The Role of the Fusarium oxysporum FTF2 Transcription Factor in Host Colonization and Virulence in Common Bean Plants (Phaseolus vulgaris L.).

The FTF (Fusarium Transcription Factor) gene family is composed of two members (FTF1 and FTF2) with high-sequence homology that encode transcription factors involved in the modulation of virulence in the F. oxysporum species complex (FOSC). While FTF1 is a multicopy gene exclusive of highly virulent strains of FOSC and is located in the accessory genome, FTF2 is a single-copy gene, located in the core genome, and well-conserved in all filamentous ascomycete fungi, except yeast. The involvement of FTF1 in the colonization of the vascular system and regulation of the expression of SIX effectors has been stablished. To address the role of FTF2, we generated and characterized mutants defective in FTF2 in a F. oxysporum f. sp. phaseoli weakly virulent strain and analyzed them together with the equivalent mutants formerly obtained in a highly virulent strain. The results obtained highlight a role for FTF2 as a negative regulator of the production of macroconidia and demonstrate that it is required for full virulence and the positive regulation of SIX effectors. In addition, gene expression analyses provided compelling evidence that FTF2 is involved in the regulation of hydrophobins likely required for plant colonization.

Nitric Oxide Metabolism Affects Germination in Botrytiscinerea and Is Connected to Nitrate Assimilation.

Nitric oxide regulates numerous physiological processes in species from all taxonomic groups. Here, its role in the early developmental stages of the fungal necrotroph Botrytis cinerea was investigated. Pharmacological analysis demonstrated that NO modulated germination, germ tube elongation and nuclear division rate. Experimental evidence indicates that exogenous NO exerts an immediate but transitory negative effect, slowing down germination-associated processes, and that this effect is largely dependent on the flavohemoglobin BCFHG1. The fungus exhibited a "biphasic response" to NO, being more sensitive to low and high concentrations than to intermediate levels of the NO donor. Global gene expression analysis in the wild-type and ΔBcfhg1 strains indicated a situation of strong nitrosative and oxidative stress determined by exogenous NO, which was much more intense in the mutant strain, that the cells tried to alleviate by upregulating several defense mechanisms, including the simultaneous upregulation of the genes encoding the flavohemoglobin BCFHG1, a nitronate monooxygenase (NMO) and a cyanide hydratase. Genetic evidence suggests the coordinated expression of Bcfhg1 and the NMO coding gene, both adjacent and divergently arranged, in response to NO. Nitrate assimilation genes were upregulated upon exposure to NO, and BCFHG1 appeared to be the main enzymatic system involved in the generation of the signal triggering their induction. Comparative expression analysis also showed the influence of NO on other cellular processes, such as mitochondrial respiration or primary and secondary metabolism, whose response could have been mediated by NmrA-like domain proteins.

In Planta Gene Expression Analysis and Colonization of Fusarium oxysporum.

In planta gene expression analysis and GFP-based confocal microscopy are two powerful techniques that may be coupled to assess the extent and dynamics of plant colonization by a fungal pathogen. Here we describe methods to prepare common bean plants for inoculation with a highly virulent strain of Fusarium oxysporum f. sp. phaseoli, quantify the extent of colonization by RT-qPCR, and visualize the colonized tissues by confocal microscopy.

A Major Effect Gene Controlling Development and Pathogenicity in Botrytis cinerea Identified Through Genetic Analysis of Natural Mycelial Non-pathogenic Isolates.

Botrytis cinerea is a necrotrophic plant pathogenic fungus with a wide host range. Its natural populations are phenotypically and genetically very diverse. A survey of B. cinerea isolates causing gray mold in the vineyards of Castilla y León, Spain, was carried out and as a result eight non-pathogenic natural variants were identified. Phenotypically these isolates belong to two groups. The first group consists of seven isolates displaying a characteristic mycelial morphotype, which do not sporulate and is unable to produce sclerotia. The second group includes one isolate, which sporulates profusely and does not produce sclerotia. All of them are unresponsive to light. Crosses between a representative mycelial non-pathogenic isolate and a highly aggressive field isolate revealed that the phenotypic differences regarding pathogenicity, sporulation and production of sclerotia cosegregated in the progeny and are determined by a single genetic locus. By applying a bulked segregant analysis strategy based on the comparison of the two parental genomes the locus was mapped to a 110 kb region in chromosome 4. Subcloning and transformation experiments revealed that the polymorphism is an SNP affecting gene Bcin04g03490 in the reference genome of B. cinerea. Genetic complementation analysis and sequencing of the Bcin04g03490 alleles demonstrated that the mutations in the mycelial isolates are allelic and informed about the nature of the alterations causing the phenotypes observed. Integration of the allele of the pathogenic isolate into the non-pathogenic isolate fully restored the ability to infect, to sporulate and to produce sclerotia. Therefore, it is concluded that a major effect gene controlling differentiation and developmental processes as well as pathogenicity has been identified in B. cinerea. It encodes a protein with a GAL4-like Zn(II)2Cys6 binuclear cluster DNA binding domain and an acetyltransferase domain, suggesting a role in regulation of gene expression through a mechanism involving acetylation of specific substrates.

The FTF gene family regulates virulence and expression of SIX effectors in Fusarium oxysporum.

The FTF (Fusarium transcription factor) gene family comprises a single copy gene, FTF2, which is present in all the filamentous ascomycetes analysed, and several copies of a close relative, FTF1, which is exclusive to Fusarium oxysporum. An RNA-mediated gene silencing system was developed to target mRNA produced by all the FTF genes, and tested in two formae speciales: F. oxysporum f. sp. phaseoli (whose host is common bean) and F. oxysporum f. sp. lycopersici (whose host is tomato). Quantification of the mRNA levels showed knockdown of FTF1 and FTF2 in randomly isolated transformants of both formae speciales. The attenuation of FTF expression resulted in a marked reduction in virulence, a reduced expression of several SIX (Secreted In Xylem) genes, the best studied family of effectors in F. oxysporum, and lower levels of SGE1 (Six Gene Expression 1) mRNA, the presumptive regulator of SIX expression. Moreover, the knockdown mutants showed a pattern of colonization of the host plant similar to that displayed by strains devoid of FTF1 copies (weakly virulent strains). Gene knockout of FTF2 also resulted in a reduction in virulence, but to a lesser extent. These results demonstrate the role of the FTF gene expansion, mostly the FTF1 paralogues, as a regulator of virulence in F. oxysporum and suggest that the control of effector expression is the mechanism involved.

Gene expression patterns and dynamics of the colonization of common bean (Phaseolus vulgaris L.) by highly virulent and weakly virulent strains of Fusarium oxysporum.

The dynamics of root and hypocotyl colonization, and the gene expression patterns of several fungal virulence factors and plant defense factors have been analyzed and compared in the interaction of two Fusarium oxysporum f. sp. phaseoli strains displaying clear differences in virulence, with a susceptible common bean cultivar. The growth of the two strains on the root surface and the colonization of the root was quantitatively similar although the highly virulent (HV) strain was more efficient reaching the central root cylinder. The main differences between both strains were found in the temporal and spatial dynamics of crown root and hypocotyl colonization. The increase of fungal biomass in the crown root was considerably larger for the HV strain, which, after an initial stage of global colonization of both the vascular cylinder and the parenchymal cells, restricted its growth to the newly differentiated xylem vessels. The weakly virulent (WV) strain was a much slower and less efficient colonizer of the xylem vessels, showing also growth in the intercellular spaces of the parenchyma. Most of the virulence genes analyzed showed similar expression patterns in both strains, except SIX1, SIX6 and the gene encoding the transcription factor FTF1, which were highly upregulated in root crown and hypocotyl. The response induced in the infected plant showed interesting differences for both strains. The WV strain induced an early and strong transcription of the PR1 gene, involved in SAR response, while the HV strain preferentially induced the early expression of the ethylene responsive factor ERF2.

New virulence groups in Fusarium oxysporum f. sp. phaseoli: the expression of the gene coding for the transcription factor ftf1 correlates with virulence.

Fusarium oxysporum f. sp. phaseoli strains isolated from runner bean plants showing Fusarium wilt symptoms were characterized. The analysis of the genetic diversity of these strains and the comparison with strains formerly isolated from diseased common bean plants grown in the same region of Spain indicated a close genetic similarity among them. Pathogenicity assays carried out on runner bean plants showed virulence differences that allowed the classification of these strains into three groups: super virulent, highly virulent, and weakly virulent. However, all the analyzed strains behaved as highly virulent when inoculated on common bean plants, indicating that virulence is specific of the host-pathogen interaction. We also analyzed the number of copies and expression of the gene encoding the transcription factor ftf1, which has been shown to be specific of virulent F. oxysporum strains and highly up-regulated during plant infection. In planta real-time quantitative polymerase chain reaction expression analysis showed that expression of ftf1 was correlated with the degree of virulence. The comparative analysis of the polymorphic copies of ftf1 detected in the strains here characterized and those detected in the genome sequence of F. oxysporum f. sp. lycopersici strain 4287 indicates that some of the copies are likely nonfunctional.

fost12, the Fusarium oxysporum homolog of the transcription factor Ste12, is upregulated during plant infection and required for virulence.

We have identified a Fusarium oxysporum homolog of the Ste12 transcription factor that regulates mating and filamentation in Saccharomyces cerevisiae. The corresponding gene, fost12, from a highly virulent strain of F. oxysporum f. sp. phaseoli, was confirmed to share a high level of similarity and possessed the STE and C2H2 domains characteristic of the fungal Ste12 transcription factor family of proteins. Disruption of fost12 resulted in no visible alterations of colony morphology or in vitro growth characteristics. However, the disruption mutants showed a substantial reduction in virulence when inoculated in common bean seedlings. In planta transcription of fost12 is drastically increased between 12 and 24h after inoculation, as detected by real-time RT-PCR. The results of the transcriptional analyses carried out in several F. oxysporum strains during axenic growth suggest that the fost12 gene product is a virulence factor required to deal with the nutritional stress confronted by the pathogen during host plant colonization.

The gene coding for a new transcription factor (ftf1) of Fusarium oxysporum is only expressed during infection of common bean.

We report the isolation and analysis of the gene encoding ftf1 (Fusarium transcription factor 1), a previously undescribed putative transcription factor from highly virulent strains of Fusarium oxysporum f.sp. phaseoli that is transcribed specifically during early stages of infection of its host common bean (Phaseolus vulgaris L.). The predicted 1080 amino acid ftf1 protein contains a Zn(II)2-Cys6 binuclear cluster DNA-binding motif. ftf1 expression during axenic growth in culture was not detected by either Northern or RT-PCR. On the contrary, in planta transcription of ftf1 is increased about 24h after plant inoculation, as detected by real-time RT-PCR. This result suggests that ftf1 has a role in the establishment of the fungus within the plant and/or the progress of the disease. Multiple copies of ftf1 are present in highly virulent strains of F. oxysporum f.sp. phaseoli.

A DNA-Based Procedure for In Planta Detection of Fusarium oxysporum f. sp. phaseoli.

ABSTRACT We have characterized strains of Fusarium oxysporum from common bean fields in Spain that were nonpathogenic on common bean, as well as F. oxysporum strains (F. oxysporum f. sp. phaseoli) pathogenic to common bean by random amplified polymorphic DNA (RAPD) analysis. We identified a RAPD marker (RAPD 4.12) specific for the highly virulent pathogenic strains of the seven races of F. oxysporum f. sp. phaseoli. Sequence analysis of RAPD 4.12 allowed the design of oligonucleotides that amplify a 609-bp sequence characterized amplified region (SCAR) marker (SCAR-B310A280). Under controlled environmental and greenhouse conditions, detection of the pathogen by polymerase chain reaction was 100% successful in root samples of infected but still symptomless plants and in stem samples of plants with disease severity of >/=4 in the Centro Internacional de Agricultura Tropical (CIAT; Cali, Colombia) scale. The diagnostic procedure can be completed in 5 h and allows the detection of all known races of the pathogen in plant samples at early stages of the disease with no visible symptoms.