{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,28]],"date-time":"2026-02-28T04:26:28Z","timestamp":1772252788597,"version":"3.50.1"},"reference-count":44,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2021,2,25]],"date-time":"2021-02-25T00:00:00Z","timestamp":1614211200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003329","name":"Ministerio de Econom\u00eda y Competitividad","doi-asserted-by":"publisher","award":["BFU2015-65501-P"],"award-info":[{"award-number":["BFU2015-65501-P"]}],"id":[{"id":"10.13039\/501100003329","id-type":"DOI","asserted-by":"publisher"}]},{"name":"FEDER, and RYC-2014-15844","award":["2014-15844"],"award-info":[{"award-number":["2014-15844"]}]},{"name":"Fundaci\u00f3n S\u00e9neca-Agencia de Ciencia y Tecnolog\u00eda de la Regi\u00f3n de Murcia, Spain","award":["19339\/PI\/14"],"award-info":[{"award-number":["19339\/PI\/14"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Mucormycosis is a lethal disease caused by Mucorales, which are emerging as human causes that explain the high mortality for this disease. Consequently, the research community is searching for virulence determinants that could be repurposed as targets to develop new treatments against mucormycosis. Our work explores an RNA interference (RNAi)-based approach to find targets involved in the virulence of Mucorales. A transcriptomewide analysis compared sRNAs and their target mRNAs in two Mucor lusitanicus different pathotypes, virulent and avirulent, generating a list of 75 loci selected by their differential sRNA accumulation in these strains. As a proof of concept and validity, an experimental approach characterized two loci showing opposite behavior, confirming that RNAi activity causes their differential expression in the two pathotypes. We generated deletion mutants for two loci and a knockin-strain overexpressing for one of these loci. Their functional analysis in murine virulence assays identified the gene wex1, a putative DEDDy exonuclease with RNase domains, as an essential factor for virulence. The identification of wex1 showed the potential of our approach to discover virulence factors not only in Mucorales but also in any other fungal model with an active RNAi machinery. More importantly, it adds a new layer to the biological processes controlled by RNAi in M. lusitanicus, confirming that the Dicer-dependent RNAi pathway can silence gene expression to promote virulence.<\/jats:p>","DOI":"10.3390\/ijms22052282","type":"journal-article","created":{"date-parts":[[2021,2,26]],"date-time":"2021-02-26T02:08:17Z","timestamp":1614305297000},"page":"2282","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["The RNAi Mechanism Regulates a New Exonuclease Gene Involved in the Virulence of Mucorales"],"prefix":"10.3390","volume":"22","author":[{"given":"Carlos","family":"P\u00e9rez-Arques","sequence":"first","affiliation":[{"name":"Departmento de Gen\u00e9tica y Microbiolog\u00eda, Facultad de Biolog\u00eda, Universidad de Murcia, 30100 Murcia, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3560-6155","authenticated-orcid":false,"given":"Mar\u00eda Isabel","family":"Navarro-Mendoza","sequence":"additional","affiliation":[{"name":"Departmento de Gen\u00e9tica y Microbiolog\u00eda, Facultad de Biolog\u00eda, Universidad de Murcia, 30100 Murcia, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7768-0989","authenticated-orcid":false,"given":"Laura","family":"Murcia","sequence":"additional","affiliation":[{"name":"Departmento de Gen\u00e9tica y Microbiolog\u00eda, Facultad de Biolog\u00eda, Universidad de Murcia, 30100 Murcia, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3091-4378","authenticated-orcid":false,"given":"Eusebio","family":"Navarro","sequence":"additional","affiliation":[{"name":"Departmento de Gen\u00e9tica y Microbiolog\u00eda, Facultad de Biolog\u00eda, Universidad de Murcia, 30100 Murcia, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7605-1726","authenticated-orcid":false,"given":"Victoriano","family":"Garre","sequence":"additional","affiliation":[{"name":"Departmento de Gen\u00e9tica y Microbiolog\u00eda, Facultad de Biolog\u00eda, Universidad de Murcia, 30100 Murcia, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3762-5971","authenticated-orcid":false,"given":"Francisco Esteban","family":"Nicol\u00e1s","sequence":"additional","affiliation":[{"name":"Departmento de Gen\u00e9tica y Microbiolog\u00eda, Facultad de Biolog\u00eda, Universidad de Murcia, 30100 Murcia, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,2,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1016\/j.ijantimicag.2017.08.010","article-title":"Antifungal resistance in mucorales","volume":"50","author":"Dannaoui","year":"2017","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Kontoyiannis, D.P., Yang, H., Song, J., Kelkar, S.S., Yang, X., Azie, N., Harrington, R., Fan, A., Lee, E., and Spalding, J.R. (2016). Prevalence, clinical and economic burden of mucormycosis-related hospitalizations in the United States: A retrospective study. BMC Infect. Dis., 16.","DOI":"10.1186\/s12879-016-2023-z"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Prakash, H., and Chakrabarti, A. (2019). Global epidemiology of mucormycosis. J. Fungi, 5.","DOI":"10.3390\/jof5010026"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"15898","DOI":"10.1038\/s41598-017-16123-9","article-title":"Intrinsic short-tailed azole resistance in mucormycetes is due to an evolutionary conserved aminoacid substitution of the lanosterol 14\u03b1-demethylase","volume":"7","author":"Caramalho","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1007\/s10096-006-0107-1","article-title":"Zygomycosis: The re-emerging fungal infection","volume":"25","author":"Chayakulkeeree","year":"2006","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1038\/nature13575","article-title":"Antifungal drug resistance evoked via RNAi-dependent epimutations","volume":"513","author":"Calo","year":"2014","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Li, C.H., Cervantes, M., Springer, D.J., Boekhout, T., Ruiz-Vazquez, R.M., Torres-Martinez, S.R., Heitman, J., and Lee, S.C. (2011). Sporangiospore size dimorphism is linked to virulence of Mucor circinelloides. PLoS Pathog., 7.","DOI":"10.1371\/journal.ppat.1002086"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1111\/1469-0691.12466","article-title":"Why is mucormycosis more difficult to cure than more common mycoses?","volume":"20","author":"Katragkou","year":"2014","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Nishimoto, A.T., Sharma, C., and Rogers, P.D. (2019). Molecular and genetic basis of azole antifungal resistance in the opportunistic pathogenic fungus Candida albicans. J. Antimicrob. Chemother., 75.","DOI":"10.1093\/jac\/dkz400"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2101","DOI":"10.1111\/brv.12550","article-title":"Fungal evolution: Diversity, taxonomy and phylogeny of the Fungi","volume":"94","year":"2019","journal-title":"Biol. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1577","DOI":"10.1016\/j.cub.2016.04.038","article-title":"Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication","volume":"26","author":"Corrochano","year":"2016","journal-title":"Curr. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1128\/CMR.13.2.236","article-title":"Zygomycetes in human disease","volume":"13","author":"Ribes","year":"2000","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3791","DOI":"10.1016\/j.cub.2019.09.024","article-title":"Early Diverging Fungus Mucor circinelloides Lacks Centromeric Histone CENP-A and Displays a Mosaic of Point and Regional Centromeres","volume":"29","author":"Panchal","year":"2019","journal-title":"Curr. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Nicol\u00e1s, F.E., Vila, A., Moxon, S., Cascales, M.D., Torres-Mart\u00ednez, S., Ruiz-V\u00e1zquez, R.M., and Garre, V. (2015). The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides. BMC Genom., 16.","DOI":"10.1186\/s12864-015-1443-2"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Lax, C., Tahiri, G., Pati\u00f1o-Medina, J.A., C\u00e1novas-M\u00e1rquez, J.T., P\u00e9rez-Ruiz, J.A., Osorio-Concepci\u00f3n, M., Navarro, E., and Calo, S. (2020). The evolutionary significance of rnai in the fungal kingdom. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21249348"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Lax, C., P\u00e9rez-arques, C., Navarro-mendoza, M.I., C\u00e1novas-m\u00e1rquez, J.T., Tahiri, G., P\u00e9rez-ruiz, J.A., Osorio-concepci\u00f3n, M., Murcia-flores, L., Navarro, E., and Garre, V. (2020). Genes, pathways, and mechanisms involved in the virulence of mucorales. Genes, 11.","DOI":"10.3390\/genes11030317"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"15348","DOI":"10.3390\/ijms140815348","article-title":"Functional Diversity of RNAi-Associated sRNAs in Fungi","volume":"14","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"5535","DOI":"10.1093\/nar\/gkq301","article-title":"Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides","volume":"38","author":"Nicolas","year":"2010","journal-title":"Nucleic Acids Res."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Chang, Z., Billmyre, R.B., Lee, S.C., and Heitman, J. (2019). Broad antifungal resistance mediated by RNAi-dependent epimutation in the basal human fungal pathogen Mucor circinelloides. PLoS Genet., 15.","DOI":"10.1101\/526459"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Chang, Z., and Heitman, J. (2019). Drug-resistant epimutants exhibit organ-specific stability and induction during murine infections caused by the human fungal pathogen Mucor circinelloides. MBio, 10.","DOI":"10.1128\/mBio.02579-19"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Trieu, T.A., Calo, S., Nicol\u00e1s, F.E., Vila, A., Moxon, S., Dalmay, T., Torres-Mart\u00ednez, S., Garre, V., and Ruiz-V\u00e1zquez, R.M. (2015). A Non-canonical RNA Silencing Pathway Promotes mRNA Degradation in Basal Fungi. PLOS Genet., 11.","DOI":"10.1371\/journal.pgen.1005168"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"P\u00e9rez-Arques, C., Navarro-Mendoza, M.I., Murcia, L., Navarro, E., Garre, V., and Nicol\u00e1s, F.E. (2020). A non-canonical RNAi pathway controls virulence and genome stability in Mucorales. PLoS Genet., 16.","DOI":"10.1101\/2020.01.14.906289"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Calo, S., Nicol\u00e1s, F.E., Lee, S.C., Vila, A., Cervantes, M., Torres-Martinez, S., Ruiz-Vazquez, R.M., Cardenas, M.E., and Heitman, J. (2017). A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides. PLoS Genet., 13.","DOI":"10.1371\/journal.pgen.1006686"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Nicol\u00e1s, F.E., Murcia, L., Navarro, E., Navarro-Mendoza, M.I., P\u00e9rez-Arques, C., and Garre, V. (2020). Mucorales species and macrophages. J. Fungi, 6.","DOI":"10.3390\/jof6020094"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"P\u00e9rez-Arques, C., Navarro-Mendoza, M.I., Murcia, L., Lax, C., Mart\u00ednez-Garc\u00eda, P., Heitman, J., Nicol\u00e1s, F.E., and Garre, V. (2019). Mucor circinelloides Thrives inside the Phagosome through an Atf-Mediated Germination Pathway. MBio, 10.","DOI":"10.1128\/mBio.02765-18"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1080\/21505594.2018.1435249","article-title":"Understanding Mucor circinelloides pathogenesis by comparative genomics and phenotypical studies","volume":"9","author":"Sanchis","year":"2018","journal-title":"Virulence"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1016\/j.fgb.2006.09.003","article-title":"Mutants defective in a Mucor circinelloides dicer-like gene are not compromised in siRNA silencing but display developmental defects","volume":"44","author":"Nicolas","year":"2007","journal-title":"Fungal. Genet. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1486","DOI":"10.1128\/EC.00191-09","article-title":"A single dicer gene is required for efficient gene silencing associated with two classes of small antisense RNAs in Mucor circinelloides","volume":"8","author":"Calo","year":"2009","journal-title":"Eukaryot. Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1017","DOI":"10.1093\/nar\/29.5.1017","article-title":"Exoribonuclease superfamilies: Structural analysis and phylogenetic distribution","volume":"29","author":"Zuo","year":"2001","journal-title":"Nucleic Acids Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3063","DOI":"10.1007\/s00253-012-4432-2","article-title":"Malic enzyme activity is not the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides","volume":"97","author":"Garre","year":"2013","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Binder, U., Navarro-Mendoza, M.I., Naschberger, V., Bauer, I., Nicolas, F.E., Pallua, J.D., Lass-Fl\u00f6rl, C., and Garre, V. (2018). Generation of a mucor circinelloides reporter strain\u2014A promising new tool to study antifungal drug efficacy and mucormycosis. Genes, 9.","DOI":"10.3390\/genes9120613"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.fbr.2007.02.007","article-title":"Determinants of virulence in the pathogenic fungi","volume":"21","author":"Casadevall","year":"2007","journal-title":"Fungal. Biol. Rev."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Trieu, T.A., Navarro-Mendoza, M.I., Perez-Arques, C., Sanchis, M., Capilla, J., Navarro-Rodriguez, P., Lopez-Fernandez, L., Torres-Martinez, S., Garre, V., and Ruiz-Vazquez, R.M. (2017). RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis. PLoS Pathog., 13.","DOI":"10.1371\/journal.ppat.1006150"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3983","DOI":"10.1093\/emboj\/cdg384","article-title":"Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes","volume":"22","author":"Nicolas","year":"2003","journal-title":"EMBO J."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Nicol\u00e1s, F.E., Torres-Mart\u00ednez, S., and Ruiz-V\u00e1zquez, R.M. (2013). Loss and Retention of RNA Interference in Fungi and Parasites. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003089"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"5556","DOI":"10.1093\/nar\/gkm614","article-title":"The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein","volume":"35","author":"Stead","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.fgb.2014.09.007","article-title":"Distinct and redundant roles of exonucleases in Cryptococcus neoformans: Implications for virulence and mating","volume":"73","author":"Wollschlaeger","year":"2014","journal-title":"Fungal. Genet. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1007\/978-1-4939-8742-9_13","article-title":"Molecular tools for carotenogenesis analysis in the mucoral Mucor circinelloides","volume":"Volume 1852","author":"Navarro","year":"2018","journal-title":"Methods in Molecular Biology"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"907","DOI":"10.1038\/s41587-019-0201-4","article-title":"Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype","volume":"37","author":"Kim","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol., 15.","DOI":"10.1186\/s13059-014-0550-8"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"W160","DOI":"10.1093\/nar\/gkw257","article-title":"deepTools2: A next generation web server for deep-sequencing data analysis","volume":"44","author":"Ryan","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/B978-0-12-418687-3.00006-9","article-title":"Purification of DNA oligos by denaturing polyacrylamide gel electrophoresis (PAGE)","volume":"529","author":"Nicolas","year":"2013","journal-title":"Methods Enzymol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"7660","DOI":"10.1038\/s41598-018-26051-x","article-title":"Components of a new gene family of ferroxidases involved in virulence are functionally specialized in fungal dimorphism","volume":"8","author":"Murcia","year":"2018","journal-title":"Sci. Rep."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/5\/2282\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:28:32Z","timestamp":1760160512000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/5\/2282"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,2,25]]},"references-count":44,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2021,3]]}},"alternative-id":["ijms22052282"],"URL":"https:\/\/doi.org\/10.3390\/ijms22052282","relation":{"has-preprint":[{"id-type":"doi","id":"10.20944\/preprints202101.0277.v1","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,2,25]]}}}