The vascular wilt fungi Verticillium dahliae and V. albo-atrum infect over 200 plant species, causing billions of dollars in annual crop losses. The characteristic wilt symptoms are a result of colonization and proliferation of the pathogens in the xylem vessels, which undergo fluctuations in osmolarity. To gain insights into the mechanisms that confer the organisms' pathogenicity and enable them to proliferate in the unique ecological niche of the plant vascular system, we sequenced the genomes of V. dahliae and V. albo-atrum and compared them to each other, and to the genome of Fusarium oxysporum, another fungal wilt pathogen. Our analyses identified a set of proteins that are shared among all three wilt pathogens, and present in few other fungal species. One of these is a homolog of a bacterial glucosyltransferase that synthesizes virulence-related osmoregulated periplasmic glucans in bacteria. Pathogenicity tests of the corresponding V. dahliae glucosyltransferase gene deletion mutants indicate that the gene is required for full virulence in the Australian tobacco species Nicotiana benthamiana. Compared to other fungi, the two sequenced Verticillium genomes encode more pectin-degrading enzymes and other carbohydrate-active enzymes, suggesting an extraordinary capacity to degrade plant pectin barricades. The high level of synteny between the two Verticillium assemblies highlighted four flexible genomic islands in V. dahliae that are enriched for transposable elements, and contain duplicated genes and genes that are important in signaling/transcriptional regulation and iron/lipid metabolism. Coupled with an enhanced capacity to degrade plant materials, these genomic islands may contribute to the expanded genetic diversity and virulence of V. dahliae, the primary causal agent of Verticillium wilts. Significantly, our study reveals insights into the genetic mechanisms of niche adaptation of fungal wilt pathogens, advances our understanding of the evolution and development of their pathogenesis, and sheds light on potential avenues for the development of novel disease management strategies to combat destructive wilt diseases.

Plant resistance to disease is controlled by the combination of defense response pathways that are activated depending on the nature of the pathogen. We identified the Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) gene that is transcriptionally regulated by Botrytis cinerea infection. Inactivation of BIK1 causes severe susceptibility to necrotrophic fungal pathogens but enhances resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae pv tomato. The response to an avirulent bacterial strain is unchanged, limiting the role of BIK1 to basal defense rather than race-specific resistance. The jasmonate-and ethylene-regulated defense response, generally associated with resistance to necrotrophic fungi, is attenuated in the bik1 mutant based on the expression of the plant defensin PDF1.2 gene. bik1 mutants show altered root growth, producing more and longer root hairs, demonstrating that BIK1 is also required for normal plant growth and development. Whereas the pathogen responses of bik1 are mostly dependent on salicylic acid (SA) levels, the nondefense responses are independent of SA. BIK1 is membrane-localized, suggesting possible involvement in early stages of the recognition or transduction of pathogen response. Our data suggest that BIK1 modulates the signaling of cellular factors required for defense responses to pathogen infection and normal root hair growth, linking defense response regulation with that of growth and development.

SummaryAn interesting observation, reported for transgenic plants that have been engineered to overproduce osmolytes, is that they often exhibit impaired growth in the absence of stress. As growth reduction and accumulation of osmolytes both typically result from adaptation, we hypothesized that growth reduction may actually result from osmolyte accumulation. To examine this possibility more closely, intracellular proline level was manipulated by expressing mutated derivatives of tomPRO2 (a D 1 -pyrroline-5-carboxylate synthetase, P5CS, from tomato) in Saccharomyces cerevisiae. This was done in the presence and absence of a functional proline oxidase, followed by selection and screening for increased accumulation of proline in the absence of any stress. Here we show, in support of our hypothesis, that the level of proline accumulation and the amount of growth are inversely correlated in cells grown under normal osmotic conditions. In addition, the intracellular concentration of proline also resulted in increases in ploidy level, vacuolation and altered accumulation of several different transcripts related to cell division and gene expression control. Because these cellular modi®cations are common responses to salt stress in both yeast and plants, we propose that proline and other osmolytes may act as a signaling/regulatory molecule able to activate multiple responses that are part of the adaptation process. As in previous studies with transgenic plants that overaccumulate osmolytes, we observed some increase in relative growth of proline-overaccumulating cells in mild hyperosmotic stress.

SummaryVerticillium dahliae Klebahn is a soil-borne fungal pathogen causing vascular diseases. The pathogen penetrates the host through the roots, spreads through the xylem, and systemically colonizes both resistant and susceptible genotypes. To elucidate the genetic and molecular bases of plant±Verticillium interactions, we have developed a pathosystem utilizing Arabidopsis thaliana and an isolate of V. dahliae pathogenic to both cruciferous and non-cruciferous crops. Relative tolerance (based on symptom severity) but no immunity was found in a survey of Arabidopsis ecotypes. Anthocyanin accumulation, stunting, and chlorosis were common symptoms. Speci®c responses of the more susceptible ecotype Columbia were induction of early¯owering and dying. The more tolerant ecotype C-24 was characterized by pathogeninduced delay of transition to¯owering and mild chlorosis symptoms. Genetic analysis indicated that a single dominant locus, Verticillium dahliae-tolerance (VET1), likely functioning also as a negative regulator of the transition to¯owering, was able to convey increased tolerance. VET1 was mapped on chromosome IV. The differential symptom responses observed between ecotypes were not correlated with different rates of fungal tissue colonization or with differential transcript accumulation of PR-1 and PDF1.2 defense genes whose activation was not detected during the Arabidopsis±V. dahliae interaction. Impairment in salicylic acid (SA)-or jasmonic acid (JA)-dependent signaling did not cause hypersensitivity to the fungal infection, whereas ethylene insensitivity led to reduced chlorosis and ABA de®ciency to reduced anthocyanin accumulation. The results of this study clearly indicated that the ability of V. dahliae to induce disease symptoms is also connected to the genetic control of development and life span in Arabidopsis.

Three Botrytis-susceptible mutants bos2, bos3, and bos4 which define independent and novel genetic loci required for Arabidopsis resistance to Botrytis cinerea were isolated. The bos2 mutant is susceptible to B. cinerea but retains wild-type levels of resistance to other pathogens tested, indicative of a defect in a response pathway more specific to B. cinerea. The bos3 and bos4 mutants also show increased susceptibility to Alternaria brassicicola, another necrotrophic pathogen, suggesting a broader role for these loci in resistance. bos4 shows the broadest range of effects on resistance, being more susceptible to avirulent strain of Pseudomonas syringae pv. tomato. Interestingly, bos3 is more resistant than wild-type plants to virulent strains of the biotrophic pathogen Peronospora parasitica and the bacterial pathogen P. syringae pv. tomato. The Pathogenesis Related gene 1 (PR-1), a molecular marker of the salicylic acid (SA)-dependent resistance pathway, shows a wild-type pattern of expression in bos2, while in bos3 this gene was expressed at elevated levels, both constitutively and in response to pathogen challenge. In bos4 plants, PR-1 expression was reduced compared with wild type in response to B. cinerea and SA. In bos3, the mutant most susceptible to B. cinerea and with the highest expression of PR-1, removal of SA resulted in reduced PR-1 expression but no change to the B. cinerea response. Expression of the plant defensin gene PDF1-2 was generally lower in bos mutants compared with wild-type plants, with a particularly strong reduction in bos3. Production of the phytoalexin camalexin is another well-characterized plant defense response. The bos2 and bos4 mutants accumulate reduced levels of camalexin whereas bos3 accumulates significantly higher levels of camalexin than wild-type plants in response to B. cinerea. The BOS2, BOS3, and BOS4 loci may affect camalexin levels and responsiveness to ethylene and jasmonate. The three new mutants appear to mediate disease responses through mechanisms independent of the previously described BOS1 gene. Based on the differences in the phenotypes of the bos mutants, it appears that they affect different points in defense response pathways.