Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/4/2013
Publication Date: 9/9/2013
Citation: Duressa, D., Anchieta, A.G., Chen, D., Klimes, A., Garcia-Pedrajas, M.D., Dobinson, K.F., Klosterman, S.J. 2013. RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae. Biomed Central (BMC) Genomics. DOI: 10.1186/1471-2164-14-607. Interpretive Summary: Verticillium dahliae is a soilborne fungus, and the cause of Verticllium wilt disease in plants. Verticillium wilt is prevalent on crop and ornamental plants worldwide. The disease is especially difficult to control because the pathogen is capable of surviving in the soil for 10 to15 years as darkly pigmented microsclerotia, rendering crop rotation strategies for the control of this disease ineffective. Since the pathogen is able to survive for long periods, even in the absence of a host plant, expensive soil fumigants are often applied to kill the microsclerotia in the soil. In this study, analyses of genes expressed in cultures of V. dahliae yielded insights into the genes and their protein products that play a role in the production of pigment and morphological transition of the fungus from the filamentous form to the production of the long-lived microsclerotia. This insight sheds light on genes expressed and molecular mechanisms that enable these organisms to persist in the soil for so long, and may further yield alternative disease control strategies through the targeting of these mechanisms.
Technical Abstract: The soilborne fungus, Verticillium dahliae, causes Verticillium wilt disease in plants. Verticillium wilt is difficult to control since V. dahliae is capable of persisting in the soil for 10 to15 years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. Microsclerotia of V. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. Consequentially, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae. To shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in V. dahliae, three replicate RNA-seq libraries were prepared from 10 day-old microsclerotia (MS)-producing cultures of V. dahliae, strain VdLs.17 (ave=52.23 million reads), and those not producing microsclerotia (NoMS, ave=50.58 million reads. Analyses of these libraries for differential gene expression revealed over 200 differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase (344-fold increase) and scytalone dehydratase (231-fold increase), and additional genes located in a 48.8 kilobase melanin biosynthetic gene cluster of strain VdLs.17. Approximately 40 % of the genes identified as differentially expressed in the MS library encode hypothetical proteins. Confirmation of differential expression by RNA-seq was performed using RT-qPCR and RNA derived from several MS and NoMs culture types, including MS cultures that were stored for 6 months at 4°C, and seven day-old cultures with an intermediate number of melanized MS. These data provide additional insight into gene expression and molecular processes that govern melanin biosynthesis and MS formation in V. dahliae, and the identified gene products may represent alternative disease control targets.