{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T18:10:56Z","timestamp":1789150256912,"version":"build-2803163510"},"reference-count":28,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Open Project of Chongqing Key Laboratory of Scientific Utilization of Tobacco Resources","award":["HX20230208"],"award-info":[{"award-number":["HX20230208"]}]},{"name":"Chongqing College Student Innovation and Entrepreneurship Training Program","award":["S20241063378"],"award-info":[{"award-number":["S20241063378"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    <jats:italic>Nicotiana benthamiana<\/jats:italic>\n                    is a widely used platform for plant molecular farming, yet recombinant protein yields are frequently compromised by the host\u2019s innate defense mechanisms, particularly proteolytic degradation. While the general effects of Agroinfiltration are known, the distinct contributions of mechanical injury, bacterial perception, and product-specific stress remain poorly resolved. Here we utilized high-depth 4D-DIA proteomics to dissect the host response across three dimensions: physical stress (buffer infiltration), pathogen-associated stress (\n                    <jats:italic>Agrobacterium<\/jats:italic>\n                    ), and product-associated stress (GFP vs. the FMDV capsid precursor P1_2A). We demonstrate that buffer infiltration is not a neutral event but an independent inducer of cell wall remodeling and oxidative stress. By filtering out these background effects, we defined a core Agrobacterium-responsive proteome characterized by a growth-defense trade-off. We also expanded the known protease repertoire of\n                    <jats:italic>N. benthamiana<\/jats:italic>\n                    to 1,505 enzymes through improved genomic annotation. We found that the expression of the FMDV capsid precursor P1_2A was associated with a distinct and more pronounced protease profile compared to soluble GFP, characterized by the upregulation of subtilases and cysteine proteases. These findings suggest that host proteolytic responses vary with the recombinant cargo, a factor worth considering when designing engineering strategies for the production of complex biopharmaceuticals in plants.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0356905","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T17:43:27Z","timestamp":1789148607000},"page":"e0356905","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["4D-DIA proteomics reveals distinct proteolytic landscapes induced by mechanical stress, Agrobacterium, and a viral capsid precursor"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-0990-5145","authenticated-orcid":true,"given":"You","family":"Tang","sequence":"first","affiliation":[{"name":"Chongqing Key Laboratory of Scientific Utilization of Tobacco Resources","place":["Chongqing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoping","family":"Qiu","sequence":"additional","affiliation":[{"name":"Southwest University","place":["Chongqing, China"],"department":["School of Life Science"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinli","family":"Ye","sequence":"additional","affiliation":[{"name":"Southwest University","place":["Chongqing, China"],"department":["School of Life Science"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaorui","family":"Ma","sequence":"additional","affiliation":[{"name":"Southwest University","place":["Chongqing, China"],"department":["School of Life Science"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yilin","family":"Mao","sequence":"additional","affiliation":[{"name":"Southwest University","place":["Chongqing, China"],"department":["School of Life Science"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiwei","family":"Yang","sequence":"additional","affiliation":[{"name":"Southwest University","place":["Chongqing, China"],"department":["School of Life Science"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yike","family":"Chen","sequence":"additional","affiliation":[{"name":"Southwest University","place":["Chongqing, China"],"department":["School of Life Science"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2299-8505","authenticated-orcid":true,"given":"Yiming","family":"Sun","sequence":"additional","affiliation":[{"name":"Southwest University","place":["Chongqing, China"],"department":["School of Life Science"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lijun","family":"Zhu","sequence":"additional","affiliation":[{"name":"Chongqing Key Laboratory of Scientific Utilization of Tobacco Resources","place":["Chongqing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,9,11]]},"reference":[{"key":"pone.0356905.ref001","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1146\/annurev-phyto-080417-050141","article-title":"The rise and rise of Nicotiana benthamiana: a plant for all reasons","volume":"56","author":"J Bally","year":"2018","journal-title":"Annu Rev Phytopathol"},{"issue":"1","key":"pone.0356905.ref002","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1111\/pbi.70460","article-title":"Nicotiana benthamiana\u2019s responses to agroinfiltration, a treasure grove of new avenues to improve protein yields in plant molecular farming","volume":"24","author":"L-P Hamel","year":"2026","journal-title":"Plant Biotechnol J"},{"key":"pone.0356905.ref003","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.copbio.2019.10.006","article-title":"Proteases of Nicotiana benthamiana: an emerging battle for molecular 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benthamiana","volume":"16","author":"F Grosse-Holz","year":"2018","journal-title":"Plant Biotechnol J"},{"issue":"3","key":"pone.0356905.ref007","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1007\/s11248-021-00256-9","article-title":"Metabolic effects of agro-infiltration on N. benthamiana accessions","volume":"30","author":"M Drapal","year":"2021","journal-title":"Transgenic Res"},{"issue":"5","key":"pone.0356905.ref008","doi-asserted-by":"crossref","first-page":"1146","DOI":"10.1111\/pbi.14252","article-title":"Heterologous expression of influenza haemagglutinin leads to early and transient activation of the unfolded protein response in Nicotiana benthamiana","volume":"22","author":"L-P Hamel","year":"2024","journal-title":"Plant Biotechnol J"},{"issue":"1","key":"pone.0356905.ref009","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1111\/pbi.70075","article-title":"Molecular changes in agroinfiltrated leaves of Nicotiana\u2009benthamiana expressing suppressor of silencing P19 and coronavirus-like particles","volume":"24","author":"L-P Hamel","year":"2026","journal-title":"Plant Biotechnol J"},{"issue":"3","key":"pone.0356905.ref010","doi-asserted-by":"crossref","first-page":"740","DOI":"10.1111\/jipb.13841","article-title":"Navigating the landscape of plant proteomics","volume":"67","author":"T Sang","year":"2025","journal-title":"J Integr Plant Biol"},{"key":"pone.0356905.ref011","doi-asserted-by":"crossref","first-page":"633706","DOI":"10.3389\/fvets.2021.633706","article-title":"The high immunity induced by the virus-like particles of foot-and-mouth disease virus serotype O","volume":"8","author":"Y Xiao","year":"2021","journal-title":"Front Vet Sci"},{"issue":"9","key":"pone.0356905.ref012","article-title":"Improvement and application of vacuum-infiltration system in tomato","volume":"11","author":"X Yao","year":"2024","journal-title":"Hortic 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Kourelis","year":"2019","journal-title":"BMC Genomics"},{"issue":"4","key":"pone.0356905.ref016","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"KJ Livak","year":"2001","journal-title":"Methods"},{"issue":"10","key":"pone.0356905.ref017","doi-asserted-by":"crossref","first-page":"3319","DOI":"10.1002\/bit.28781","article-title":"Synergetic light and cytokinin treatments mitigate the recombinant protein yield depression induced by high-density cultivation of hydroponically-grown Nicotiana benthamiana","volume":"121","author":"S Bilotta","year":"2024","journal-title":"Biotechnol Bioeng"},{"issue":"4","key":"pone.0356905.ref018","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1016\/j.cell.2006.03.037","article-title":"Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation","volume":"125","author":"C Zipfel","year":"2006","journal-title":"Cell"},{"issue":"8","key":"pone.0356905.ref019","doi-asserted-by":"crossref","first-page":"2248","DOI":"10.1111\/pbi.14342","article-title":"Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming","volume":"22","author":"N Prudhomme","year":"2024","journal-title":"Plant Biotechnol J"},{"issue":"5","key":"pone.0356905.ref020","doi-asserted-by":"crossref","first-page":"1068","DOI":"10.1111\/pbi.12852","article-title":"The transcriptome, extracellular proteome and active secretome of agroinfiltrated Nicotiana benthamiana uncover a large, diverse protease repertoire","volume":"16","author":"F Grosse-Holz","year":"2018","journal-title":"Plant Biotechnol J"},{"key":"pone.0356905.ref021","doi-asserted-by":"crossref","DOI":"10.1093\/nar\/gkt953","article-title":"MEROPS: the database of proteolytic enzymes, their substrates and inhibitors","volume":"42","author":"ND Rawlings","year":"2014","journal-title":"Nucleic Acids Res"},{"issue":"8","key":"pone.0356905.ref022","doi-asserted-by":"crossref","first-page":"1670","DOI":"10.1111\/pbi.13092","article-title":"Activity-based proteomics reveals nine target proteases for the recombinant protein-stabilizing inhibitor SlCYS8 in Nicotiana benthamiana","volume":"17","author":"PV Jutras","year":"2019","journal-title":"Plant Biotechnol J"},{"key":"pone.0356905.ref023","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/j.biochi.2015.06.017","article-title":"Nicotiana benthamiana cathepsin B displays distinct enzymatic features which differ from its human relative and aleurain-like protease","volume":"122","author":"M Niemer","year":"2016","journal-title":"Biochimie"},{"issue":"5685","key":"pone.0356905.ref024","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1126\/science.1099859","article-title":"A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death","volume":"305","author":"N Hatsugai","year":"2004","journal-title":"Science"},{"key":"pone.0356905.ref025","unstructured":"Thomas EL. Protein processing in Nicotiana benthamiana by vacuolar processing enzymes and other proteases. University of Oxford; 2020."},{"issue":"3","key":"pone.0356905.ref026","doi-asserted-by":"crossref","first-page":"846","DOI":"10.1111\/nph.19894","article-title":"Improving transient protein expression in agroinfiltrated Nicotiana benthamiana","volume":"243","author":"K Beritza","year":"2024","journal-title":"New Phytol"},{"key":"pone.0356905.ref027","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1387\/ijdb.130199bl","article-title":"The roles of bacterial and host plant factors in Agrobacterium-mediated genetic transformation","volume":"57","author":"B Lacroix","year":"2013","journal-title":"Int J Dev Biol"},{"issue":"1","key":"pone.0356905.ref028","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1104\/pp.112.200212","article-title":"Formation of complex extrachromosomal T-DNA structures in Agrobacterium tumefaciens-infected plants","volume":"160","author":"K Singer","year":"2012","journal-title":"Plant Physiol"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0356905","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T17:43:32Z","timestamp":1789148612000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0356905"}},"subtitle":[],"editor":[{"given":"Vitaly","family":"Citovsky","sequence":"first","affiliation":[{"name":"Stony Brook University","place":["UNITED STATES OF AMERICA"]}],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,9,11]]},"references-count":28,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9,11]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0356905","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,11]]}}}