Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

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.

). † These authors contributed equally to this study. SummaryThe expression profiles of Botrytis-inoculated Arabidopsis plants were studied to determine the nature of the defense transcriptome and to identify genes involved in host responses to the pathogen. Normally resistant Arabidopsis wild-type plants were compared with coi1, ein2, and nahG plants that are defective in various defense responses and/or show increased susceptibility to Botrytis. In wild-type plants, the expression of 621 genes representing approximately 0.48% of the Arabidopsis transcriptome was induced greater than or equal to twofold after infection. Of these 621 Botrytis-induced genes (BIGs), 462 were induced at or before 36 h postinoculation, and may be involved in resistance to the pathogen. The expression of 181 BIGs was dependent on a functional COI1 gene required for jasmonate signaling, whereas the expression of 63 and 80 BIGs were dependent on ethylene (ET) signaling or salicylic acid accumulation, respectively, based on results from ein2 and nahG plants. BIGs encode diverse regulatory and structural proteins implicated in pathogen defense and abiotic and oxidative-stress responses. Thirty BIGs encode putative DNA-binding proteins that belong to ET response, zinc-finger, MYB, WRKY, and HD-ZIP family transcription-factor proteins. Fourteen BIGs were studied in detail to determine their role in resistance to Botrytis. T-DNA insertion alleles of ZFAR1 (At2G40140), the gene encoding a putative zinc-finger protein with ankyrin-repeat domains, showed increased local susceptibility to Botrytis and sensitivity to germination in the presence of abscisic acid (ABA), supporting the role of ABA in mediating responses to Botrytis infection. In addition, two independent T-DNA insertion alleles in the WRKY70 gene showed increased susceptibility to Botrytis. The transcriptional activation of genes involved in plant hormone signaling and synthesis, removal of reactive oxygen species, and defense and abiotic-stress responses, coupled with the susceptibility of the wrky70 and zfar1 mutants, highlights the complex genetic network underlying defense responses to Botrytis in Arabidopsis.

Fusarium graminearum is a ubiquitous pathogen of cereal crops, including wheat, barley, and maize. Diseases caused by F. graminearum are of particular concern because harvested grains frequently are contaminated with harmful mycotoxins such as deoxynivalenol (DON). In this study, we explored the role of Ras GTPases in pathogenesis. The genome of F. graminearum contains two putative Ras GTPase-encoding genes. The two genes (RAS1 and RAS2) showed different patterns of expression under different conditions of nutrient availability and in various mutant backgrounds. RAS2 was dispensable for survival but, when disrupted, caused a variety of morphological defects, including slower growth on solid media, delayed spore germination, and significant reductions in virulence on wheat heads and maize silks. Intracellular cAMP levels were not affected by deletion of RAS2 and exogenous treatment of the ras2 mutant with cAMP did not affect phenotypic abnormalities, thus indicating that RAS2 plays a minor or no role in cAMP signaling. However, phosphorylation of the mitogen-activated protein (MAP) kinase Gpmk1 and expression of a secreted lipase (FGL1) required for infection were reduced significantly in the ras2 mutant. Based on these observations, we hypothesize that RAS2 regulates growth and virulence in F. graminearum by regulating the Gpmk1 MAP kinase pathway.

Fumonisins are a group of mycotoxins that contaminate maize and cause leukoencephalomalacia in equine, pulmonary edema in swine, and promote cancer in mice. Fumonisin biosynthesis in Fusarium verticillioides is repressed by nitrogen and alkaline pH. We cloned a PACC-like gene (PAC1) from F. verticillioides. PACC genes encode the major transcriptional regulators of several pH-responsive pathways in other filamentous fungi. In Northern blot analyses, a PAC1 probe hybridized to a 2.2-kb transcript present in F. verticillioides grown at alkaline pH. A mutant of F. verticillioides with a disrupted PAC1 gene had severely impaired growth at alkaline pH. The mutant produced more fumonisin than the wild type when grown on maize kernels and in a synthetic medium buffered at an acidic pH, 4.5. The mutant, but not the wild type, also produced fumonisin B 1 when mycelia were resuspended in medium buffered at an alkaline pH, 8.4. Transcription of FUM1, a gene involved in fumonisin biosynthesis, was correlated with fumonisin production. We conclude that PAC1 is required for growth at alkaline pH and that Pac1 may have a role as a repressor of fumonisin biosynthesis under alkaline conditions.

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Fusarium verticillioides, a fungal pathogen of maize, produces fumonisin mycotoxins that adversely affect human and animal health. Basic questions remain unanswered regarding the interactions between the host plant and the fungus that lead to the accumulation of fumonisins in maize kernels. In this study, we evaluated the role of kernel endosperm composition in regulating fumonisin B1 (FB1) biosynthesis. We found that kernels lacking starch due to physiological immaturity did not accumulate FB1. Quantitative polymerase chain reaction analysis indicated that kernel development also affected the expression of fungal genes involved in FB1 biosynthesis, starch metabolism, and nitrogen regulation. A mutant strain of F. verticillioides with a disrupted a-amylase gene was impaired in its ability to produce FB1 on starchy kernels, and both the wild-type and mutant strains produced significantly less FB1 on a high-amylose kernel mutant of maize. When grown on a defined medium with amylose as the sole carbon source, the wild-type strain produced only trace amounts of FB1, but it produced large amounts of FB1 when grown on amylopectin or dextrin, a product of amylopectin hydrolysis. We conclude that amylopectin induces FB1 production in F. verticillioides. This study provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.

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.