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Comparative Study
, 4 (6), 999-1008

The RIM101/pacC Homologue From the Basidiomycete Ustilago Maydis Is Functional in Multiple pH-sensitive Phenomena

Affiliations
Comparative Study

The RIM101/pacC Homologue From the Basidiomycete Ustilago Maydis Is Functional in Multiple pH-sensitive Phenomena

Elva T Aréchiga-Carvajal et al. Eukaryot Cell.

Abstract

A homologue of the gene encoding the transcription factor Rim101 (PacC), involved in pH signal transduction in fungi, was identified in the pathogenic basidiomycete Ustilago maydis. The gene (RIM101) encodes a protein of 827 amino acid residues, which shows highest similarity to PacC proteins from Fusarium oxysporum and Aspergillus niger. The gene had the capacity to restore protease activity to rim101 mutants from Yarrowia lipolytica, confirming its homologous function, and was expressed at both acid and neutral pH. Null Deltarim101 mutants were not affected in the in vitro pH-induced dimorphic transition, their growth rate, resistance to hypertonic sorbitol or KCl stress, and pathogenicity. However, similar to pacC (rim101) mutants in other fungi, they displayed a pleiotropic phenotype with alterations in morphogenesis, impairment in protease secretion, and increased sensitivity to Na+ and Li+ ions. Other phenotypic characteristics not previously reported in fungal pacC (rim101) mutants (morphological changes, increased sensitivity to lytic enzymes, and augmented polysaccharide secretion) were also observed in U. maydis mutants. All these modifications were alleviated by transformation with the wild-type gene, confirming that all were the result of mutation in RIM101. These data indicate that the Pal/Rim pathway is functional in U. maydis (and probably in other basidiomycetes) and plays complex roles in pH-sensing phenomena, as occurs in ascomycetes and deuteromycetes.

Figures

FIG. 1.
FIG. 1.
Confirmation analyses of RIM101 mutation. A. DNA was subjected to PCR with primers 2075 and 2078. Lanes 1 to 3, Δrim101 mutants (BMA1, BMA2, and BMA4, respectively); lane 4, FB2 wild-type strain; lane 5, plasmid pUpacH; lane 6, plasmid pUpacC4. B. EcoRI digestion of PCR products. Lane 2, BMA1; lane 4, BMA2; lane 6, FB2; lane 8, pUpacH; lane 10, pUpacC4. Lanes 1, 3, 5, 7, and 9, controls with no restriction enzyme added to BMA1, BMA2, FB2, pUpacH, and pUpacC4, respectively. C. Southern blot hybridization of HincII-digested DNA with the 1.2-kb StuI-BamHI RIM101-specific probe. Lane 1, FB1; lane 2, BMA1; lane 3, BMA7; lane 4, FB2; lane 5, BMA2; lane 6, BMA4. Note absence of hybridization in mutants compared to wild-type strains. Arrows show positions of the molecular size standards. D. Restriction map of RIM101 locus of the wild-type strain showing the primer (solid arrow) and probe (solid line) positions. E. Restriction map of RIM101 locus of the mutant allele used in the experiment showing the position of the primers (solid arrows) and the Hph gene (large open arrow).
FIG. 2.
FIG. 2.
Determination of RIM101 expression. Shown are results of Northern analysis of RIM101 gene expression in strain FB2 grown for 24 h in MM, pH 7 (lane I) or pH 3 (lane II), using the 1.2-kb StuI-BamHI RIM101-specific probe. rRNAs stained with ethidium bromide are shown as a loading control.
FIG. 3.
FIG. 3.
Effect of salt stress on U. maydis growth. Different U. maydis cell numbers (107, 106, 105, or 104 cells/aliquot) were inoculated on plates of solid MM and incubated for 96 h. Panels A and C, MM, pH 7; panels B and D, MM, pH 3. Panels A and B, 0.05 M LiCl added to the medium; panels C and D, 1 M NaCl added to the medium. Rows 1, FB2; rows 2, BMA1 (Δrim101); rows 3, BMA2 (Δrim101); rows 4, PGM1 (Δrim101::hyg/pUpacC23).
FIG. 4.
FIG. 4.
Cell morphology of U. maydis wild-type and mutant strains. Strains were grown in MM at pH 3 (A, B, and C) or pH 7 (D, E, and F) and stained with Calcofluor white. A and D, FB2 (wild type); B and E, BMA2 (Δrim101); C and F, PGM1 (Δrim101::hyg/pUpacC23). Bar, 10 μm.
FIG. 5.
FIG. 5.
Polysaccharide secretion by wild-type and mutant strains of U. maydis. A. Strains FB2 and BMA2 were grown in pH 7 CM. At different times, the polysaccharide was precipitated with ethanol, and the content of neutral sugars was measured. B. Cells were grown for 48 h in MM of different pH values. Triangles and solid lines, strain FB2; circles and broken lines, strain BMA2. Polysaccharide data are expressed in mg/ml.
FIG. 6.
FIG. 6.
Proteolytic activities of different U. maydis and Y. lipolytica strains. Strains were inoculated over skim milk plates and incubated for 48 h. A. U. maydis. I/1, FB2 wild-type strain; II/1, FB1 wild-type strain; I/2, BMA2 Δrim101 mutant; I/3, BMA4 Δrim101 mutant; II/2, PGM1 complemented strain; II/3, AAP1 complemented strain. B. Y. lipolytica. I/1, ΔFL3 mutant; I/2, ETA1 complemented strain; I/3, E121 wild-type strain.

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