RESEARCH ARTICLE Payungsak Rauyaree Æ Manuel D. Ospina-Giraldo Seogchan Kang Æ Ravindra G. Bhat Krishna V. Subbarao Æ Sandra J. Grant Katherine F. Dobinson Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae Received: 25 January 2005 / Revised: 3 May 2005 / Accepted: 5 May 2005 / Published online: 8 July 2005 Ó Springer-Verlag 2005 Abstract Verticillium dahliae is an important soil-borne fungal pathogen that causes vascular wilt diseases in a large variety of important crop plants. Due to its per- sistence in the soil, control of Verticillium wilt relies heavily on soil fumigation. The global ban on methyl bromide, a highly effective soil fumigant, poses an urgent need to develop alternative control measures against Verticillium wilt; and these might be more forthcoming with a better understanding of the molecular and cellular mechanisms that underpin the pathogenicity of V. dah- liae. In this study, we assessed the role in growth, devel- opment, and pathogenicity of VMK1, a gene encoding a mitogen-activated protein (MAP) kinase (hence, Verti- cillium MAP Kinase 1). Disruption of VMK1 via Agro- bacterium tumefaciens-mediated transformation, in two V. dahliae isolates, one from lettuce and the other from tomato, resulted in severely reduced virulence in diverse host plants, suggesting that VMK1 is essential for path- ogenicity and that the MAP kinase-mediated signaling pathway has a conserved role in fungal pathogenicity. The vmk1 mutants also exhibited reduced conidiation and microsclerotia formation, suggesting that the gene is important for multiple cellular processes. Keywords Targeted mutagenesis Æ Pathogenicity factor Introduction Verticillium dahliae (Kleb.) is a causal agent of Verticil- lium wilt disease in over 400 plant species, many of which are economically important crops (Woolliams 1966; Isaac 1967; McCain et al. 1981; Pegg and Brady 2002). This list is expanding, with new hosts succumbing to the pathogen being continually added (Bhat et al. 1999, 2003). Verti- cillium wilt is a difficult disease to control, in large part because V. dahliae forms large numbers of melanized resting-structures, called microsclerotia (Wilhelm 1955; Green 1980; Xiao 1998), in the vascular tissue of sus- ceptible hosts (DeVay et al. 1974; Davis et al. 1994) and to some extent in non-hosts (Krikun and Bernier 1990). The microsclerotia are able to remain viable for many years in the soil and under favorable environmental conditions (e.g., the presence of root exudates from host plant) they germinate and enter the host vascular tissue by directly penetrating the root cortical tissue or through wounds. The fungus subsequently causes wilt symptoms by impairing vascular function: once inside the plant, it grows upward through the xylem and can systemically colonize the host by producing a large number of conidia that are transported through the xylem transpiration stream to the aerial parts of the plant (Garas et al. 1986). Both extensive fungal growth, which clogs xylem vessels, and toxin production have been implicated in the result- ing impairment of water and mineral transport through the vascular system. Despite the global economic importance of Verticil- lium wilt, this disease has been poorly studied at the molecular level. A recent study (Dobinson et al. 2004) reported that targeted gene disruptions may be readily produced in V. dahliae, using transposon mutagenesis of a cloned gene of interest, followed by transfer of the resulting mutant allele into V. dahliae via Agrobacterium tumefaciens-mediated transformation (ATMT). These Communicated by J. Heitman P.R. and R.G.B. equally contributed to the work P. Rauyaree Æ M. D. Ospina-Giraldo Æ S. Kang (&) Department of Plant Pathology, The Pennsylvania State University, University Park, PA 16802, USA E-mail: sxk55@psu.edu Tel.: +814-863-3846 Fax: +814-863-7217 R. G. Bhat Æ K. V. Subbarao Department of Plant Pathology, c/o USDA Station, University of California–Davis, Salinas, CA 93905, USA S. J. Grant Æ K. F. Dobinson Agriculture & Agri-Food Canada, London, N5V 4T3, Canada Curr Genet (2005) 48: 109–116 DOI 10.1007/s00294-005-0586-0