Roles of the catalytic subunit of cAMP-dependent protein kinase A in virulence and development of the soilborne plant pathogen Verticillium dahliae

Abstract

Verticillium dahliae is a soilborne fungus that causes vascular wilt disease in a broad range of hosts and survives for many years in the soil in the form of microsclerotia. Although the role of cAMP-dependent protein kinase A (PKA) has been extensively studied in foliar pathogens, there is limited information about its role in soilborne fungal pathogens that infect through the root system. Genome database search revealed the presence of two PKA catalytic subunit genes in V. dahliae, named VdPKAC1 and VdPKAC2. A phylogenetic analysis showed that VdPKAC2 groups with fungal PKA catalytic subunits that appear to play a minor role in PKA activity. This gene was expressed considerably lower than that of VdPKAC1. Although disruption of VdPKAC1 did not affect the ability of V. dahliae to infect through the roots of tomato and eggplant, disease severity was significantly reduced. Since pathogen-derived ethylene is presumed to play a major role in symptom induction in vascular wilt diseases, ethylene generation was measured in fungal culture. The mutants defective in VdPKAC1 produced less ethylene than the corresponding wild type strains, suggesting a regulatory role of PKA in ethylene biosynthesis. Growth rates of these mutants were similar to those of wild type strains, while the rate of spore germination was slightly elevated and conidia production was significantly reduced. When grown on minimal media, the mutants showed greater microsclerotia production compared with the wild type strains. These results suggest multiple roles of VdPKAC1, including virulence, conidiation, microsclerotia formation, and ethylene biosynthesis, in the soilborne fungus V. dahliae.

Introduction

Verticillium dahliae is a soilborne fungal pathogen with a special infection behavior and long-term survival capability, which defies existing control strategies (Klose et al., 2007, Tjamos, 1989). This fungus does not exhibit distinct host specificity, causing wilt disease in a wide range of annual herbaceous, perennial, and woody plants (Armengol et al., 2005, Bhat et al., 2003, Levin et al., 2003, Vallad and Subbarao, 2008). The fungus differs from other vascular-colonizing fungi and other Verticillium species in that it forms microsclerotia, which are heavily melanised resting structures (Green, 1981, Perry and Evert, 1982). Microscletotia allow V. dahliae to survive in the soil for more than 10 years and serve as the main propagule that initiates infection (Schnathorst, 1981). In the presence of root exudates, microsclerotia overcome the mycostatic activity of the soil and germinate. Hyphae grow towards the nearby roots, probably being attracted by nutrient gradient (Huisman, 1982, Fradin and Thomma, 2006, Schnathorst, 1981). After the entry of the root cortex through wounds or by penetrating epidermal cells, the fungus reaches the vascular tissue by crossing the endodermis (Schnathorst, 1981, Vallad and Subbarao, 2008). Systemic invasion occurs when a large number of conidia are produced and transported through the xylem to the aerial parts of the plants (Garas et al., 1986, Vallad and Subbarao, 2008). Resulting disease symptoms have been attributed to water stress caused by the occlusion of xylem vessels and the production of phytotoxins and ethylene (Buchner et al., 1982, Cronshaw and Pegg, 1976, Cooper and Durrands, 1989, DeVay, 1989, Pegg, 1981).

The switch from the dormant stage (microsclerotia) to the parasitic phase of V. dahliae has been shown to be influenced by the presence of potential hosts and the changing nutrient status of the soil environment, caused by root exudates (Huisman, 1982, Fradin and Thomma, 2006, Schnathorst, 1981). In several fungi, adaptation to nutritional availability and stress is found to be regulated by the cyclicAMP (cAMP)-dependent signaling pathway. In addition, this pathway controls fungal differentiation, sexual development and virulence (Kronstad et al., 1998, Liebmann et al., 2004, Mehrabi and Kema, 2006; Yamauchi et al., 2004).

The cAMP-dependent protein kinase A (PKA) is the major downstream effector of the cAMP-dependent signaling pathway. Binding of cAMP, a secondary messenger produced from ATP by the action of adenylate cyclase, to each of the two PKA regulatory (R) subunits leads to the dissociation of the R subunits from the PKA complex, resulting in the activation of two monomeric catalytic (C) subunits that phosphorylate target proteins (Taylor et al., 1990).

PKA has been extensively studied in yeast (Kronstad et al., 1998, Toda et al., 1987). In Saccharomyces cerevisiae, PKA is implicated in the control of growth on several carbon sources, glycogen accumulation, stress resistance, and filamentous differentiation in response to nutrient availability (Gray et al., 2004, Pan and Heitman, 1999, Toda et al., 1987, Thevelein and de Winde, 1999). The C subunits in S. cerevisiae are encoded by three genes (TPK1, TPK2, and TPK3), while only one gene (Bcy1) codes for the R subunit. The three C subunits seem to have distinct, but also overlapping functions. Tpk2 activates pseudohyphal growth in S. cerevisiae, while Tpk1 and Tpk3 repress filamentous growth, possibly by a feedback loop that down-regulates cAMP accumulation (Pan et al., 2000).

In phytopathogenic fungi, PKA has been shown to regulate dimorphic transition and pathogenesis in Ustilago maydis (Dürrenberger et al., 1998), appressorium formation and virulence in Magnaporthe grisea and Colletotrichum trifolii (Mitchell and Dean, 1995, Xu et al., 1997, Yang and Dickman, 1999) and pathogenesis in Colletotrichum lagenarium (Yamauchi et al., 2004).

In U. maydis, two genes (adr1 and uka1) were identified to code for C subunits. Adr1 mutants displayed filamentous growth as opposed to yeast-like growth of the wild type haploid strains. Moreover, dikaryotic hyphae derived from crosses between haploid Δadr1 mutants of compatible mating type or Δadr1 homozygous diploid strains, were not able to cause disease. In contrast, disruption of the uka1 gene had little effect on mating, morphogenesis, and virulence (Dürrenberger et al., 1998).

In M. grisea, the cpkA gene is implicated in appresorium development and host penetration, whereas it is dispensable for infectious growth. Mutants defective in cpkA were delayed in appressorium formation, and resulting appresoria were smaller compared to those produced by the wild type strains and were unable to penetrate intact host tissue (Mitchell and Dean, 1995). The cpkA mutants of C. trifolli exhibited similar phenotypes during leaf tissue penetration and invasive growth, except that appressoria were formed without delay with their size being comparable to that of wild type strains (Yang and Dickman, 1999).

PKA also regulates virulence in Mycosphaerella graminicola, a fungal pathogen that does not form appresoria for infection but invades the host through stomata. Disruption of the R (MgBcy1) or the C (MgTpk2) subunit of PKA resulted in mutants that were impaired in virulence and were incapable of producing pycnidia in infected leaves (Mehrabi and Kema, 2006). Increased levels of PKA activity correlated with initiation of sclerotia production in Sclerotinia sclerotiorum (white sclerotium stage), while in melanized sclerotia PKA activity is reduced (Harel et al., 2005). However, disruption of a gene encoding a C subunit in S. sclerotiorum had no effect on pathogenicity or microsclerotia production (Jurick et al., 2004).

Similar to M. graminicola, V. dahliae is not known to form appressoria for infection but invades the host through natural openings or wounds on the root surface. Despite the extensive research that has been performed in the past decades to elucidate factors involved in V. dahliae pathogenicity (Buchner et al., 1982, Cronshaw and Pegg, 1976, Cooper and Durrands, 1989, Klimes and Dobinson, 2006, Klimes et al., 2008, Rauyaree et al., 2005, Vallad and Subbarao, 2008), limited information is available about the molecular mechanisms underpinning Verticillium wilt (Fradin and Thomma, 2006).

Characterization of the MAP kinase gene VMK1, a S. cerevisiae Fus3 ortholog in V. dahliae, revealed a significant role of the MAP kinase signaling pathway in pathogenicity and microsclerotia development, as vmk1 mutants caused no visible symptoms in various hosts and produced less microsclerotia compared to the wild type strain (Rauyaree et al., 2005). Also, the gene VDH1, encoding a hydrophobin in V. dahliae, is implicated in microsclerotia development, since disruption of VDH1 caused reduction of microsclerotia formation (Klimes and Dobinson, 2006). Overexpression of VDH1 resulted in reduced disease symptoms relative to wild type strains, suggesting a role of VDH1 in pathogenicity or plant–pathogen recognition (Klimes et al., 2008).

In order to gain knowledge on the function of the cAMP signaling pathway in pathogenicity and microsclerotia production of V. dahliae, a gene encoding a C subunit of PKA (VdPKAC1) was cloned and disrupted. Results of this study suggest that this gene plays important roles in virulence, microsclerotia formation, conidiation and ethylene production in V. dahliae.