Vascular Wilt Fungus Fusarium Oxysporum Research Articles

Molecular Dialogue During Host Manipulation by the Vascular Wilt Fungus Fusarium oxysporum.

Vascular wilt fungi are a group of hemibiotrophic phytopathogens that infect diverse crop plants. These pathogens have adapted to thrive in the nutrient-deprived niche of the plant xylem. Identification and functional characterization of effectors and their role in the establishment of compatibility across multiple hosts, suppression of plant defense, host reprogramming, and interaction with surrounding microbes have been studied mainly in model vascular wilt pathogens Fusarium oxysporum and Verticillium dahliae. Comparative analysis of genomes from fungal isolates has accelerated our understanding of genome compartmentalization and its role in effector evolution. Also, advances in recent years have shed light on the cross talk of root-infecting fungi across multiple scales from the cellular to the ecosystem level, covering their interaction with the plant microbiome as well as their interkingdom signaling. This review elaborates on our current understanding of the cross talk between vascular wilt fungi and the host plant, which eventually leads to a specialized lifestyle in the xylem. We particularly focus on recent findings in F. oxysporum, including multihost associations, and how they have contributed to understanding the biology of fungal adaptation to the xylem. In addition, we discuss emerging research areas and highlight open questions and future challenges.

Fusarium oxysporum Casein Kinase 1, a Negative Regulator of the Plasma Membrane H+-ATPase Pma1, Is Required for Development and Pathogenicity.

Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pHc) and mitogen-activated protein kinase (MAPK) signaling. In fungi, pHc is mainly controlled by the essential plasma membrane H+-ATPase Pma1. Here we created mutants of F. oxysporum lacking casein kinase 1 (Ck1), a known negative regulator of Pma1. We found that the ck1Δ mutants have constitutively high Pma1 activity and exhibit reduced alkalinization of the surrounding medium as well as decreased hyphal growth and conidiation. Importantly, the ck1Δ mutants exhibit defects in hyphal chemotropism towards plant roots and in pathogenicity on tomato plants. Thus, Ck1 is a key regulator of the development and virulence of F. oxysporum.

Marchantia polymorpha model reveals conserved infection mechanisms in the vascular wilt fungal pathogen Fusarium oxysporum.

Root-infecting vascular fungi cause wilt diseases and provoke devastating losses in hundreds of crops. It is currently unknown how these pathogens evolved and whether they can also infect nonvascular plants, which diverged from vascular plants over 450 million years ago. We established a pathosystem between the nonvascular plant Marchantia polymorpha (Mp) and the root-infecting vascular wilt fungus Fusarium oxysporum (Fo). On angiosperms, Fo exhibits exquisite adaptation to the plant xylem niche as well as host-specific pathogenicity, both of which are conferred by effectors encoded on lineage-specific chromosomes. Fo isolates displaying contrasting lifestyles on angiosperms - pathogenic vs endophytic - are able to infect Mp and cause tissue maceration and host cell killing. Using isogenic fungal mutants we define a set of conserved fungal pathogenicity factors, including mitogen activated protein kinases, transcriptional regulators and cell wall remodelling enzymes, that are required for infection of both vascular and nonvascular plants. Markedly, two host-specific effectors and a morphogenetic regulator, which contribute to vascular colonisation and virulence on tomato plants are dispensable on Mp. Collectively, these findings suggest that vascular wilt fungi employ conserved infection strategies on nonvascular and vascular plant lineages but also have specific mechanisms to access the vascular niche of angiosperms.

NADPH oxidase regulates chemotropic growth of the fungal pathogen Fusarium oxysporum towards the host plant.

Soil-inhabiting fungal pathogens use chemical signals to locate and colonise the host plant. In the vascular wilt fungus Fusarium oxysporum, hyphal chemotropism towards tomato roots is triggered by secreted plant peroxidases (Prx), which catalyse the reductive cleavage of reactive oxygen species (ROS). Here we show that this chemotropic response requires the regulated synthesis of ROS by the conserved fungal NADPH oxidase B (NoxB) complex, and their transformation into hydrogen peroxide (H2 O2 ) by superoxide dismutase (SOD). Deletion of NoxB or the regulatory subunit NoxR, or pharmacological inhibition of SOD, specifically abolished chemotropism of F.oxysporum towards Prx gradients. Addition of isotropic concentrations of H2 O2 rescued chemotropic growth in the noxBΔ and noxRΔ mutants, but not in a mutant lacking the G protein-coupled receptor Ste2. Prx-triggered rapid Nox- and Ste2-dependent phosphorylation of the cell wall integrity mitogen-activated protein kinase (CWI MAPK) Mpk1, an essential component of the chemotropic response. These results suggest that Ste2 and the CWI MAPK cascade function downstream of NoxB in Prx chemosensing. Our findings reveal a new role for Nox enzymes in directed hyphal growth of a filamentous pathogen towards its host and might be of broad interest for chemotropic interactions between plants and root-colonising fungi.

Arabidopsis thaliana RESISTANCE TO FUSARIUM OXYSPORUM 2 Implicates Tyrosine-Sulfated Peptide Signaling in Susceptibility and Resistance to Root Infection

In the plant Arabidopsis thaliana, multiple quantitative trait loci (QTLs), including RFO2, account for the strong resistance of accession Columbia-0 (Col-0) and relative susceptibility of Taynuilt-0 (Ty-0) to the vascular wilt fungus Fusarium oxysporum forma specialis matthioli. We find that RFO2 corresponds to diversity in receptor-like protein (RLP) genes. In Col-0, there is a tandem pair of RLP genes: RFO2/At1g17250 confers resistance while RLP2 does not. In Ty-0, the highly diverged RFO2 locus has one RLP gene conferring weaker resistance. While the endogenous RFO2 makes a modest contribution to resistance, transgenic RFO2 provides strong pathogen-specific resistance. The extracellular leucine-rich repeats (eLRRs) in RFO2 and RLP2 are interchangeable for resistance and remarkably similar to eLRRs in the receptor-like kinase PSY1R, which perceives tyrosine-sulfated peptide PSY1. Reduced infection in psy1r and mutants of related phytosulfokine (PSK) receptor genes PSKR1 and PSKR2 shows that tyrosine-sulfated peptide signaling promotes susceptibility. The related eLRRs in RFO2 and PSY1R are not interchangeable; and expression of the RLP nPcR, in which eLRRs in RFO2 are replaced with eLRRs in PSY1R, results in constitutive resistance. Counterintuitively, PSY1 signaling suppresses nPcR because psy1r nPcR is lethal. The fact that PSK signaling does not similarly affect nPcR argues that PSY1 signaling directly downregulates the expression of nPcR. Our results support a speculative but intriguing model to explain RFO2's role in resistance. We propose that F. oxysporum produces an effector that inhibits the normal negative feedback regulation of PSY1R, which stabilizes PSY1 signaling and induces susceptibility. However, RFO2, acting as a decoy receptor for PSY1R, is also stabilized by the effector and instead induces host immunity. Overall, the quantitative resistance of RFO2 is reminiscent of the better-studied monogenic resistance traits.

HapX-Mediated Iron Homeostasis Is Essential for Rhizosphere Competence and Virulence of the Soilborne Pathogen Fusarium oxysporum

Soilborne fungal pathogens cause devastating yield losses and are highly persistent and difficult to control. During the infection process, these organisms must cope with limited availability of iron. Here we show that the bZIP protein HapX functions as a key regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. Deletion of hapX does not affect iron uptake but causes derepression of genes involved in iron-consuming pathways, leading to impaired growth under iron-depleted conditions. F. oxysporum strains lacking HapX are reduced in their capacity to invade and kill tomato (Solanum lycopersicum) plants and immunodepressed mice. The virulence defect of ΔhapX on tomato plants is exacerbated by coinoculation of roots with a biocontrol strain of Pseudomonas putida, but not with a siderophore-deficient mutant, indicating that HapX contributes to iron competition of F. oxysporum in the tomato rhizosphere. These results establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals.

Role of new bacterial surfactins in the antifungal interaction between Bacillus amyloliquefaciens and Fusarium oxysporum

The aim of this work was to investigate the major mechanisms involved in antagonism of BO7, a novel strain of Bacillus amyloliquefaciens isolated from orchard soil, against the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol). BO7 markedly reduced the incidence of Fol vascular wilt on tomato plants and displayed strong in vitro inhibitory activity against Fol. Scanning electron microscopy demonstrated the ability of BO7 cells to adhere to fungal hyphae and to efficiently colonize tomato roots. The low molecular weight fraction of BO7 culture filtrate displayed high antifungal activity against Fol, resulting in growth inhibition and dramatic alterations of hyphal morphology. Three structurally related surfactin lipopeptides, identified as the major components of the bioactive fraction, were assayed for their inhibitory activity against Fol. One of these compounds exerted a strong and uncommon antifungal activity for lipopeptides of the surfactin family, accounting for most of the inhibitory activity of the BO7 culture filtrate. Among a collection of Fol knockout isolates tested, mutants altered in cell wall structure showed increased sensitivity to the bacterial compounds. These results suggest that the fungal cell wall might have a key role in the sensitivity of Fol towards bacterial surfactins from B. amyloliquefaciens.

Fusarium oxysporum Adh1 has dual fermentative and oxidative functions and is involved in fungal virulence in tomato plants

An alcohol dehydrogenase gene, adh1, has been identified in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that adh1 is highly expressed in mycelia grown in potato dextrose liquid medium (PDB) under hypoxic conditions, as compared to mycelia grown under aerobic conditions. One spontaneous allyl alcohol-resistant (Ally R) mutant exhibited insertion of an incomplete F. oxysporum transposable element, while another mutant contained a short (13 nucleotide) deletion, in both cases interrupting the coding region of the adh1 gene. These mutations caused deficiency in Adh activity due to loss of the main constitutive isoform of Adh1, as well as alteration of different physiological parameters related to carbon and energy metabolism, including the ability to use ethanol as a carbon source under aerobic conditions; impaired growth under hypoxic conditions with glucose as the carbon source; and diminished production of ethanol in glucose-containing medium. Interestingly, the adh1 mutations resulted in a significant delay in fungal disease development in tomato plants. Complementation with the wild-type adh1 allele repaired all defects caused by mutation, indicating that the product of the adh1 gene has dual enzymatic functions (fermentative and oxidative), depending on culture conditions, and is also required for full fungal virulence.

The Membrane Mucin Msb2 Regulates Invasive Growth and Plant Infection in Fusarium oxysporum

Fungal pathogenicity in plants requires a conserved mitogen-activated protein kinase (MAPK) cascade homologous to the yeast filamentous growth pathway. How this signaling cascade is activated during infection remains poorly understood. In the soil-borne vascular wilt fungus Fusarium oxysporum, the orthologous MAPK Fmk1 (Fusarium MAPK1) is essential for root penetration and pathogenicity in tomato (Solanum lycopersicum) plants. Here, we show that Msb2, a highly glycosylated transmembrane protein, is required for surface-induced phosphorylation of Fmk1 and contributes to a subset of Fmk1-regulated functions related to invasive growth and virulence. Mutants lacking Msb2 share characteristic phenotypes with the Δfmk1 mutant, including defects in cellophane invasion, penetration of the root surface, and induction of vascular wilt symptoms in tomato plants. In contrast with Δfmk1, Δmsb2 mutants were hypersensitive to cell wall targeting compounds, a phenotype that was exacerbated in a Δmsb2 Δfmk1 double mutant. These results suggest that the membrane mucin Msb2 promotes invasive growth and plant infection upstream of Fmk1 while contributing to cell integrity through a distinct pathway.

Biological management of vascular wilt of tomato caused by Fusarium oxysporum f.sp. lycospersici by plant growth-promoting rhizobacterial mixture

Two plant growth promoting rhizobacterial (PGPR) strains, GIBC-Jamog ( Bacillus subtilis) and TEPF-Sungal ( Burkholderia cepacia) and PGPR strain mixtures, S2BC-1 ( B. subtilis)+GIBC-Jamog ( B. subtilis) and S2BC-2 ( Bacillus atrophaeus)+TEPF-Sungal ( Burkholderia cepacia) which inhibited the mycelial growth of vascular wilt fungus Fusarium oxysporum f.sp. lycopersici were selected for assessing vascular wilt control in tomato by localized and induced systemic resistance (ISR) experiments in the greenhouse. Seed bacterization and soil application of S2BC-1+GIBC-Jamog challenge-inoculated with F. oxysporum f.sp. lycopersici resulted in significantly lower incidence (54.9% and 53.8% reduction) relative to the non-bacterized pathogen control in localized and split-root experiments, respectively. In localized studies, the lower disease incidence coincided with increases in the plant vigor index and fresh weight of 44.1% and 183.4%, respectively, relative to the pathogen control. Analysis of root samples in localized and split-root experiments also indicated increased induction of chitinase (2.2- and 2.6-fold, respectively) and β-1,3-glucanase (1.4- and 1.1-fold, respectively). In native gel activity assay, S2BC-1+GIBC-Jamog with challenge-inoculation, expressed high intensity peroxidase isoforms in localized and ISR experiments. It is presumed that the induced chitinase, β-1,3-glucanase and peroxidase in localized and split-root experiments may be involved in the reduction of vascular wilt development in tomato. Reduced disease severity coupled with enhanced enzyme production elicited by S2BC-1+GIBC-Jamog in localized and split-root experiments indicate that its mode of action for vascular wilt suppression in tomato is through both direct biocontrol and ISR.

A nitrogen response pathway regulates virulence in plant pathogenic fungi

Virulence in plant pathogenic fungi is controlled through a variety of cellular pathways in response to the host environment. Nitrogen limitation has been proposed to act as a key signal to trigger the in planta expression of virulence genes. Moreover, a conserved Pathogenicity mitogen activated protein kinase (MAPK) cascade is strictly required for plant infection in a wide range of pathogens. We investigated the relationship between nitrogen signaling and the Pathogenicity MAPK cascade in controlling infectious growth of the vascular wilt fungus Fusarium oxysporum. Several MAPK-activated virulence functions such as invasive growth, vegetative hyphal fusion and host adhesion were strongly repressed in the presence of the preferred nitrogen source ammonium. Repression of these functions by ammonium was abolished by L-Methionine sulfoximine (MSX) or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR (Target Of Rapamycin), respectively, and was dependent on the bZIP protein MeaB. Supplying tomato plants with ammonium rather than nitrate resulted in a significant delay of vascular wilt symptoms caused by the F. oxysporum wild type strain, but not by the ΔmeaB mutant. Ammonium also repressed invasive growth in two other pathogens, the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. Our results suggest the presence of a conserved nitrogen-responsive pathway that operates via TOR and MeaB to control infectious growth in plant pathogenic fungi.

A nitrogen response pathway regulates virulence functions in Fusarium oxysporum via the protein kinase TOR and the bZIP protein MeaB.

During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine sulfoximine or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR, respectively. Deletion of the bZIP protein MeaB also resulted in nitrogen source-independent activation of virulence mechanisms. Activation of these functions did not require the global nitrogen regulator AreA, suggesting that MeaB-mediated repression of virulence functions does not act through inhibition of AreA. Tomato plants (Solanum lycopersicum) supplied with ammonium rather than nitrate showed a significant reduction in vascular wilt symptoms when infected with the wild type but not with the DeltameaB strain. Nitrogen source also affected invasive growth in the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. We propose that a conserved nitrogen-responsive pathway might operate via TOR and MeaB to control virulence in plant pathogenic fungi.

The effector protein Avr2 of the xylem‐colonizing fungus Fusarium oxysporum activates the tomato resistance protein I‐2 intracellularly

To promote host colonization, many plant pathogens secrete effector proteins that either suppress or counteract host defences. However, when these effectors are recognized by the host's innate immune system, they trigger resistance rather than promoting virulence. Effectors are therefore key molecules in determining disease susceptibility or resistance. We show here that Avr2, secreted by the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol), shows both activities: it is required for full virulence in a susceptible host and also triggers resistance in tomato plants carrying the resistance gene I-2. Point mutations in AVR2, causing single amino acid changes, are associated with I-2-breakingFol strains. These point mutations prevent recognition by I-2, both in tomato and when both genes are co-expressed in leaves of Nicotiana benthamiana. Fol strains carrying the Avr2 variants are equally virulent, showing that virulence and avirulence functions can be uncoupled. Although Avr2 is secreted into the xylem sap when Fol colonizes tomato, the Avr2 protein can be recognized intracellularly by I-2, implying uptake by host cells.

Possible involvement of G-proteins and cAMP in the induction of progesterone hydroxylating enzyme system in the vascular wilt fungus Fusarium oxysporum

Fungi present the ability to hydroxylate steroids. In some filamentous fungi, progesterone induces an enzyme system which converts the compound into a less toxic hydroxylated product. We investigated the progesterone response in the vascular wilt pathogen Fusarium oxysporum, using mass spectrometry and high performance liquid chromatography (HPLC). Progesterone was mainly transformed into 15α-hydroxyprogesterone, which was found predominantly in the extracellular medium. The role of two conserved fungal signaling cascades in the induction of the progesterone-transforming enzyme system was studied, using knockout mutants lacking the mitogen-activated protein kinase Fmk1 or the heterotrimeric G-protein β subunit Fgb1 functioning upstream of the cyclic adenosine monophosphate (cAMP) pathway. No steroid hydroxylation was induced in the Δ fgb1 strain, suggesting a role for the G-protein β subunit in progesterone signaling. Exogenous cAMP restored the induction of progesterone-transforming activity in the Δ fgb1 strain, suggesting that steroid signaling in F. oxysporum is mediated by the cAMP-PKA pathway.

Identification of virulence genes in Fusarium oxysporum f. sp. lycopersici by large‐scale transposon tagging

Forward genetic screens are efficient tools for the dissection of complex biological processes, such as fungal pathogenicity. A transposon tagging system was developed in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici by inserting the novel modified impala element imp160::gfp upstream of the Aspergillus nidulans niaD gene, followed by transactivation with a constitutively expressed transposase. A collection of 2072 Nia(+) revertants was obtained from reporter strain T12 and screened for alterations in virulence, using a rapid assay for invasive growth on apple slices. Seven strains exhibited reduced virulence on both apple slices and intact tomato plants. Five of these were true revertants showing the re-insertion of imp160::gfp within or upstream of predicted coding regions, whereas the other two showed either excision without re-insertion or no excision. Linkage between imp160::gfp insertion and virulence phenotype was determined in four transposon-tagged loci using targeted deletion in the wild-type strain. Knockout mutants in one of the genes, FOXG_00016, displayed significantly reduced virulence, and complementation of the original revertant with the wild-type FOXG_00016 allele fully restored virulence. FOXG_00016 has homology to the velvet gene family of A. nidulans. The high rate of untagged virulence mutations in the T12 reporter strain appears to be associated with increased genetic instability, possibly as a result of the transactivation of endogenous transposable elements by the constitutively expressed transposase.

Influence of the chloride channel of Fusarium oxysporum on extracellular laccase activity and virulence on tomato plants

CLC-type voltage-gated chloride channels are a family of proteins which mediate chloride transport across the plasma and intracellular membranes. A clc1 gene from the vascular wilt fungus Fusarium oxysporum was characterized and disrupted. The predicted Clc1 protein contained highly conserved transmembrane and CBS domains of this protein family and showed significant identities to the Saccharomyces cerevisiae GEF1 and the Cryptococcus neoformans CLC-A chloride channels. Inactivation of clc1 caused a deficiency in laccase activity which was more severe than that found in any of the structural laccase mutants previously described. The addition of copper sulphate to the growth medium resulted in total recovery of extracellular laccase activity in Deltaclc1 mutants, although it did not activate transcription of any laccase genes. The pleiotropic phenotype displayed by the Fusarium chloride channel-deficient mutants included a significant delay in the development of disease on tomato plants, with a higher sensitivity to oxidative stress compounds as well as a significant decrease in laccase activity, thus suggesting a possible connection between virulence and the two processes. Nevertheless, we cannot rule out that additional phenotypes present in the Deltaclc1 mutants could play an essential role in the full virulence of Fusarium.

Functional Analyses of Laccase Genes from Fusarium oxysporum

Six laccase genes, lcc1, lcc2, lcc3, lcc4, lcc5, and lcc9, of the vascular wilt fungus Fusarium oxysporum were isolated and characterized. All genes have the characteristic conserved domains for copper binding of phenol oxidase enzymes. Targeted inactivation of lcc1, lcc3, and lcc5 resulted in a significant decrease of extracellular laccase activity. Reverse transcription-polymerase chain reaction showed that lcc1, lcc2, and lcc9 were constitutively expressed in culture, whereas lcc3 and lcc5 appeared down and up-regulated, respectively, by PacC. Oxidative stress conditions and phenolic compounds altered the growth rate of the Deltalcc3 mutant compared with the wild-type. lcc1, lcc3, and lcc9 were expressed in roots and stems during the infection process. However, inactivation of lcc1, lcc3, and lcc5 had no detectable effects on virulence on tomato plants.

Ctf1, a transcriptional activator of cutinase and lipase genes in Fusarium oxysporum is dispensable for virulence

Cutinolytic enzymes are secreted by fungal pathogens attacking the aerial parts of the plant, to facilitate penetration of the outermost cuticular barrier of the host. The role of cutinases in soil-borne root pathogens has not been studied thus far. Here we report the characterization of the zinc finger transcription factor Ctf1 from the vascular wilt fungus Fusarium oxysporum, a functional orthologue of CTF1alpha that controls expression of cutinase genes and virulence in the pea stem pathogen Fusarium solani f. sp. pisi. Mutants carrying a Deltactf1 loss-of-function allele grown on inducing substrates failed to activate extracellular cutinolytic activity and expression of the cut1 and lip1 genes, encoding a putative cutinase and lipase, respectively, whereas strains harbouring a ctf1(C) allele in which the ctf1 coding region was fused to the strong constitutive Aspergillus nidulans gpdA promoter showed increased induction of cutinase activity and gene expression. These results suggest that F. oxysporum Ctf1 mediates expression of genes involved in fatty acid hydrolysis. However, expression of lip1 during root infection was not dependent on Ctf1, and virulence of the ctf1 mutants on tomato plants and fruits was indistinguishable from that of the wild-type. Thus, in contrast to the stem pathogen F. solani, Ctf1 is not essential for virulence in the root pathogen F. oxysporum.

Vegetative Hyphal Fusion Is Not Essential for Plant Infection by Fusarium oxysporum

Vegetative hyphal fusion (VHF) is a ubiquitous phenomenon in filamentous fungi whose biological role is poorly understood. In Neurospora crassa, the mitogen-activated protein kinase (MAPK) Mak-2 and the WW domain protein So are required for efficient VHF. A MAPK orthologous to Mak-2, Fmk1, was previously shown to be essential for root penetration and pathogenicity of the vascular wilt fungus Fusarium oxysporum. Here we took a genetic approach to test two hypotheses, that (i) VHF and plant infection have signaling mechanisms in common and (ii) VHF is required for efficient plant infection. F. oxysporum mutants lacking either Fmk1 or Fso1, an orthologue of N. crassa So, were impaired in the fusion of vegetative hyphae and microconidial germ tubes. Deltafmk1 Deltafso1 double mutants exhibited a more severe fusion phenotype than either single mutant, indicating that the two components function in distinct pathways. Both Deltafso1 and Deltafmk1 strains were impaired in the formation of hyphal networks on the root surface, a process associated with extensive VHF. The Deltafso1 mutants exhibited slightly reduced virulence in tomato fruit infection assays but, in contrast to Deltafmk1 strains, were still able to perform functions associated with invasive growth, such as secretion of pectinolytic enzymes or penetration of cellophane sheets, and to infect tomato plants. Thus, although VHF per se is not essential for plant infection, both processes have some signaling components in common, suggesting an evolutionary relationship between the underlying cellular mechanisms.

The Fusarium oxysporum sti35 gene functions in thiamine biosynthesis and oxidative stress response

The sti35 gene of the vascular wilt fungus Fusarium oxysporum was originally identified based on induced expression under stress conditions. In this study, the transcriptional regulation and biological function of sti35 were examined in the tomato pathogen F. oxysporum f.sp . lycopersici. Expression of sti35 was repressed by thiamine and induced by high temperatures. Sti35 transcripts were detected both during early and late stages of infection of tomato plants by F. oxysporum. Heterologous expression of the sti35 cDNA restored thiamine prototrophy in a Saccharomyces cerevisiae thi4 mutant and increased UV tolerance in a uvr − mutant of Escherichia coli. Targeted Δ sti35 knockout mutants of F. oxysporum exhibited a thiamine auxotrophic phenotype and reduced tolerance to the superoxide-generating agent menadione, indicating that Sti35 has a dual role in thiamine biosynthesis and oxidative stress response. RT-PCR analysis revealed the presence of differential RNA splicing of the second 5′-UTR intron, suggesting that thiamine may regulate sti35 expression via a post-transcriptional mechanism. F. oxysporum transformants carrying a transcriptional fusion of the sti35 promoter to the lacZ reporter gene produced high levels of β-galactosidase activity when grown in the absence, but not in the presence of thiamine. Thus, the sti35 promoter represents a useful tool for the controlled expression of genes of interest in F. oxysporum.