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Antifungal proteins (<jats:styled-content style=\"fixed-case\">AFP<\/jats:styled-content>s) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy\u2010to\u2010use, open access viral vector based on <jats:italic>Tobacco mosaic virus<\/jats:italic> (<jats:styled-content style=\"fixed-case\">TMV<\/jats:styled-content>). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated <jats:styled-content style=\"fixed-case\">TMV<\/jats:styled-content> fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different <jats:styled-content style=\"fixed-case\">AFP<\/jats:styled-content>s in <jats:italic>Nicotiana benthamiana<\/jats:italic> leaves, namely the <jats:italic>Aspergillus giganteus <\/jats:italic><jats:styled-content style=\"fixed-case\">AFP<\/jats:styled-content> and the <jats:italic>Penicillium digitatum<\/jats:italic> AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when <jats:styled-content style=\"fixed-case\">AFP<\/jats:styled-content>s were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders <jats:styled-content style=\"fixed-case\">AFP<\/jats:styled-content>s fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from <jats:italic>Botrytis cinerea<\/jats:italic> infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.<\/jats:p>","DOI":"10.1111\/pbi.13038","type":"journal-article","created":{"date-parts":[[2018,12,6]],"date-time":"2018-12-06T15:36:56Z","timestamp":1544110616000},"page":"1069-1080","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":48,"title":["Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus"],"prefix":"10.1111","volume":"17","author":[{"given":"Xiaoqing","family":"Shi","sequence":"first","affiliation":[{"name":"Centre for Research in Agricultural Genomics (CRAG CSIC\u2010IRTA\u2010UAB\u2010UB) Cerdanyola del Vall\u00e8s Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Teresa","family":"Cordero","sequence":"additional","affiliation":[{"name":"Instituto de Biolog\u00eda Molecular y Celular de Plantas (IBMCP CSIC\u2010Universitat Polit\u00e8cnica de Val\u00e8ncia) Valencia Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Garrigues","sequence":"additional","affiliation":[{"name":"Instituto de Agroqu\u00edmica y Tecnolog\u00eda de Alimentos (IATA, CSIC) Paterna Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose F.","family":"Marcos","sequence":"additional","affiliation":[{"name":"Instituto de Agroqu\u00edmica y Tecnolog\u00eda de Alimentos (IATA, CSIC) Paterna Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9\u2010Antonio","family":"Dar\u00f2s","sequence":"additional","affiliation":[{"name":"Instituto de Biolog\u00eda Molecular y Celular de Plantas (IBMCP CSIC\u2010Universitat Polit\u00e8cnica de Val\u00e8ncia) Valencia Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5578-3175","authenticated-orcid":false,"given":"Mar\u00eda","family":"Coca","sequence":"additional","affiliation":[{"name":"Centre for Research in Agricultural Genomics (CRAG CSIC\u2010IRTA\u2010UAB\u2010UB) Cerdanyola del Vall\u00e8s Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2018,12,6]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1742-4658.2009.07011.x"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.fgb.2014.10.012"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.3390\/jof3040057"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2229-14-102"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00009a032"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bcp.2016.11.019"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.plaphy.2017.12.037"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2015.01.028"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1023\/B:PLAN.0000028791.34706.80"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00425-005-0069-z"},{"key":"e_1_2_7_12_1","first-page":"1","volume-title":"Plant Viral Vectors","author":"Dawson W.O.","year":"2014"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1364-3703.2011.00783.x"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.88.16.7204"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature10947"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.2016.0332"},{"key":"e_1_2_7_17_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-015-7110-3"},{"key":"e_1_2_7_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-15277-w"},{"key":"e_1_2_7_19_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2018.02370"},{"key":"e_1_2_7_20_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1318"},{"key":"e_1_2_7_21_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11248-006-0001-8"},{"key":"e_1_2_7_22_1","doi-asserted-by":"publisher","DOI":"10.1111\/pbi.12299"},{"key":"e_1_2_7_23_1","doi-asserted-by":"publisher","DOI":"10.3390\/biomedicines5030044"},{"key":"e_1_2_7_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.fbr.2012.07.002"},{"key":"e_1_2_7_25_1","doi-asserted-by":"publisher","DOI":"10.1023\/A:1006496308160"},{"key":"e_1_2_7_26_1","first-page":"1","article-title":"Molecular farming in barley: development of a novel production platform to produce human antimicrobial peptide LL\u201037","volume":"1700628","author":"Hol\u00e1skov\u00e1 E.","year":"2018","journal-title":"Biotechnol. 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