health and disease. Science, 2009, 324: 750-752

[17] Gu J, Sun J, Liu N, Sun X, Liu C, Wu L, Liu G, Zeng F, Hou C, Han S, Zhen W, Wang D. A novel cysteine-rich receptor-like kinase gene, TaCRK2, contributes to leaf rust resistance in wheat. Mol Plant Pathol, 2020, 21: 732-746

[18] Gupta YK, Marcelino-Guimarães FC, Lorrain C, Farmer A, Haridas S, Ferreira EGC, Lopes-Caitar VS, Oliveira LS, Morin E, Widdison S, Cameron C, Inoue Y, Thor K, Robinson K, Drula E, Henrissat B, LaButti K, Bini AMR, Paget E, Singan V, Daum C, Dorme C, van Hoek M, Janssen A, Chandat L, Tarriotte Y, Richardson J, Melo B, Wittenberg AHJ, Schneiders H, Peyrard S, Zanardo LG, Holtman VC, Coulombier-Chauvel F, Link TI, Balmer D, Müller AN, Kind S, Bohnert S, Wirtz L, Chen C, Yan M, Ng V, Gautier P, Meyer MC, Voegele RT, Liu Q, Grigoriev IV, Conrath U, Brommonschenkel SH, Loehrer M, Schaffrath U, Sirven C, Scalliet G, Duplessis S, van Esse HP. Major proliferation of transposable elements shaped the genome of the soybean rust pathogen Phakopsora pachyrhizi. Nat Commun, 2023, 14: 1835

[19] Hossain MZ, Ishiga Y, Yamanaka N, Ogiso-Tanaka E, Yamaoka Y. Soybean leaves transcriptomic data dissects the phenylpropanoid pathway genes as a defence response against Phakopsora pachyrhizi. Plant Physiol Biochem, 2018, 132: 424-433

[20] Ishiga Y, Uppalapati SR, Gill US, Huhman D, Tang Y, Mysore KS. Transcriptomic and metabolomic analyses identify a role for chlorophyll catabolism and phytoalexin during Medicago nonhost resistance against Asian soybean rust. Sci Rep, 2015, 5 13061

[21] Jiang B, Su C, Wang Y, Xu X, Li Y, Ma D. Genome-wide identification of glutathione peroxidase (GPX) family genes and silencing TaGPX3.2A reduced disease resistance in wheat. Plant Physiol Biochem, 2023, 204 108139

[22] Jones JDG, Dangl JL. The plant immune system. Nature, 2006, 444: 323-329

[23] Juliatti BCM, Monteiro ACA, dos Santos Botelho DM, Leal FDS, de Resende MLV, Juliatti FC, Pozza EA. The value of chemical and physical barriers against Asian rust in soybean genotypes with partial resistance. Eur J Plant Pathol, 2025

[24] Kadam SB, Barvkar VT. COI1-mediated jasmonic acid signalling regulates mycorrhizal colonisation intensity and is an indispensable component of mycorrhiza-induced resistance. Physiol Plant, 2024, 176 e14643

[25] Klosowski AC, Mio LLMD, Miessner S, Rodrigues R, Stammler G. Detection of the F129L mutation in the cytochrome b gene in Phakopsora pachyrhizi. Pest Manag Sci, 2016, 72: 1211-1215

[26] Langenbach C, Campe R, Beyer SF, Mueller AN, Conrath U. Fighting Asian soybean rust. Front Plant Sci, 2016, 7 797

[27] Li G, Newman M, Yu H, Rashidzade M, Martínez-Soto D, Caicedo A, Allen KS, Ma LJ. Fungal effectors: past, present, and future. Curr Opin Microbiol, 2024, 81 102526

[28] Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell, 2002, 110: 213-222

[29] Li N, Zhao M, Liu T, Dong L, Cheng Q, Wu J, Wang L, Chen X, Zhang C, Lu W, Xu P, Zhang S. A novel soybean dirigent gene GmDIR22 contributes to promotion of lignan biosynthesis and enhances resistance to Phytophthora sojae. Front Plant Sci, 2017, 8: 1185

[30] Li Y, Zheng X, Wang C, Hou D, Li T, Li D, Ma C, Sun Z, Tian Y. Pear xyloglucan endotransglucosylase/hydrolases PcBRU1 promotes stem growth through regulating cell wall elongation. Plant Sci, 2021, 312 111026

[31] Liu Q, Liu Y, Tang Y, Chen J, Ding W. Overexpression of NtWRKY50 increases resistance to Ralstonia solanacearum and alters salicylic acid and jasmonic acid production in tobacco. Front Plant Sci, 2017, 8: 1710

[32] Luan Q, Chen C, Liu M, Li Q, Wang L, Ren Z. CsWRKY50 mediates defense responses to Pseudoperonospora cubensis infection in Cucumis sativus. Plant Sci, 2019, 279: 59-69

[33] Luna E, Pastor V, Robert J, Flors V, Mauch-Mani B, Ton J. Callose deposition: a multifaceted plant defense response. Mol Plant Microbe Interact, 2011, 24: 183-193

[34] Michalski A, Cox J, Mann M. More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS. J Proteome Res, 2011, 10: 1785-1793

[35] Miedes E, Zarra I, Hoson T, Herbers K, Sonnewald U, Lorences EP. Xyloglucan endotransglucosylase and cell wall extensibility. J Plant Physiol, 2011, 168: 196-203

[36] Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, Kaku H, Shibuya N. CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci U S A, 2007, 104: 19613-19618

[37] Monteiro F, Nishimura MT. Structural, functional, and genomic diversity of plant NLR proteins: an evolved resource for rational wngineering of plant immunity. Annu Rev Phytopathol, 2018, 56: 243-267

[38] Navarro L, Bari R, Achard P, Lisón P, Nemri A, Harberd NP, Jones JD. DELLAs control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling. Curr Biol, 2008, 18: 650-655

[39] Ouyang H, Sun G, Li K, Wang R, Lv X, Zhang Z, Zhao R, Wang Y, Shu H, Jiang H, Zhang S, Wu J, Zhang Q, Chen X, Liu T, Ye W, Wang Y, Wang Y. Profiling of Phakopsora pachyrhizi transcriptome revealed co-expressed virulence effectors as prospective RNA interference targets for soybean rust management. J Integr Plant Biol, 2024, 66(11): 2543-2560

[40] Pallas V, García JA. How do plant viruses induce disease? Interactions and interference with host components. J Gen Virol, 2011, 92: 2691-2705

[41] Pandey SP, Somssich IE. The role of WRKY transcription factors in plant immunity. Plant Physiol, 2009, 150: 1648-1655

[42] Panthee DR, Marois JJ, Wright DL, Narváez D, Yuan JS, Stewart CN. Differential expression of genes in soybean in response to the causal agent of Asian soybean rust (Phakopsora pachyrhizi Sydow) is soybean growth stage-specific. Theor Appl Genet, 2009, 118: 359-370

[43] Panthee DR, Yuan JS, Wright DL, Marois JJ, Mailhot D, Stewart CNJr. Gene expression analysis in soybean in response to the causal agent of Asian soybean rust (Phakopsora pachyrhizi Sydow) in an early growth stage. Funct Integr Genomics, 2007, 7(4): 291-301

[44] Peng Y, Yang J, Li X, Zhang Y. Salicylic acid: biosynthesis and signaling. Annu Rev Plant Biol, 2021, 72: 761-791

[45] Planas A. Peptidoglycan deacetylases in bacterial cell wall remodeling and pathogenesis. Curr Med Chem, 2022, 29: 1293-1312

[46] Ratnaparkhe MB, Marmat N, Kumawat G, Shivakumar M, Kamble VG, Nataraj V, Ramesh SV, Deshmukh MP, Singh AK, Sonah H, Deshmukh RK, Prasad M, Chand S, Gupta S. Whole genome re-sequencing of soybean accession EC241780 providing genomic landscape of candidate genes involved in rust resistance. Curr Genomics, 2020, 21(7): 504-511

[47] Saintenac C, Cambon F, Aouini L, Verstappen E, Ghaffary SMT, Poucet T, Marande W, Berges H, Xu S, Jaouannet M, Favery B, Alassimone J, Sánchez-Vallet A, Faris J, Kema G, Robert O, Langin T. A wheat cysteine-rich receptor-like kinase confers broad-spectrum resistance against Septoria tritici blotch. Nat Commun, 2021, 12 433

[48] Shan ZH, Liu Y, Ba HP, Shan LM, Chen HF, Sha AH, Qiu DZ, Yang ZL, Chen SL, Zhou XA. New soybean germplasm resistance to Phakopsora pachyrhizi Syd. Chin J Oil Crop Sci, 2012, 34: 188-192

[49] Shuken SR. An introduction to mass spectrometry-based proteomics. J Proteome Res, 2023, 22: 2151-2171

[50] Silva E, Perez da Graça J, Porto C, Martin do Prado R, Nunes E, Corrêa Marcelino-Guimarães F, Conrado Meyer M, Jorge Pilau E,. Untargeted metabolomics analysis by UHPLC-MS/MS of soybean plant in a compatible response to Phakopsora pachyrhizi infection. Metabolites, 2021, 11(3): 179

[51] Sohn SI, Pandian S, Oh YJ, Kang HJ, Cho WS, Cho YS. Metabolic engineering of isoflavones: an updated overview. Front Plant Sci, 2021, 12 670103

[52] Soria-Guerra RE, Rosales-Mendoza S, Chang S, Haudenshield JS, Padmanaban A, Rodriguez-Zas S, Hartman GL, Ghabrial SA, Korban SS. Transcriptome analysis of resistant and susceptible genotypes of Glycine tomentella during Phakopsora pachyrhizi infection reveals novel rust resistance genes. Theor Appl Genet, 2010, 120(71315-1333

[53] Sun T, Zhang Y. Map kinase cascades in plant development and immune signaling. EMBO Rep, 2022, 23 e53817

[54] Sun TP, Gubler F. Molecular mechanism of gibberellin signaling in plants. Annu Rev Plant Biol, 2004, 55: 197-223

[55] Torres MA, Jones JD, Dangl JL. Reactive oxygen species signaling in response to pathogens. Plant Physiol, 2006, 141: 373-378

[56] Tremblay A, Hosseini P, Li S, Alkharouf NW, Matthews BF. Analysis of Phakopsora pachyrhizi transcript abundance in critical pathways at four time-points during infection of a susceptible soybean cultivar using deep sequencing. BMC Genomics, 2013, 14 614

[57] Vlot AC, Dempsey DA, Klessig DF. Salicylic acid, a multifaceted hormone to combat disease. Annu Rev Phytopathol, 2009, 47: 177-206

[58] Wang J, Wang J, Shang H, Chen X, Xu X, Hu X. TaXa21, a leucine-rich repeat receptor-like kinase gene associated with TaWRKY76 and TaWRKY62, plays positive roles in wheat high-temperature seedling plant resistance to Puccinia striiformis f. sp. tritici. Mol Plant Microbe Interact, 2019, 32: 1526-1535

[59] Wu Y, Wen J, Xia Y, Zhang L, Du H. Evolution and functional diversification of R2R3-MYB transcription factors in plants. Hortic Res, 2022, 9 uhac058

[60] Yang S, Zhang Q, Li T, Du J, Yang S, Yang C. AtSIA1, an ABC1-like kinase, regulates salt response in Arabidopsis. Biol, 2012, 67: 1107-1111

[61] Yorinori JT, Paiva WM, Frederick RD, Costamilan LM, Bertagnolli PF, Hartman GE, Godoy CV, Nunes JJr. Epidemics of soybean rust (Phakopsora pachyrhizi) in Brazil and Paraguay from 2001 to 2003. Plant Dis, 2005, 89: 675-677

[62] Zaynab M, Fatima M, Abbas S, Sharif Y, Umair M, Zafar MH, Bahadar K. Role of secondary metabolites in plant defense against pathogens. Microb Pathog, 2018, 124: 198-202

[63] Zhang L, Zhu Q, Tan Y, Deng M, Zhang L, Cao Y, Guo X. Mitogen-activated protein kinases MPK3 and MPK6 phosphorylate receptor-like cytoplasmic kinase CDL1 to regulate soybean basal immunity. Plant Cell, 2024, 36: 963-986

[64] Zhang S, Klessig DF. Mapk cascades in plant defense signaling. Trends Plant Sci, 2001, 6: 520-527

[65] Zhang Y, Tian H, Chen D, Zhang H, Sun M, Chen S, Qin Z, Ding Z, Dai S. Cysteine-rich receptor-like protein kinases: emerging regulators of plant stress responses. Trends Plant Sci, 2023, 28: 776-794

[66] Zhao ZX, Xu YJ, Lei Y, Li Q, Zhao JQ, Li Y, Fan J, Xiao S, Wang WM. Annexin 8 negatively regulates RPW8.1-mediated cell death and disease resistance in Arabidopsis. J Integr Plant Biol, 2021, 63: 378-392

Funding

National Key Research and Development Program of China(2022YFF1001504)

Key Laboratory of Agricultural Information Service Technology(CAAS-ASTIP-2022-OCRI)

Hubei Province Technological Innovation Program(2024BBB003)

RIGHTS & PERMISSIONS

The Author(s)