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.

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

BACKGROUND: It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. RESULTS: Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species. CONCLUSIONS: This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms.

Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

KEY MESSAGE: Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

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.