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

Abstract

BackgroundThe 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.ResultsTo 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.ConclusionCollectively, 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.

Phylogenic analysis revealed an expanded C2H2-homeobox subfamily and expression profiles of C2H2 zinc finger gene family in Verticillium dahliae

“…Microsclerotia (MS) play a crucial role in Verticillium disease cycle, and many gene families are involved in MS development, including some C 2 H 2 genes and melanin biosynthesis genes that are differentially expressed during MS formation (Duressa et al, 2013;Xiong et al, 2014). Xiong et al found that most of the genes located in the four LS regions annotated in VdLs.17 were expressed at low or undetectable levels in V. dahliae XS11 during MS formation (Klosterman et al, 2011;Xiong et al, 2014).…”

Genomics Spurs Rapid Advances in Our Understanding of the Biology of Vascular Wilt Pathogens in the Genus Verticillium

“…Although light and electron microscopic examinations have defined the morphological sequence of events in the microsclerotial developmental pathway (see précis in Reference 70 and Figure 1), the molecular mechanisms that direct microsclerotial development remain for the most part uncharacterized. However, with genomics-based studies now coming to the fore, genes involved in this developmental process are coming to light through transcriptional profiling and genome-scale random mutagenesis (19,13,41,70,72).…”

VdCrz1 is involved in microsclerotia formation and required for full virulence in Verticillium dahliae

“…6A). The expression levels of the hydrophobin gene VDH1, involved in early stage of microsclerotia formation (Klimes and Dobinson, 2006) and five genes sharing strong homology to fungal melanin biosynthesis genes and expressed in developing microsclerotia (Duressa et al, 2013;Xiong et al, 2014) were examined by reverse transcription quantitative PCR analyses. As expected, the expression level of VDH1 was significantly reduced in the DVdCrz1-2 strain but was comparable between wild-type and DVdCrz1_C strains (Fig.…”