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The fungal genus Plectosphaerella comprises species and strains with different lifestyles on plants, such as P. cucumerina, which has served as model for the characterization of Arabidopsis thaliana basal and nonhost resistance to necrotrophic fungi. We have sequenced, annotated, and compared the genomes and transcriptomes of three Plectosphaerella strains with different lifestyles on A. thaliana, namely, PcBMM, a natural pathogen of wild-type plants (Col-0), Pc2127, a nonpathogenic strain on Col-0 but pathogenic on the immunocompromised cyp79B2 cyp79B3 mutant, and P0831, which was isolated from a natural population of A. thaliana and is shown here to be nonpathogenic and to grow epiphytically on Col-0 and cyp79B2 cyp79B3 plants. The genomes of these Plectosphaerella strains are very similar and do not differ in the number of genes with pathogenesis-related functions, with the exception of secreted carbohydrate-active enzymes (CAZymes), which are up to five times more abundant in the pathogenic strain PcBMM. Analysis of the fungal transcriptomes in inoculated Col-0 and cyp79B2 cyp79B3 plants at initial colonization stages confirm the key role of secreted CAZymes in the necrotrophic interaction, since PcBMM expresses more genes encoding secreted CAZymes than Pc2127 and P0831. We also show that P0831 epiphytic growth on A. thaliana involves the transcription of specific repertoires of fungal genes, which might be necessary for epiphytic growth adaptation. Overall, these results suggest that in-planta expression of specific sets of fungal genes at early stages of colonization determine the diverse lifestyles and pathogenicity of Plectosphaerella strains.

[EN] 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 RNAmediated 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.

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (Fom), is one of the most widespread and devastating melon diseases. This vascular disease is caused by the colonization of melon xylem vessels by any of the four Fom races reported (r0, r1, r2 and r1,2, subdivided into r1,2w and r1,2y). The macroscopic evaluation of disease symptoms (disease rating, DR) at several days post-inoculation (dpi) with Fom spores has been the traditional method to determine the resistance of melon accessions to this fungal pathogen. In this study, one isolate from each Fom race was transformed by Agrobacterium tumefaciens to constitutively express the green fluorescent protein (GFP). Fom-GFP transformants, as virulent as the corresponding wildtype races, were selected to develop an inoculation assay based on the non-invasive evaluation of the fluorescence emitted by Fom-GFP. It was determined that melon root neck was the appropriate area to follow Fom-GFP and a fluorescence signal rating (FSR) was established in parallel to DR determination. This method allowed the evaluation of GFP signal in the root neck of inoculated melon seedlings at 11–15 dpi. The GFP signal was scored in 62 melon accessions/breeding lines inoculated with different Fom-GFP, followed by evaluation of the macroscopic DR in the aerial part of melon seedlings at 20–28 dpi. Correlation analysis demonstrated a direct and significant relationship between FSR and DR. This method has shown to be an effective and reliable tool that can assist Fom resistance breeding programmes in melon.

Plant resistance to necrotrophic fungi is regulated by a complex set of signaling pathways that includes those mediated by the hormones salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and abscisic acid (ABA). The role of ABA in plant resistance remains controversial, as positive and negative regulatory functions have been described depending on the plant-pathogen interaction analyzed. Here, we show that ABA signaling negatively regulates Arabidopsis (Arabidopsis thaliana) resistance to the necrotrophic fungus Plectosphaerella cucumerina. Arabidopsis plants impaired in ABA biosynthesis, such as the aba1-6 mutant, or in ABA signaling, like the quadruple pyr/pyl mutant (pyr1pyl1pyl2pyl4), were more resistant to P. cucumerina than wild-type plants. In contrast, the hab1-1abi1-2abi2-2 mutant impaired in three phosphatases that negatively regulate ABA signaling displayed an enhanced susceptibility phenotype to this fungus. Comparative transcriptomic analyses of aba1-6 and wild-type plants revealed that the ABA pathway negatively regulates defense genes, many of which are controlled by the SA, JA, or ET pathway. In line with these data, we found that aba1-6 resistance to P. cucumerina was partially compromised when the SA, JA, or ET pathway was disrupted in this mutant. Additionally, in the aba1-6 plants, some genes encoding cell wall-related proteins were misregulated. Fourier transform infrared spectroscopy and biochemical analyses of cell walls from aba1-6 and wild-type plants revealed significant differences in their Fourier transform infrared spectratypes and uronic acid and cellulose contents. All these data suggest that ABA signaling has a complex function in Arabidopsis basal resistance, negatively regulating SA/JA/ET- mediated resistance to necrotrophic fungi. Fil: Sánchez Vallet, Andrea. Universidad Politécnica de Madrid; España Fil: López, Gemma. Universidad Politécnica de Madrid; España Fil: Ramos, Brisa. Universidad Politécnica de Madrid; España Fil: Delgado Cerezo, Magdalena. Universidad Politécnica de Madrid; España Fil: Riviere, Marie Pierre. Universidad Politécnica de Madrid; España Fil: Llorente, Francisco. Universidad Politécnica de Madrid; España Fil: Fernández, Paula Virginia. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Miedes, Eva. Universidad Politécnica de Madrid; España Fil: Estevez, Jose Manuel. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Grant, Murray. University of Exeter; Reino Unido Fil: Molina, Antonio. Universidad Politécnica de Madrid; España

Summary The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs. To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens-mediated transformation. We generated PcBMM-GFP and Pc2127-GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T-DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM-GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers. By comparing the colonization of Arabidopsis wild-type plants and hypersusceptible (agb1-1 and cyp79B2cyp79B3) and resistant (irx1-6) mutants by PcBMM-GFP or Pc2127-GFP, we found that the plant immune response was already mounted at 12–18 h post-inoculation, and that Arabidopsis resistance to these fungi correlated with the time course of spore germination and hyphal growth on the leaf surface. The virulence of a subset of the PcBMM T-DNA insertional transformants was determined in Arabidopsis wild-type plants and agb1-1 mutant, and several transformants were identified that showed altered virulence in these genotypes in comparison with that of untransformed PcBMM. The T-DNA flanking regions in these fungal mutants were successfully sequenced, further supporting the utility of these functional genomics tools in the molecular characterization of the pathogenicity of necrotrophic fungi.

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.

The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs. To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens‐mediated transformation. We generated PcBMM‐GFP and Pc2127‐GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T‐DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM‐GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers. By comparing the colonization of Arabidopsis wild‐type plants and hypersusceptible (agb1‐1 and cyp79B2cyp79B3) and resistant (irx1‐6) mutants by PcBMM‐GFP or Pc2127‐GFP, we found that the plant immune response was already mounted at 12–18 h post‐inoculation, and that Arabidopsis resistance to these fungi correlated with the time course of spore germination and hyphal growth on the leaf surface. The virulence of a subset of the PcBMM T‐DNA insertional transformants was determined in Arabidopsis wild‐type plants and agb1‐1 mutant, and several transformants were identified that showed altered virulence in these genotypes in comparison with that of untransformed PcBMM. The T‐DNA flanking regions in these fungal mutants were successfully sequenced, further supporting the utility of these functional genomics tools in the molecular characterization of the pathogenicity of necrotrophic fungi.

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.

A defence pathway contributing to non-host resistance to biotrophic fungi in Arabidopsis involves the synthesis and targeted delivery of the tryptophan (trp)-derived metabolites indol glucosinolates (IGs) and camalexin at pathogen contact sites. We have examined whether these metabolites are also rate-limiting for colonization by necrotrophic fungi. Inoculation of Arabidopsis with adapted or non-adapted isolates of the ascomycete Plectosphaerella cucumerina triggers the accumulation of trp-derived metabolites. We found that their depletion in cyp79B2 cyp79B3 mutants renders Arabidopsis fully susceptible to each of three tested non-adapted P. cucumerina isolates, and super-susceptible to an adapted P. cucumerina isolate. This assigns a key role to trp-derived secondary metabolites in limiting the growth of both non-adapted and adapted necrotrophic fungi. However, 4-methoxy-indol-3-ylmethylglucosinolate, which is generated by the P450 monooxygenase CYP81F2, and hydrolyzed by PEN2 myrosinase, together with the antimicrobial camalexin play a minor role in restricting the growth of the non-adapted necrotrophs. This contrasts with a major role of these two trp-derived phytochemicals in limiting invasive growth of non-adapted biotrophic powdery mildew fungi, thereby implying the existence of other unknown trp-derived metabolites in resistance responses to non-adapted necrotrophic P. cucumerina. Impaired defence to non-adapted P. cucumerina, but not to the non-adapted biotrophic fungus Erysiphe pisi, on cyp79B2 cyp79B3 plants is largely restored in the irx1 background, which shows a constitutive accumulation of antimicrobial peptides. Our findings imply differential contributions of antimicrobials in non-host resistance to necrotrophic and biotrophic pathogens.

In plants, resistance to necrotrophic pathogens depends on the interplay between different hormone systems, such as those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene, and abscisic acid. Repression of auxin signaling by the SA pathway was recently shown to contribute to antibacterial resistance. Here, we demonstrate that Arabidopsis auxin signaling mutants axr1, axr2, and axr6 that have defects in the auxin-stimulated SCF (Skp1¿Cullin¿ F-box) ubiquitination pathway exhibit increased susceptibility to the necrotrophic fungi Plectosphaerella cucumerina and Botrytis cinerea. Also, stabilization of the auxin transcriptional repressor AXR3 that is normally targeted for removal by the SCF-ubiquitin/proteasome machinery occurs upon P. cucumerina infection. Pharmacological inhibition of auxin transport or proteasome function each compromise necrotroph resistance of wild-type plants to a similar extent as in non-treated auxin response mutants. These results suggest that auxin signaling is important for resistance to the necrotrophic fungi P. cucumerina and B. cinerea. SGT1b (one of two Arabidopsis SGT1 genes encoding HSP90/HSC70 co-chaperones) promotes the functions of SCF E3-ubiquitin ligase complexes in auxin and JA responses and resistance conditioned by certain Resistance (R) genes to biotrophic pathogens. We find that sgt1b mutants are as resistant to P. cucumerina as wild-type plants. Conversely, auxin/SCF signaling mutants are uncompromised in RPP4-triggered resistance to the obligate biotrophic oomycete, Hyaloperonospora parasitica. Thus, the predominant action of SGT1b in R gene-conditioned resistance to oomycetes appears to be at a site other than assisting SCF E3-ubiquitin ligases. However, genetic additivity of sgt1b axr1 double mutants in susceptibility to H. parasitica suggests that SCF-mediated ubiquitination contributes to limiting biotrophic pathogen colonization once plant¿pathogen compatibility is established.