Vayg1 is required for microsclerotium formation and melanin production in Verticillium dahliae

Highlights

• Vayg1 is involved in melanin production and microsclerotium formation in V. dahliae.

• Deletion of Vayg1 reduced the virulence of V. dahliae on cotton seedlings.

Abstract

The fungus Verticillium dahliae causes vascular wilt disease on many plant species, including economically important crop and ornamental plants worldwide. It produces darkly pigmented resting structures known as microsclerotia, which are able to survive for up to 14 years in soil, and represent one of the defining characteristics of this species. The pigment produced in V. dahliae is dihydroxynaphthalene (DHN)-melanin, a form of melanin common among fungi and named so for the intermediary of this melanin biosynthetic pathway. In this study, we characterized the function of the V. dahliae Vayg1 gene, whose homologs were involved in melanin biosynthesis in Exophiala dermatitidis (Wayg1) and Aspergillus fumigatus (Aayg1), by deletion and complementation of the gene and co-incubating deletion mutant with wild-type strain. Results showed that melanin production and microsclerotial formation in deletion mutants are inhibited. The Vayg1 deletion mutant also exhibited reduced pathogenicity. These results showed that Vayg1 is necessary for melanin and microsclerotium production, and we may thus hypothesize that the Vayg1 product may catalyze two different precursors, one of which is essential for DHN melanin production and the other one is involved in a signal network for microsclerotial formation in V. dahliae.

Introduction

Verticillium dahliae Kleb. is a soilborne plant pathogenic fungus classified in the Sordariomycetes of the Ascomycota, although a sexual reproductive phase has not been identified yet (Inderbitzin et al., 2011). It causes Verticillium wilt on more than 200 plant species, including many agriculturally important crops (Pegg and Brady, 2002). The list of plant hosts affected by V. dahliae is continually expanding as new hosts are identified (Atallah et al., 2011, Bhat and Subbarao, 1999, Lu et al., 2013).

The disease cycle of V. dahliae consists of a parasitic stage in which the plant is colonized, and a brief saprophytic stage where the pathogen can decompose plant tissues, followed by a dormant stage in which the pathogen can survive for years in the soil (Klosterman et al., 2009). The dormant survival structures that enable V. dahliae to survive up to 14 years, even in the absence of a host (Wilhelm, 1955), are known as microsclerotia that are resistant to extreme temperatures, desiccation and other environmental stresses (Bell and Wheeler, 1986, Butler and Day, 1998). Primary infections are typically initiated in the root by germinating microsclerotia, which produce infectious hyphae that directly penetrate the root (Klosterman et al., 2009). Following a period of colonization and growth in the xylem, large numbers of microsclerotia are produced on senescent infected plants (Powelson, 1970), and released into soil with decomposed plant debris. Thus, microsclerotia play a key role in the disease cycle of V. dahliae.

Microsclerotia in V. dahliae are composed of compact masses of thick-walled, darkly pigmented (melanized) cells, which originate from swollen, septate hyphae by a process of budding (Brandt, 1964, Fradin and Thomma, 2006). The cell walls of the microsclerotia are impregnated with melanin as the propagules mature (Brandt, 1964). Most fungal melanin has been identified as dihydroxynaphthalene (DHN)-melanin, named for the pathway intermediary, 1,8-DHN. DHN-melanin biosynthesis starts with a polyketide synthase (PKS) to produce 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), which is reduced to scytalone via a reductase (T4HR). Then with a series of downstream reactions, 1,8-DHN is produced.

Melanin is essential in V. dahliae for the development of fully functional microsclerotia (Bell and Wheeler, 1986). Production of melanin may be critical for long-term survival, as melanin enables V. dahliae and other fungi to ward off damage from UV irradiation, extreme temperatures and enzymatic degradation from β-1,3 glucanases and chitinases present in soil microorganisms (Bell et al., 1976, Butler and Day, 1998). In wild-type V. dahliae, the presence of melanin implies the existence of microsclerotia and its absence implies lack of microsclerotia (Brandt, 1964, Duressa et al., 2013). Nevertheless, melanin biosynthesis and microsclerotial development are not inextricably linked and melanin or products from later stages of the biosynthetic pathway are not necessary for formation of microsclerotia in V. dahliae (Bell et al., 1976).

In view of the importance of microsclerotia in the disease cycle of V. dahliae (Klosterman et al., 2009), an increased understanding of the genetics and molecular mechanisms that regulate their formation, along with DHN-melanin biosynthesis, may lead to novel disease control targets or approaches. Previously, a V. dahliae homolog of Aspergillus fumigatus Aayg1 (Fujii et al., 2004) was expressed at 165-fold higher levels in melanized microsclerotium-producing cultures than in non-pigmented amicrosclerotial cultures by RNA-Seq analyses (Duressa et al., 2013). The A. fumigatus Aayg1 gene is responsible for polyketide chain-length shortening, and catalyzing the conversion of the heptaketide naphthopyrone (YWA1) to 1,3,6,8-THN (Fujii et al., 2004), a key initial step during melanin biosynthesis in some fungi. However, homologs of Aayg1 are not present in all fungal species, including several species in the Fusarium genus (Klosterman et al., 2011).

There is limited amount of published information on the function of Aayg1 and its homologs. Only its homologs in Exophiala dermatitidis (Wayg1) and Botrytis cinerea (Bcygh1) have been shown to be required for catalyzing 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (AT4HN) derived from PKS1 to melanin in E. dermatitidis or catalyzing one PKS product in conidia pigment biosynthesis pathway in B. cinerea, respectively (Schumacher, 2016, Wheeler et al., 2008). Though functions of Aayg1 homologs in melanin biosynthesis pathway are quite conserved, a couple of unique features have been identified. For example, in contrast to the wild-type conidia that are covered with rodlet structure, some patches of organized rodlet layers were observed on the conidial surface of Aayg1 deletion mutants in A. fumigatus, indicating that the cell surface defects resulted in defective virulence (Bayry et al., 2014). The roles of melanin production related genes in pathogenicity have been well studied for many plant fungal pathogens, including Magnaporthe oryzae and Colletotrichum species (Chumley and Valent, 1990, Howard and Ferrari, 1989). However, the role of Aayg1 homolog has not been elucidated in V. dahliae. Although several genes have been identified to be involved in microsclerotial formation or infection process in V. dahliae (Gao et al., 2010, Rauyaree et al., 2005, Tzima et al., 2011), mechanism of microsclerotial formation still remains unclear.

In the present study, we demonstrated the involvement of Vayg1 in melanin production and microsclerotial formation by characterization of the phenotypes in deletion mutants of the single Aayg1 gene homolog in V. dahliae (Vayg1) and its complementation and co-incubation the deletion mutant with the wild-type strain. Results showed that deletion of Vayg1 prevented not only the melanin production but also microsclerotial formation. Given the dual requirement of Vayg1 for both microsclerotium production and melanin biosynthesis, we hypothesize that Vayg1 may catalyze two precursors, one of which is like YWA1 or At4HN that is essential for melanin production and the other is involved in the signal network for microsclerotial formation in V. dahliae. In addition we have obtained data which suggested that the Vayg1 deletion mutant also had reduced virulence.