HOY1, a Homeo Gene Required for Hyphal Formation inYarrowia lipolytica

Abstract

The dimorphic fungus Yarrowia lipolytica grows to form hyphae either in rich media or in media with GlcNAc as a carbon source. A visual screening, called FIL (filamentation minus), for Y. lipolytica yeast growth mutants has been developed. The FIL screen was used to identify three Y. lipolytica genes that abolish hypha formation in all media assayed. Y. lipolytica HOY1, a gene whose deletion prevents the yeast-hypha transition both in liquid and solid media, was characterized. HOY1 is predicted to encode a 509-amino-acid protein with a homeodomain homologous to that found in the chicken Hox4.8 gene. Analysis of the protein predicts a nuclear location. These observations suggest that Hoy1p may function as a transcriptional regulatory protein. In disrupted strains, reintroduction of HOY1 restored the capacity for hypha formation. Northern blot hybridization revealed the HOY1 transcript to be approximately 1.6 kb. Expression of this gene was detected when Y. lipolytica grew as a budding yeast, but an increase in its expression was observed by 1 h after cells had been induced to form hyphae. The possible functions of HOY1 in hyphal growth and the uses of the FIL screen to identify morphogenetic regulatory genes from heterologous organisms are discussed.The yeast-to-hypha morphological transition (dimorphism) is typical of many pathogenic fungi (62). The dimorphic transition is a freely reversible process that can be induced by changes in many parameters (56). Much attention has been focused on Candida albicans as a model for analyzing dimorphism because it is the most frequently isolated fungal pathogen in humans. However, although several groups have reported the isolation of mutants which persist in either the yeast (10) or hyphal (21, 30) form, analysis of these mutants is hampered by the difficulties inherent to genetic manipulations of this asexual diploid organism.Two approaches have been successfully used to carry out the analysis of dimorphism. With one of them, differential hybridization screening (7), several genes that are differentially expressed during morphogenesis have been isolated in C. albicans (i.e., ECE1, expressed in association with cell elongation, and PHR1, which is differentially regulated in response to the pH of the growth medium). Deletion of PHR1 results in a pH-conditional defect in morphogenesis (59).The second approach consists of cloning Candida homologs of Saccharomyces cerevisiae genes known to regulate pseudofilamentous growth. Under conditions of nutrient limitation (i.e., nitrogen starvation), S. cerevisiae undergoes a dimorphic transition to growth of pseudohyphae, linear chains of elongated cells in which the daughters remain attached to the mothers (23, 24). In this context, several C. albicans genes that are members of a Candida map kinase (MAPK) cascade, i.e., CPH1 (40) or ACPR (43), HST7 (11), and CST20 (36, 39), have been isolated by complementation of the corresponding Saccharomyces mutants, i.e., STE12, STE7, and STE20, respectively (11,36,39,40,43). When C. albicans st...

A Copper-responsive Transcription Factor, CRF1, Mediates Copper and Cadmium Resistance in Yarrowia lipolytica

“…The flanking sequences of the proposed initiation and termination codons are consistent with the Y. lipolytica consensus for translation initiation and polyadenylation signals, respectively (24,31). A putative TATA box was located at position Ϫ412 to Ϫ419, and several tandem repeats of the CAAT element in the context of a 5Ј-CAAT(A)5GGAG(A)5-6GA-3Ј sequence were found between bases Ϫ881 and Ϫ941.…”

“…Total RNAs were prepared from each sample as described previously (31). RNA samples (5 g) were denatured and separated on 1.2% agarose gels containing 2.2 M formaldehyde (32).…”

“…Complementation Analysis-Strain MP73 was transformed with a Y. lipolytica gene bank constructed in plasmid pINA62 (26,31). The library was linearized with NotI for integration, and leucine prototrophs were selected on SC agar lacking uracil.…”

Tagging Morphogenetic Genes by Insertional Mutagenesis in the Yeast Yarrowia lipolytica

“…Third, the YlTup1p transcriptional factor appears to act as a repressor of the yeast form, like in C. albicans, but unlike in S. cerevisiae, in which Tup1p is required for pseudomyceliation (10,17). Because two transcriptional factors (Hoy1p and Mhy1p) that do not have any clear homologs in C. albicans or in S. cerevisiae have been found to be essential for the dimorphic transition in Y. lipolytica (32,66), it was unknown whether a conserved pathway controlling morphogenesis was conserved in the three yeasts.…”

YlBMH1 encodes a 14-3-3 protein that promotes filamentous growth in the dimorphic yeast Yarrowia lipolytica a aThe GenBank accession numbers for the YlBMH1 and YlBMH2 sequences reported in this study are AY090661 and AY090662, respectively.

“…Accordingly, additional studies are required in other organisms to acquire a more complete understanding of the overall phenomenon of fungal dimorphism (Odds, 1988 ;Shepherd, 1988 ;Maresca & Kobayashi, 1989 ;Gow, 1994 ;Banuett, 1995 ;Lo et al, 1997 ;Madhani & Fink, 1998 ;Bolker, 2001). Due to its ability to alternate between a unicellular yeast form and different filamentous forms (hyphae and pseudohyphae), its amenability to genetic and molecular biological analyses and its ability to reproduce sexually, the yeast Yarrowia lipolytica has emerged as an excellent alternative model for the investigation of the molecular aspects of fungal morphogenesis (Enderlin & Ogrydziak, 1994 ;Barth & Gaillardin, 1996 ;Torres-Guzman & Dominguez, 1997 ;Hurtado & Rachubinski, 1999 ;Hurtado et al, 2000 ;Richard et al, 2001 ;Szabo, 2001).…”