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Open Access Highly Accessed Research article

RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae

Dechassa Duressa1, Amy Anchieta1, Dongquan Chen2, Anna Klimes345, Maria D Garcia-Pedrajas6, Katherine F Dobinson34 and Steven J Klosterman1*

Author Affiliations

1 United States Department of Agriculture – Agricultural Research Service, Salinas, CA, USA

2 Comprehensive Cancer Center & Division of Preventive Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

3 Department of Biology, University of Western Ontario, London, ON, Canada

4 Agriculture and Agri-Food Canada, London, ON, Canada

5 Department of Physiological and Biological Science, Western New England University, Springfield, MA, USA

6 Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Estación Experimental “La Mayora”, Algarrobo-Costa, Málaga, Spain

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BMC Genomics 2013, 14:607  doi:10.1186/1471-2164-14-607

Published: 9 September 2013

Abstract

Background

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 to 15 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. Consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae.

Results

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 (average = 52.23 million reads), and those not producing microsclerotia (NoMS, average = 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. Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins. Additional comparative analyses of gene expression in V. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with RNA derived from V. dahliae strain Dvd-T5, and from the amicrosclerotial vdh1 strain. Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations.

Conclusion

Collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during MS biogenesis and maturation in V. dahliae. The identified gene products could therefore potentially represent new targets for disease control through prevention of survival structure development.

Keywords:
Verticillium dahliae; Morphogenesis; Microsclerotia; RNA-seq; Gene expression