Verticillium dahliae is a soilborne fungus causing vascular wilt in a diverse array of plant species. Its virulence has been attributed, among other factors, to the activity of hydrolytic cell wall-degrading enzymes (CWDE). The sucrose nonfermenting 1 gene (VdSNF1), which regulates catabolic repression, was disrupted in V. dahliae tomato race 1. Expression of CWDE in the resulting mutants was not induced in inductive medium and in simulated xylem fluid medium. Growth of the mutants was significantly reduced when grown with pectin or galactose as a carbon source whereas, with glucose, sucrose, and xylose, they grew similarly to wild-type and ectopic transformants. The mutants were severely impaired in virulence on tomato and eggplant (final disease severity reduced by an average of 87%). Microscopic observation of the infection behavior of a green fluorescent protein (gfp)-labeled VdSNF1 mutant (70ΔSF-gfp1) showed that it was defective in initial colonization of roots. Cross sections of tomato stem at the cotyledonary level showed that 70ΔSF-gfp1 colonized xylem vessels considerably less than the wild-type strain. The wild-type strain heavily colonized xylem vessels and adjacent parenchyma cells. Quantification of fungal biomass in plant tissues further confirmed reduced colonization of roots, stems, and cotyledons by 70ΔSF-gfp1 relative to that by the wild-type strain.
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 persistence 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. dahliae. In this study, we assessed the role in growth, development, and pathogenicity of VMK1, a gene encoding a mitogen-activated protein (MAP) kinase (hence, Verticillium MAP Kinase 1). Disruption of VMK1 via Agrobacterium 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 pathogenicity 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.
summary A system-wide approach was adopted to further elucidate mechanisms regulating disease outcome between rice and the fungal pathogen Magnaporthe grisea. First, a cDNA library was constructed from M. grisea infected rice at 48 h post-inoculation. The 5' end-sequencing of 619 randomly selected clones revealed 359 expressed sequence tags (ESTs) that had not previously been described. A total of 124 from 260 ESTs with high and moderate similarity scores, based on BlastX, were organized into categories according to their putative function. The largest category of sequences (21%) contained stress or defence response genes. Eleven per cent of identified ESTs were redundant. In a second approach, differential hybridization analysis of the cDNA library using high-density filters resulted in the identification of novel genes and previously characterized M. grisea genes, including several that had previously been implicated in the infection process. A survey of up-regulated cDNA clones revealed clone 29003, which corresponded to the rice peroxidase POX22.3. This gene is known to be expressed in rice upon infection with Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen. Importantly, this approach demonstrates the utility of gene discovery, through ESTs, for revealing novel genes in addition to those previously characterized as being potentially implicated in host-pathogen interactions.
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