Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development

Abstract

The Candida albicans genes, CST20 and HST7, were cloned by their ability to suppress the mating defects of Saccharomyces cerevisiae mutants in the ste20 and ste7 genes, which code for elements of the mating mitogenactivated protein (MAP) kinase pathway. These Candida genes are both structural and functional homologs of the cognate Saccharomyces genes. The pattern of suppression in Saccharomyces is related to their presumptive position in the MAP kinase cascade. Null alleles of these genes were constructed in Candida. The Candida homozygous null mutants are defective in hyphal formation on some media, but are still induced to form hyphae by serum, showing that serum induction of hyphae is independent of the MAP kinase cascade. The Candida heterozygotes CST20/cst20 and HST7/hst7 are also defective in hyphal formation. This lack of dominance of the wild-type allele suggests that gene dosage is important in Candida.

Figure 1

Cst20 and Ste20 share homologous Cdc42-binding domains and catalytic domains. Translation such that CTG codes for serine (19). (A) Cdc42 binding domains. (B) Kinase domains.

Figure 2

(A) Candida genes suppress the mating defects in haploid S. cerevisiae ste mutant strains. Saccharomyces strains were transformed with the 2-micron vector B2205 (column 1), and with plasmids based on this vector: pJK25 carrying the CST20 gene (column 2), pJK7 carrying HST7 (column 3), and pHL14 carrying CPH1 (column 4). Haploid Saccharomyces strains are JKY40 (ste20), JKY1 (ste7), and JKY 87 (ste12). Patches of transformants were grown on selective medium to maintain the plasmid, replica plated to YPD plates covered with lawns of the mating tester strain JBY311, incubated for 4 h, replica plated to minimal medium, and incubated for 2 days. (B) Candida genes complement pseudohyphal growth defects in diploid ste/ste S. cerevisiae strains containing mutations in genes encoding components of the mating MAPK cascade. Ste⁺ (L5366), ste20/ste20 (L5624), ste11/ste11 (L5625), ste7/ste7 (L5626), and ste12/ste12 (L5627) were transformed with Candida genes on 2-micron plasmids, as indicated above the columns. The same plasmids were used as in the mating patch assays. Transformants were streaked on SLAD medium and incubated for 5 days. (Bar = 1 mm.)

Figure 3

(A) HST7 deletion construct: map of pJK41. The BglII site, with which the hisG-URA3-hisG cassette was inserted, and the SphI site were created by site-directed mutagenesis and therefore not present in the wild-type gene. (B) Southern blot of HST7 deletion mutants: Candida genomic DNA digested with BglII. Lane 1, wild type. The 6.8-kb band results from digestion at the BglII site in the HST7 open reading frame and a site on the chromosome upstream of the gene, the 2.9-kb band results from digestion the BglII site in the open reading frame and a site on the chromosome downstream of it. Lane 2, HST7/hst7::hisG-URA3-hisG. Integration of the disruption construct at one of the two copies of HST7 results in loss of the BglII site in the open reading frame and gain of a new site in the construct, creating a 6.2-kb band between the new BglII site and the 3′ chromosomal BglII site, and a 5.7-kb band between the 5′ chromosomal site and the new BglII site. Lane 3, HST7/hst7::hisG. Eviction of URA3 and one hisG repeat results in a decrease in size of the fragment between the new BglII site and the 3′ site from 6.2 to 4 kb. Lane 4, hst7::hisG-URA3-hisG/hst7::hisG. Integration of the disruption construct into the remaining HST7 allele results in loss of the wild-type bands. Lane 5, hst7::hisG/hst7::hisG. Arrows indicate kilobases. Fragment sizes are approximate. (C) CST20 deletion construct: map of pJK51. The construct lacks the first 0.3 kb of the open reading frame. (D) Southern blot of CST20 mutants: Candida genomic DNA digested with NsiI. Lane 1, wild type. The band results from digestion at the NsiI site in the open reading frame and a chromosomal NsiI site 4.5 kb downstream of it. Lane 2. CST20/cst20::hisG-URA3-hisG. Lane 3, CST20/cst20::hisG. Lane 4, cst20::hisG-URA3-hisG/cst20::hisG. Lane 5, cst20::hisG/cst20::hisG. Arrows indicate kilobases. Fragment sizes are approximate.

Figure 4

Filamentous growth defects of HST7/hst7 heterozygotes. The extent of filamentation varies among strains and from colony to colony. Incubation for 5 days on Spider medium. (A) Wild type SC5314. (B) JKC48. (C) JKC63. (D) JKC52. (Bar = 5 mm.)

Figure 5

HST7 and CST20 are required for hyphal growth on Spider medium. (A) Wild-type strain SC5314 (CST20/CST20 HST7/HST7 URA3/URA3). (B–D) CST20 mutants. (E–G) HST7 mutants. (B) CST20/cst20::hisG-URA3-hisG (JKC91). (C) cst20::hisG/cst20::hisG-URA3-hisG (JKC97). (D) cst20::hisG/cst20::hisG/cst20-ura3 (JKC 178). (E) HST7/hst7::hisG-URA3-hisG (JKC63). (F) hst7::hisG/hst7::hisG-URA3-hisG (JKC131). (G) hst7::hisG/hst7::hisG/HST7-URA3 (JKC173). Candida strains were incubated for 5 days on Spider medium. (Bar = 2 mm.)