{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T03:23:24Z","timestamp":1760239404190,"version":"build-2065373602"},"reference-count":45,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2020,11,3]],"date-time":"2020-11-03T00:00:00Z","timestamp":1604361600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Aspergillus flavus is a saprophytic cosmopolitan fungus, capable of infecting crops both pre- and post-harvest and exploiting different secondary metabolites, including aflatoxins. Aflatoxins are known carcinogens to animals and humans, but display no clear effect in host plants such as maize. In a previous study, we mined the genome of A. flavus to identify secondary metabolite clusters putatively involving the pathogenesis process in maize. We now focus on cluster 32, encoding for fungal effectors such as salicylate hydroxylase (SalOH), and necrosis- and ethylene-inducing proteins (npp1 domain protein) whose expression is triggered upon kernel contact. In order to understand the role of this genetic cluster in maize kernel infection, mutants of A. flavus, impaired or enhanced in specific functions (e.g., cluster 32 overexpression), were studied for their ability to cause disease. Within this frame, we conducted histological and histochemical experiments to verify the expression of specific genes within the cluster (e.g., SalOH, npp1), the production of salicylate, and the presence of its dehydroxylated form. Results suggest that the initial phase of fungal infection (2 days) of the living tissues of maize kernels (e.g., aleuron) coincides with a significant increase of fungal effectors such as SalOH and Npp1 that appear to be instrumental in eluding host defences and colonising the starch-enriched tissues, and therefore suggest a role of cluster 32 to the onset of infection.<\/jats:p>","DOI":"10.3390\/ijms21218213","type":"journal-article","created":{"date-parts":[[2020,11,3]],"date-time":"2020-11-03T09:09:32Z","timestamp":1604394572000},"page":"8213","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Aspergillus flavus Exploits Maize Kernels Using an \u201cOrphan\u201d Secondary Metabolite Cluster"],"prefix":"10.3390","volume":"21","author":[{"given":"Ludovica","family":"Antiga","sequence":"first","affiliation":[{"name":"Department of Environmental Biology, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Roma, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sonia Roberta","family":"La Starza","sequence":"additional","affiliation":[{"name":"Department of Environmental Biology, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Roma, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cecilia","family":"Miccoli","sequence":"additional","affiliation":[{"name":"CREA-OFA, Via di Fioranello 52, 00134 Rome, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Simone","family":"D\u2019Angeli","sequence":"additional","affiliation":[{"name":"Department of Environmental Biology, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Roma, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9383-8766","authenticated-orcid":false,"given":"Valeria","family":"Scala","sequence":"additional","affiliation":[{"name":"CREA-DC, via C.G. Bertero 22, 00156 Roma, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marco","family":"Zaccaria","sequence":"additional","affiliation":[{"name":"Department of Biology, Boston College, 140 Commonwealth avenue, Chestnut Hill, MA 02467, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaomei","family":"Shu","sequence":"additional","affiliation":[{"name":"Center for Biotechnology and Genomics, Texas Tech University, 2500 Broadway, Lubbock, TX 79410, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gregory","family":"Obrian","sequence":"additional","affiliation":[{"name":"Department of Plant Pathology, North Carolina State University, Raleigh, CA 27607, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5901-1386","authenticated-orcid":false,"given":"Marzia","family":"Beccaccioli","sequence":"additional","affiliation":[{"name":"Department of Environmental Biology, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Roma, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gary A.","family":"Payne","sequence":"additional","affiliation":[{"name":"Department of Plant Pathology, North Carolina State University, Raleigh, CA 27607, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3671-6783","authenticated-orcid":false,"given":"Massimo","family":"Reverberi","sequence":"additional","affiliation":[{"name":"Department of Environmental Biology, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Roma, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,11,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1146\/annurev-phyto-072910-095221","article-title":"Aspergillus flavus","volume":"49","author":"Amaike","year":"2011","journal-title":"Annu. Rev. Phytopathol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1677","DOI":"10.1099\/mic.0.2007\/007641-0","article-title":"Aspergillus flavus: Human pathogen, allergen and mycotoxin producer","volume":"153","author":"Hedayati","year":"2007","journal-title":"Microbiology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1088","DOI":"10.1080\/02652030701510012","article-title":"Biodiversity of Aspergillus section Flavi in the United States: A review","volume":"24","author":"Horn","year":"2007","journal-title":"Food Addit. Contam."},{"key":"ref_4","unstructured":"Dowd, P.F. (1998). Involvement of Arthropods in the Establishment of Mycotoxigenic Fungi under Field Conditions, Marcel Dekker."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1146\/annurev.phyto.36.1.329","article-title":"Genetics and physiology of aflatoxin biosynthesis","volume":"36","author":"Payne","year":"1998","journal-title":"Annu. Rev. Phytopathol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"167","DOI":"10.4315\/0362-028X-65.1.167","article-title":"Restriction fragment length polymorphism assessment of the heterogeneous nature of maize population GT-MAS: Gk and field evaluation of resistance to aflatoxin production by Aspergillus flavus","volume":"65","author":"Guo","year":"2002","journal-title":"J. Food Prot."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"754","DOI":"10.3389\/fmicb.2020.00754","article-title":"Host induced gene silencing targeting Aspergillus flavus aflM reduced aflatoxin contamination in transgenic maize under field conditions","volume":"11","author":"Raruang","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/j.1439-0523.1991.tb00522.x","article-title":"Preharvest Aflatoxin Contamination in Maize: Resistance and Genetics","volume":"107","author":"Gorman","year":"1991","journal-title":"Plant Breed."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"EFSA Panel on Contaminants in the Food Chain (CONTAM), Schrenk, D., Bignami, M., Bodin, L., Chipman, J.K., del Mazo, J., Grasl-Kraupp, B., Hogstrand, C., Hoogenboom, L., and Leblanc, J.C. (2020). Risk assessment of aflatoxins in food. EFSA J., 18, e06040.","DOI":"10.2903\/j.efsa.2020.6040"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"50","DOI":"10.3389\/fmicb.2014.00050","article-title":"Non-aflatoxigenic Aspergillus flavus to prevent aflatoxin contamination in crops: Advantages and limitations","volume":"5","author":"Ehrlich","year":"2014","journal-title":"Front. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"24328","DOI":"10.1038\/srep24328","article-title":"Aflatoxin B 1 contamination in maize in Europe increases due to climate change","volume":"6","author":"Battilani","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"62","DOI":"10.3389\/fmicb.2015.00062","article-title":"Association of fungal secondary metabolism and sclerotial biology","volume":"6","author":"Calvo","year":"2015","journal-title":"Front. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Soni, P., Gangurde, S.S., Ortega-Beltran, A., Kumar, R., Parmar, S., Sudini, H.K., Lei, Y., Ni, X., Huai, D., and Fountain, J.C. (2020). Functional Biology and Molecular Mechanisms of Host-Pathogen Interactions for Aflatoxin Contamination in Groundnut (Arachis hypogaea L.) and Maize (Zea mays L.). Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.00227"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"937","DOI":"10.1038\/nrmicro1286","article-title":"Fungal secondary metabolism\u2014From biochemistry to genomics","volume":"3","author":"Keller","year":"2005","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"736","DOI":"10.1016\/j.fgb.2010.06.003","article-title":"SMURF: Genomic mapping of fungal secondary metabolite clusters","volume":"47","author":"Khaldi","year":"2010","journal-title":"Fungal Genet. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1111\/j.1364-3703.2009.00594.x","article-title":"Beyond aflatoxin: Four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters","volume":"11","author":"Georgianna","year":"2010","journal-title":"Mol. Plant Pathol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Reverberi, M., Punelli, M., Scala, V., Scarpari, M., Uva, P., Mentzen, W.I., Dolezal, A.L., Woloshuk, C., Pinzari, F., and Fabbri, A.A. (2013). Genotypic and phenotypic versatility of Aspergillus flavus during maize exploitation. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0068735"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1289\/ehp.8562459","article-title":"Aflatrem: A tremorgenic mycotoxin with acute neurotoxic effects","volume":"62","author":"Valdes","year":"1985","journal-title":"Environ. Health Perspect."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1916","DOI":"10.3390\/toxins6061916","article-title":"Comparison of expression of secondary metabolite biosynthesis cluster genes in Aspergillus flavus, A. parasiticus, and A. oryzae","volume":"6","author":"Ehrlich","year":"2014","journal-title":"Toxins (Basel)"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1610","DOI":"10.1104\/pp.102.019836","article-title":"Altered gene expression in three plant species in response to treatment with Nep1, a fungal protein that causes necrosis","volume":"132","author":"Keates","year":"2003","journal-title":"Plant Physiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1111\/j.1364-3703.2007.00382.x","article-title":"Functional analysis of NLP genes from Botrytis elliptica","volume":"8","author":"Staats","year":"2007","journal-title":"Mol. Plant Pathol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Laluk, K., and Mengiste, T. (2010). Necrotroph attacks on plants: Wanton destruction or covert extortion?. Arab. Book\/Am. Soc. Plant Biol., 8.","DOI":"10.1199\/tab.0136"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e02241-17","DOI":"10.1128\/AEM.02241-17","article-title":"The 14-3-3 protein homolog ArtA regulates development and secondary metabolism in the opportunistic plant pathogen Aspergillus flavus","volume":"84","author":"Ibarra","year":"2018","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4289","DOI":"10.1007\/s00253-013-4865-2","article-title":"Genome-wide analysis of the Zn (II) 2 Cys 6 zinc cluster-encoding gene family in Aspergillus flavus","volume":"97","author":"Chang","year":"2013","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1007\/s10658-014-0401-1","article-title":"ROS and 9-oxylipins are correlated with deoxynivalenol accumulation in the germinating caryopses of Triticum aestivum after Fusarium graminearum infection","volume":"139","author":"Nobili","year":"2014","journal-title":"Eur. J. Plant Pathol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-017-09260-8","article-title":"Aspergillus flavus infection triggered immune responses and host-pathogen cross-talks in groundnut during in-vitro seed colonization","volume":"7","author":"Nayak","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Qi, P.-F., Zhang, Y.-Z., Liu, C.-H., Chen, Q., Guo, Z.-R., Wang, Y., Xu, B.-J., Jiang, Y.-F., Zheng, T., and Gong, X. (2019). Functional analysis of FgNahG clarifies the contribution of salicylic acid to wheat (Triticum aestivum) resistance against Fusarium head blight. Toxins (Basel), 11.","DOI":"10.3390\/toxins11020059"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3219","DOI":"10.3389\/fmicb.2018.03219","article-title":"Characterization of a Fusarium graminearum Salicylate Hydroxylase","volume":"9","author":"Hao","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"964","DOI":"10.1094\/MPMI-12-11-0319","article-title":"Molecular characterization and functional analysis of a necrosis-and ethylene-inducing, protein-encoding gene family from Verticillium dahliae","volume":"25","author":"Zhou","year":"2012","journal-title":"Mol. Plant-Microbe Interact."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Feng, B.-Z., Zhu, X.-P., Fu, L., Lv, R.-F., Storey, D., Tooley, P., and Zhang, X.-G. (2014). Characterization of necrosis-inducing NLP proteins in Phytophthora capsici. BMC Plant Biol., 14.","DOI":"10.1186\/1471-2229-14-126"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Cobos, R., Calvo-Pe\u00f1a, C., \u00c1lvarez-P\u00e9rez, J.M., Ib\u00e1\u00f1ez, A., Diez-Gal\u00e1n, A., Gonz\u00e1lez-Garc\u00eda, S., Garc\u00eda-Angulo, P., Acebes, J.L., and Coque, J.J.R. (2019). Necrotic and cytolytic activity on grapevine leaves produced by Nep1-like proteins of Diplodia seriata. Front. Plant Sci., 10.","DOI":"10.3389\/fpls.2019.01282"},{"key":"ref_32","first-page":"23","article-title":"Role of phenolics in the resistance mechanisms of plants against fungal pathogens and insects","volume":"661","author":"Lattanzio","year":"2006","journal-title":"Phytochem. Adv. Res."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Tungmunnithum, D., Thongboonyou, A., Pholboon, A., and Yangsabai, A. (2018). Flavonoids and Other Phenolic Compounds from Medicinal Plants for Pharmaceutical and Medical Aspects: An Overview. Medicines (Basel Switz.), 5.","DOI":"10.3390\/medicines5030093"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ijfoodmicro.2014.07.029","article-title":"Buckwheat achenes antioxidant profile modulates Aspergillus flavus growth and aflatoxin production","volume":"189","author":"Chitarrini","year":"2014","journal-title":"Int. J. Food Microbiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4815","DOI":"10.1038\/srep04815","article-title":"Relating significance and relations of differentially expressed genes in response to Aspergillus flavus infection in maize","volume":"4","author":"Asters","year":"2014","journal-title":"Sci. Rep."},{"key":"ref_36","first-page":"14","article-title":"Analysis of a mycotoxin gene cluster in Aspergillus nidulans","volume":"8","author":"Keller","year":"1995","journal-title":"SAAS Bull. Biochem. Biotechnol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1094\/Phyto-84-483","article-title":"A visual pattern of mycotoxin production in maize kernels by Aspergillus spp.","volume":"84","author":"Keller","year":"1994","journal-title":"Phytopathology"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1007\/s00253-002-1199-x","article-title":"Molecular genetic analysis and regulation of aflatoxin biosynthesis","volume":"61","author":"Bhatnagar","year":"2003","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_39","unstructured":"Payne, G.A., Yu, J., Nierman, W.C., Machida, M., Bhatnagar, D., Cleveland, T.E., and Dean, R.A. (2007). A First Glance into the Genome Sequence of Aspergillus fl avus. The Aspergilli, CRC Press."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1128\/AEM.65.2.834-836.1999","article-title":"Green fluorescent protein as a reporter to monitor gene expression and food colonization by Aspergillus flavus","volume":"65","author":"Du","year":"1999","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"He, Z.-M., Price, M.S., OBrian, G.R., Georgianna, D.R., and Payne, G.A. (2007). Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus. BMC Microbiol., 7.","DOI":"10.1186\/1471-2180-7-104"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Scala, V., Pietricola, C., Farina, V., Beccaccioli, M., Zjalic, S., Quaranta, F., Fornara, M., Zaccaria, M., Momeni, B., and Reverberi, M. (2020). Tramesan Elicits Durum Wheat Defense against the Septoria Disease Complex. Biomolecules, 10.","DOI":"10.20944\/preprints202003.0090.v1"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1016\/j.mib.2008.10.007","article-title":"Secondary metabolism: Regulation and role in fungal biology","volume":"11","author":"Fox","year":"2008","journal-title":"Curr. Opin. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/S1087-1845(03)00014-8","article-title":"Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus","volume":"39","author":"OBrian","year":"2003","journal-title":"Fungal Genet. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Caceres, I., Khoury, A.A., Khoury, R.E., Lorber, S., Oswald, I.P., Khoury, A.E., Atoui, A., Puel, O., and Bailly, J.D. (2020). Aflatoxin Biosynthesis and Genetic Regulation: A Review. Toxins (Basel), 12.","DOI":"10.3390\/toxins12030150"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/21\/8213\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:28:25Z","timestamp":1760178505000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/21\/8213"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,11,3]]},"references-count":45,"journal-issue":{"issue":"21","published-online":{"date-parts":[[2020,11]]}},"alternative-id":["ijms21218213"],"URL":"https:\/\/doi.org\/10.3390\/ijms21218213","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2020,11,3]]}}}