The pep4 gene encoding proteinase A is involved in dimorphism and pathogenesis of Ustilago maydis

Abstract

Vacuole proteases have important functions in different physiological processes in fungi. Taking this aspect into consideration, and as a continuation of our studies on the analysis of the proteolytic system of Ustilago maydis, a phytopathogenic member of the Basidiomycota, we have analysed the role of the pep4 gene encoding the vacuolar acid proteinase PrA in the pathogenesis and morphogenesis of the fungus. After confirmation of the location of the protease in the vacuole using fluorescent probes, we obtained deletion mutants of the gene in sexually compatible strains of U. maydis (FB1 and FB2), and analysed their phenotypes. It was observed that the yeast to mycelium dimorphic transition induced by a pH change in the medium, or the use of a fatty acid as sole carbon source, was severely reduced in Δpep4 mutants. In addition, the virulence of the mutants in maize seedlings was reduced, as revealed by the lower proportion of plants infected and the reduction in size of the tumours induced by the pathogen, when compared with wild-type strains. All of these phenotypic alterations were reversed by complementation of the mutant strains with the wild-type gene. These results provide evidence of the importance of the pep4 gene for the morphogenesis and virulence of U. maydis.

Keywords: Ustilago maydis; fungal dimorphism; plant pathogenesis; proteinase A; vacuole proteinases.

Figure 1

Ustilago maydis proteinase A (PrA) is targeted to the vacuole. The C‐terminal PrA of U. maydis was fused to enhanced green fluorescent protein (eGFP) or the monomeric form of red fluorescent protein (mRFP) and expressed in haploid U. maydis cells. (A, B) One cell of the PrA‐mRFP‐expressing U. maydis  CS8 transformant grown on complete medium (MC), incubated at 28 °C for 24 h and observed by bright field or fluorescence microscopy, respectively. (C–E) One cell of the PrA‐eGFP‐expressing U. maydis  CS7 transformant grown on MC, incubated at 28 °C for 24 h and observed by bright field or fluorescence microscopy, or stained with the fluorescent dye 7‐amino‐4‐choromethylcoumarin (CMAC), respectively. (F, G) Large vacuoles observed in CS8 PrA‐RFP cells grown in minimal medium (MM) without nitrogen source for 72 h and observed by bright field or fluorescence microscopy, respectively. Stained vacuoles are observed only in cells at the correct focal plane. Scale bars: 10 μm.

Figure 2

Mutation of pep4 gene. (A) Strategy for replacement of the pep4 open reading frame (ORF) with the hygromycin resistance cassette. (B) Southern blot hybridization of wild‐type and pep4 mutant strains of Ustilago maydis whose genomic DNA was digested with SacI‐EcoRV and probed with an ORF  pep4 fragment. The absence of hybridizing fragments in the OR1 and OR2 mutant strains indicated the deletion of the gene (lanes 2 and 4), whereas the fragment was present in the FB1 and FB2 wild‐type strains (lanes 1 and 3). (C) Southern blot hybridization of DNA digested with PvuII and probed with a 3′ untranslated region (UTR) 848‐bp fragment of the region corresponding to the terminator of the pep4 gene. A 3.6‐kb fragment was observed in the wild‐type strain (lanes 1 and 3), whereas a 5.1‐kb fragment carrying the pep4 mutant copy was identified in the mutant strain (lanes 2 and 4).

Figure 3

Proteolytic activity of wild‐type (WT) and mutant strains (Δpep4::Hyg^R). Specific proteinase A activity in wild‐type and mutant strains of cells grown in complete medium (MC) for 24 h. Error bars indicate standard deviation (n = 9).

Figure 4

Induction of yeast to mycelium dimorphism by acid pH. Strains were grown in minimal medium (MM) at pH 3 for 16 h; the cells were stained with lactophenol blue and observed by microscopy. (A, B) Wild‐type strains FB1 and FB2, respectively. (C, D) OR1 (a1b1 Δpep4::Hyg^R) and OR2 (a2b2 Δpep4::Hyg^R) mutant strains, respectively. (E, F) Complemented strains CS49 and CS1 respectively. Scale bars, 10 μm.

Figure 5

Induction of yeast to mycelium dimorphism by palmitic acid. Strains were grown in minimal medium (MM) at pH 7 with 1% palmitic acid as carbon source for 5 days; the cells were stained with lactophenol blue and observed by microscopy. (A, B) Ustilago maydis wild‐type strains FB1 and FB2, respectively. (C, D) OR1 and OR2 mutant strains, respectively. (E, F) Complemented strains CS49 and CS1, respectively. Scale bars, 10 μm.

Figure 6

Development of Ustilago maydis in inoculated maize seedlings. Sexually compatible mixtures of the different strains were inoculated in maize seedlings; sections were obtained at different days post‐inoculation (dpi), stained with lactophenol–cotton blue and observed by microscopy. (A–C) Sections from the inoculation zone of maize seedlings infected with mixtures of wild‐type strains FB1 and FB2 obtained after 3, 6 and 8 dpi, respectively. (D–F) Sections from the inoculation zone of maize seedlings infected with mixtures of OR1 and OR2 mutant strains obtained after 3, 6 and 8 dpi respectively. Some branches are marked by arrows. Scale bars, 10 μm.

Figure 7

Symptoms developed in maize seedlings inoculated with Ustilago maydis. Maize seedlings were inoculated as described in Fig. 6 and symptoms were observed at 14 days post‐inoculation (dpi). (A) Infection symptoms of maize seedlings: 1, plants injected with sterile water; 2, plants infected with wild‐type FB1 (a1b1) and FB2 (a2b2) strains; 3, plants infected with CS49 and CS1 complemented strains; 4, plants infected with OR1 and OR2 mutant strains. (B, C) Large tumour formation in seedlings inoculated with mixtures of FB1 and FB2 wild‐type or CS49 and CS1 complemented strains, respectively. (D) Small tumour formation in seedlings inoculated with OR1 and OR2 mutant strains.

Figure 8

Virulence assay in fully grown maize plants. Maturing maize ears were inoculated with a suspension of mixed sporidia from sexually compatible Ustilago maydis strains. The symptoms were evaluated after 2 weeks. (A) Tumour development in maize ears inoculated with a mixture of FB1 and FB2 wild‐type strains. (B) Tumour development in maize ears inoculated with a mixture of CS49 and CS1 complemented strains. (C) Reduced numbers of tumours in ears co‐inoculated with a mixture of sexually compatible Δpep4 mutants (OR1 and OR2).