http://www.fgsc.net/fus.htm, http://www-genome.wi.mit.edu/annotation/fungi/fusarium/, http://www.cbs.knaw.nl/fusarium/database.html.

SummaryGene expression in fungi by ambient pH is regulated via a conserved signalling cascade whose terminal component is the zinc finger transcription factor PacC/Rim1p. We have identified a pacC orthologue in the vascular wilt pathogen Fusarium oxysporum that binds the consensus 5 ¢ ¢ ¢ ¢ -GCCAAG-3 ¢ ¢ ¢ ¢ sequence and is proteolytically processed in a similar way to PacC from Aspergillus nidulans. pacC transcript levels were elevated in F. oxysporum grown in alkaline conditions and almost undetectable at extreme acidic growth conditions. PacC + + + + /-loss-of-function mutants displayed an acidity-mimicking phenotype resulting in poor growth at alkaline pH, increased acid protease activity and higher transcript levels of acid-expressed polygalacturonase genes. Reintroduction of a functional pacC copy into a pacC + + + + /-mutant restored the wild-type phenotype. Conversely, F. oxysporum merodiploids carrying a dominant activating pacC c allele had increased pacC transcript and protein levels and displayed an alkalinity-mimicking phenotype with reduced acid phosphatase and increased alkaline protease activities. PacC + + + + /-mutants were more virulent than the wild-type strain in root infection assays with tomato plants, whereas pacC c strains were significantly reduced in virulence. We propose that F. oxysporum PacC acts as a negative regulator of virulence to plants, possibly by preventing transcription of acid-expressed genes important for infection.

Fungal pathogens cause disease in plant and animal hosts. The extent to which infection mechanisms are conserved between both classes of hosts is unknown. We present a dual plant-animal infection system based on a single strain of Fusarium oxysporum, the causal agent of vascular wilt disease in plants and an emerging opportunistic human pathogen. Injection of microconidia of a well-characterized tomato pathogenic isolate (isolate 4287) into the lateral tail vein of immunodepressed mice resulted in disseminated infection of multiple organs and death of the animals. Knockout mutants in genes encoding a mitogen-activated protein kinase, a pH response transcription factor, or a class V chitin synthase previously shown to be implicated in virulence on tomato plants were tested in the mouse model. The results indicate that some of these virulence factors play functionally distinct roles during the infection of tomato plants and mice. Thus, a single F. oxysporum strain can be used to study fungal virulence mechanisms in plant and mammalian pathogenesis.

Glycosylphosphatidylinositol-anchored (beta)-1,3-glucanosyltransferases play active roles in fungal cell wall biosynthesis and morphogenesis and have been implicated in virulence on mammals. The role of beta-1,3-glucanosyltransferases in pathogenesis to plants has not been explored so far. Here, we report the cloning and mutational analysis of the gas1 gene encoding a putative beta-1,3-glucanosyltransferase from the vascular wilt fungus Fusarium oxysporum. In contrast to Candida albicans, expression of gas1 in F. oxysporum was independent of ambient pH and of the pH response transcription factor PacC. Gene knockout mutants lacking a functional gas1 allele grew in a way similar to the wildtype strain in submerged culture but exhibited restricted colony growth on solid substrates. The restricted growth phenotype was relieved by the osmotic stabilizer sorbitol, indicating that it may be related to structural alterations in the cell wall. Consistent with this hypothesis, deltagas1 mutants exhibited enhanced resistance to cell wall-degrading enzymes and increased transcript levels of chsV and rho1, encoding a class V chitin synthase and a small monomeric G protein, respectively. The deltagas1 mutants showed dramatically reduced virulence on tomato, both in a root infection assay and in a fruit tissue-invasion model, thus providing the first evidence for an essential role of fungal beta-1,3-glucanosyltransferases during plant infection.

Fungi possess efficient mechanisms of pH and ion homeostasis, allowing them to grow over a wide range of environmental conditions. In this study, we addressed the role of the pH response transcription factor PacC in salt tolerance of the vascular wilt pathogen Fusarium oxysporum. Loss-of-function pacC ؉/؊ mutants showed increased sensitivity to Li ؉ and Na ؉ and accumulated higher levels of these cations than the wild type. In contrast, strains expressing a dominant activating pacC c allele were more salt tolerant and had lower intracellular Li ؉ and Na ؉ concentrations. Although the kinetics of Li ؉ influx were not altered by mutations in pacC, we found that Li ؉ efflux at an alkaline, but not at an acidic, ambient pH was significantly reduced in pacCloss-of-function mutants. To explore the presence of a PacC-dependent efflux mechanism in F. oxysporum, we cloned ena1 encoding an orthologue of the yeast P-type Na ؉ -ATPase ENA1. Northern analysis revealed that efficient transcriptional activation of ena1 in F. oxysporum required the presence of high Na ؉ concentrations and alkaline ambient pH and was dependent on PacC function. We propose a model in which PacC controls ion homeostasis in F. oxysporum at a high pH by activating expression of ena1 coordinately with a second Na ؉ -responsive signaling pathway.Fungi are a versatile class of organisms that have successfully occupied numerous ecological niches, including those of plant and animal pathogenesis. A striking property of fungi and a major determinant of their evolutionary success is their capacity to adapt to an extremely wide range of environmental conditions. This ability depends crucially on the presence of cellular sensory networks that monitor the environment and mediate changes in gene expression in response to shifts in the external conditions. We use the vascular wilt pathogen Fusarium oxysporum as a model to understand how environmental signals regulate gene expression in fungi and how these regulatory mechanisms determine fungal virulence.A key factor in fungal growth and development is ambient pH. Fungi grow over a wide range of pH conditions and must thus be able to tailor gene expression to the particular pH of their growth environment. A conserved signaling cascade integrated by the products of the pal genes, whose terminal component is the zinc-finger transcription factor PacC/ Rim101p, regulates gene expression in response to ambient pH (18). Upon shift to alkaline pH, an inactive PacC precursor is posttranscriptionally activated by proteolytic processing into a shorter functional form that activates genes preferentially expressed at alkaline pH and represses genes expressed under acidic growth conditions (18). The pacC orthologue of F. oxysporum was recently cloned, and the encoded protein was shown to regulate pH-dependent gene expression and to function as a negative regulator of virulence on plants (11). Thus, pacC ϩ/Ϫ loss-of-function mutants of F. oxysporum mimic growth at acidic ambient pH and exhibit increased virulence, whereas pac...

Summary• Geminiviruses are plant viruses with circular, single-stranded (ss) DNA genomes that infect a wide range of species and cause important losses in agriculture. Geminiviruses do not encode their own DNA polymerase, and rely on the host cell machinery for their replication.• Here, we identify a positive effect of the curtovirus Beet curly top virus (BCTV) on the begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) infection in Nicotiana benthamiana plants.• Our results show that this positive effect is caused by the promotion of TYLCSV replication by BCTV C2. Transcriptomic analyses of plants expressing C2 unveil an up-regulation of cell cycle-related genes induced on cell cycle re-entry; experiments with two mutated versions of C2 indicate that this function resides in the N-terminal part of C2, which is also sufficient to enhance geminiviral replication. Moreover, C2 expression promotes the replication of other geminiviral species, but not of RNA viruses.• We conclude that BCTV C2 has a novel function in the promotion of viral replication, probably by restoring the DNA replication competency of the infected cells and thus creating a favourable cell environment for viral spread. Because C2 seems to have a broad impact on the replication of geminiviruses, this mechanism might have important epidemiological implications.

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PG1, the major endopolygalacturonase of the vascular wilt pathogenFusarium oxysporum, was secreted during growth on pectin by 10 of 12 isolates belonging to seven formae speciales, as determined with isoelectric focusing zymograms and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. A Southern analysis of genomic DNA and PCR performed with gene-specific primers revealed that the pg1 locus was highly conserved structurally in most isolates. Two PG1-deficient isolates were identified; one lacked the encoding gene, and the other carried a pg1 allele disrupted by a 3.2-kb insertion with sequence homology to hATtransposases. The virulence for muskmelon of different F. oxysporum f. sp. melonis isolates was not correlated with PG1 production in vitro. We concluded that PG1 is widely distributed in F. oxysporum and that it is not essential for pathogenicity.