The FRP1 F-box gene has different functions in sexuality, pathogenicity and metabolism in three fungal pathogens

Abstract

Plant-pathogenic fungi employ a variety of infection strategies; as a result, fungi probably rely on different sets of proteins for successful infection. The F-box protein Frp1, only present in filamentous fungi belonging to the Sordariomycetes, Leotiomycetes and Dothideomycetes, is required for nonsugar carbon catabolism and pathogenicity in the root-infecting fungus Fusarium oxysporum. To assess the role of Frp1 in other plant-pathogenic fungi, FRP1 deletion mutants were generated in Fusarium graminearum and Botrytis cinerea, and their phenotypes were analysed. Deletion of FgFRP1 in F. graminearum led to impaired infection of barley roots, but not of aerial plant parts. Deletion of BcFRP1 in B. cinerea did not show any effect on pathogenicity. Sexual reproduction, however, was impaired in both F. graminearum and B. cinerea FRP1 deletion mutants. The mutants of all three fungi displayed different phenotypes when grown on an array of carbon sources. The F. oxysporum and B. cinerea deletion mutants showed opposite growth phenotypes on sugar and nonsugar carbon sources. Replacement of FoFRP1 in F. oxysporum with the B. cinerea BcFRP1 resulted in the restoration of pathogenicity, but also in a switch from impaired growth on nonsugar carbon sources to impaired growth on sugar carbon sources. This effect could be ascribed in part to the B. cinerea BcFRP1 promoter sequence. In conclusion, the function of the F-box protein Frp1, despite its high sequence conservation, is not conserved between different fungi, leading to differential requirements for pathogenicity and carbon source utilization.

Figure 1

Occurrence of Frp1 homologues in various fungi. (A) A phylogenetic tree was created with MegAlign software using amino acids 175–278 from the middle domain of the Frp1 homologues, which includes the F‐box domain. The fungal classes to which the respective fungi belong are given next to the tree. Frp1 homologues of the following organisms were used in this alignment: Ab, Alternaria brassicicola (AB01633.1); Bc, Botrytis cinerea; Cg, Cheatomium globosum (CHGG_00736.1); Fg, Fusarium graminearum (FGSG_01326.3); Fo, Fusarium oxysporum (FOXG_00058.2); Fs, Fusarium solani (Nectria haematococca) protein ID 103049; Fv, Fusarium verticillioides (FVEG_01458.3); Hj, Hypocrea jecorina (Trichoderma reeesei) protein ID 120583; Mg, Magnaporthe grisea (MGG_06351); Nc, Neurospora crassa (NCU09899); Pa, Podospora anserine (Pa_1‐12760); Ptr, Pyrenophora tritici‐repentis (PTRG_0644.1); Sn, Stagonospora nodorum (SNOG_14560.1); Ss, Sclerotinia sclerotiorum (SS1G_14401). For B. cinerea Frp1, an alternative gene model was used. (B) An alignment of the F. oxysporum, F. graminearum and B. cinerea Frp1 proteins was created using MacVector software. The F‐box domain is underlined.

Figure 2

The Δfrp1 mutant strains of Fusarium graminearum and Botrytis cinerea are not affected in the infection of commonly infected plant organs. (A) Fusarium graminearum wild‐type (WT), ectopic mutant and two ΔFgfrp1 mutant strains were assessed in a wheat infection assay. Bars indicate the total amount of infected spikes after 2 weeks. (B) Botrytis cinerea WT and two ΔBcfrp1 mutants were assessed on primary leaves of bean plants. Photographs were taken 72 h post‐inoculation (hpi). (C) Botrytis cinerea WT and mutant ΔBcfrp1#4 were assessed in leaf and fruit infection assays. Bars indicate the lesion diameter.

Figure 3

Fusarium graminearumΔFgfrp1 mutants show impaired infection of barley roots. Fusarium graminearum wild‐type (WT), an ectopic transformant and two ΔFgfrp1 strains were assessed in a barley root infection assay. Bars indicate the percentage of roots containing lesions. The two ΔFgfrp1 mutant strains show a significantly smaller number of diseased roots when compared with the wild‐type.

Figure 4

The Δfrp1 mutants from Fusarium graminearum and Botrytis cinerea are sterile. (A) The ΔFgfrp1 mutant from F. graminearum does not produce perithecia when placed on carrot agar, whereas the wild‐type (WT) strain does. (B) Botrytis cinerea WT and ΔBcfrp1 mutant strains are able to produce sclerotia that are morphologically slightly different (left panels). Apothecia are produced when wild‐type sclerotia are fertilized with microconidia of B. cinerea wild‐type or ΔBcfrp1 mutant strain (male, top right panel). Sclerotia of the wild‐type B. cinerea strain can produce apothecia after fertilization with microconidia from a wild‐type strain, whereas sclerotia of ΔBcfrp1 mutant strain are female sterile after fertilization with microconidia from a wild‐type strain (female, bottom right panel). Note the slimy appearance of the mutant sclerotia, which produce copious amounts of extracellular polysaccharide.

Figure 5

Growth of the Fusarium oxysporum, Fusarium graminearum and Botrytis cinereaΔfrp1 mutants on various carbon sources. Growth is indicated by the absorbance at 600 nm (OD₆₀₀) of: (A) F. oxysporum wild‐type (WT), ΔFofrp1 and ΔFofrp1 complemented with FRP1; (B) F. graminearum and two independent ΔFgfrp1 mutants; and (C) B. cinerea and two independent ΔBcfrp1 mutants.

Figure 6

Growth of the Fusarium oxysporumΔFofrp1 mutant is restored by transformation with FgFRP1, but not with BcFRP1, controlled by its native promoter. (A) Growth indicated by the absorbance at 600 nm (OD₆₀₀) of: F. oxysporum wild‐type (WT), ΔFofrp1 and transformants bearing the FgFRP1 gene, the BcFRP1 gene or BcFRP1 driven by the FoFRP1 promoter (pFoFRP1‐BcFRP1) on various carbon sources. (B) Relative transcript levels of BcFRP1 in transformants bearing the BcFRP1 gene or BcFRP1 driven by the FoFRP1 promoter (pFoFRP1‐BcFRP1). FEM1 was used as reference to calculate the relative expression levels using the ΔC _t method.

Figure 7

The FgFRP1 or BcFRP1 genes restore virulence of the Fusarium oxysporumΔFofrp1 mutant. Bars indicate the disease index scores of tomato plants infected by F. oxysporum wild‐type (WT), ΔFofrp1, four transformants bearing the FgFRP1 gene and two transformants bearing the BcFRP1 gene.