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

Dechassa Duressa 0 3 Amy Anchieta 0 3 Dongquan Chen 2 Anna Klimes 1 5 6 Maria D Garcia-Pedrajas 4 Katherine F Dobinson 1 6 Steven J Klosterman 0 3 0 United States Department of Agriculture - Agricultural Research Service , Salinas, CA , USA 1 Department of Biology, University of Western Ontario , London, ON , Canada 2 Comprehensive Cancer Center & Division of Preventive Medicine, University of Alabama at Birmingham , Birmingham, AL , USA 3 United States Department of Agriculture - Agricultural Research Service , Salinas, CA , USA 4 Instituto de Hortofruticultura Subtropical y Mediterranea La Mayora-Consejo Superior de Investigaciones Cientificas (IHSM-UMA-CSIC), Estacion Experimental La Mayora , Algarrobo-Costa, Malaga , Spain 5 Department of Physiological and Biological Science, Western New England University , Springfield, MA , USA 6 Agriculture and Agri-Food Canada , London, ON , Canada 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. - Background Verticillium dahliae is a soilborne, plant pathogenic fungus that causes wilt disease on over 200 plant species worldwide. Verticillium wilt diseases are also referred to as vascular wilts since the fungus invades the plant xylem, disrupting water transport and causing characteristic leaf wilt symptoms. Control of Verticillium wilt diseases is complicated by the lack of genetic resistance in many plant hosts, and also by the persistence of V. dahliae in the soil [1]. Verticillium dahliae produces melanized survival structures, microsclerotia, that are able to survive in the soil for at least 10 years [2,3]. Root exudates provide a signal for microsclerotial germination [4,5], and upon germination infectious hyphae emerge from the microsclerotia to initiate plant root infection. Following penetration through the plant root epidermis, and an extended period of colonization in which plants are asymptomatic, microsclerotia are produced in large quantities within the dying and necrotic plant tissues, and are subsequently returned to the soil with plant debris to initiate a new round of the disease cycle [1]. Wild-type microsclerotia of V. dahliae are characterized by the presence of dense black melanin deposits that appear as granules within and exterior to the cell wall [6]. This melanin is proposed to act as an anti-desiccant and protect against temperature extremes and enzymatic lysis [6], and is synthesized via a dihydroxynaphthalene (DHN) melanin biosynthesis pathway [6,7]. The initial substrate molecule in the DHN pathway is 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), derived by cyclization of acetate molecules by polyketide synthase (PKS) activity. DHN monomers are generated from the 1,3,6,8-THN through alternating reduction and dehydration reactions involving several intermediates, including scytalone, 1,3,8-THN (trihydroxynaphthalene), and vermelone [6]. The DHN monomers are subsequently oxidized and polymerized into melanin by laccases [6,8]. Though the microsclerotia are typically darkly pigmented, the processes of melanin production and microsclerotial development in V. dahliae can be uncoupled [9]. Because of their central role in pathogen survival and initiating plant root infection, the microsclerotia of V. dahliae have been considered important targets for disease control [10,11]. This suggestion is supported by the correlation between reduced microsclerotial and pigment production, and reduced survival of V. dahliae [12,13]. The results of the analyses of multiple Agrobacterium tumefaciens-mediated transformation (ATMT) insertional mutants of V. dahliae also indicate an important link between microsclerotial formation and developmental processes needed for virulence [14,15]. For example, a strain of V. dahliae with mutation of the stress-responsive glutamic acid-rich protein encoding-gene (VdGarp1; VDAG_08781) shows both decreased virulence on cotton, and severely reduced microsclerotia formation under nutrient limiting conditions [14]. In contrast, however, vdpkaC1 mutants exhibit both reduced virulence, and under certain growth conditions enhanced microsclerotia production [16]. The vdh1 mutant, which exhibits an amicrosclerotial phenotype under both nutrient-rich and starvation growth conditions, is not compromised in virulence [17]. Taken together, these data indicate that microsclerotial development is not always coupled with virulence. Analyses of gene expression in Verticillium dahliae during microsclerotia formation have been carried out previously by comparisons of expressed sequence tag (EST) libraries generated from V. dahliae cultures in which microsclerotia were developing (DMS), and those generated from cultures grown in simulated xylem fluid medium (SXM), which does not stimulate microsclerotia development [18]. Those EST analyses revealed in the DMS librar (...truncated)