Our work focuses on how external signals alter gene expression in two organisms: baker’s yeast, Saccharomyces cerevisiae, and the plant, Arabidopsis thaliana. To unravel these signaling pathways we use mutants coupled with genome transcription arrays to identify the ensemble of genes responsible for adaptation to a new environment.

Fungal pathogenesis Baker’s yeast is a facile model for studying fungal disease. A switch from the yeast form to an invasive filamentous form contributes to the virulence of fungi. This yeast/filament switch is controlled by at least three signaling pathways: a MAP kinase pathway, a cAMP-regulated pathway, and a ploidy-dependent pathway. These signaling modules are also involved in the control of virulence in plant and human pathogens.

Genome-wide transcription analysis shows that the FLO11 gene is the target of the MAP kinase, the A kinase, and the ploidy pathways. FLO11 encodes a cell surface glycoprotein that is the downstream target of these pathways. Flo11p is one of a family of GPI-linked glycoproteins whose domain structure is similar to that of the adhesins of pathogenic fungi. The other members of the FLO gene family are silent: the adhesion function lacking in FLO11 mutant strains can be supplied by any one of these, but only if the FLO11 relative is turned on by an ectopic promoter. Moreover, the expression of these silent genes is unstable, resulting in frequent gene switches between on and off.

These adhesins are also required for the formation of fungal biofilms, a multicellular aggregate of microorganisms that can adhere to inert surfaces. We have found that baker’s yeast can initiate biofilm formation and form a novel structure termed a mat. These phenotypes are very similar to those described as sliding motility for Mycobacterium smegmatis. When grown in low glucose, the yeast cells adhere avidly to a number of plastic surfaces and initiate biofilm formation. On semi-solid medium they form mats, which are multicellular structures of considerable complexity comprised of yeast-form cells. The cell surface glycoprotein, Flo11p, is required for both adherence to plastic and for the formation of the mats. The ability to form mats is related to FLO11 expression levels in various mutants and wild-type strains of different ploidy. These findings provide important insights into fungal virulence as orthologs of Flo11p are important for adherence in pathogenic fungi.

Candida albicans, a diploid asexual yeast, is responsible for the majority of fungal infections in humans, which have a high mortality rate. We have developed an in vitro system in which fungal cells can be isolated from the phagolysosome of cultured mammalian macrophages. We have assayed global gene expression profiles in phagocytosed fungal cells. The predominant response is the induction of genes encoding enzymes of the glyoxylate cycle, a metabolic pathway enabling microorganisms to utilize two-carbon compounds as sources of carbon. In C. albicans, isocitrate lyase (ICL1) and malate synthase (MLS1), the principal enzymes of the glyoxylate cycle, are also upregulated upon phagocytosis. C. albicans strains lacking ICL1 are unable to utilize acetate or ethanol, but are no more sensitive to a variety of stresses than wild-type strains. In contrast, Δicl1/Δicl1 strains are markedly less virulent than the wild-type in a murine model of systemic candidiasis.

Morphogenesis of plants We have isolated mutations in the formation of roots to understand how the hormone indole acetic acid (IAA) affects cell division and gravitropism in the plant, Arabidopsis thaliana. Analysis of one lateral root defective mutant, alf5, suggests that its function is required for protection of the roots from inhibitory compounds. ALF5 has sequence homology to the multidrug and toxic compound extrusion (MATE) family, which is related to bacterial efflux transporters. Arabidopsis has at least 54 members of this family, which are often found in tandem repeats. Loss of ALF5 function results in the sensitivity of the root to a number of compounds including a contaminant of commercial agar. Moreover, expression of the Arabidopsis ALF5 cDNA in yeast confers resistance to tetramethylammonium. These phenotypes are consistent with a role for ALF5 as an efflux transporter. Both transcriptional and translational fusions of ALF5 to the GUS reporter gene show that ALF5 is strongly expressed in the root epidermis, a tissue in direct contact with the external environment. Another of these lateral root mutants, alf4, fails to make a normal primary root but not a lateral root. We are using Arabidopsis genomic arrays of alf4 and wild-type to determine what genes are required for lateral root formation.

We have isolated a miniature Arabidopsis mutant, bon1, with drastically reduced plant size due to reduction in both cell division and cell expansion. This mutant is cold sensitive: it exhibits the miniature mutant phenotype when grown at 22C, whereas growth at higher temperature (28C) results in a completely normal plant. The BON1 gene is highly homologous to the copine gene family previously found in mammals, worms, and paramecium. BON1 is required for normal cell expansion and division at low temperature and it is localized at the plasma membrane where it may be directly involved in membrane fusion.

Selected Publications

Galitski, T., Saldanha, A.J., Styles, C.A., Lander, E.S., and Fink, G.R. (1999). Ploidy regulation of gene expression. Science 285, 251-254.

Hinnen, A., Hicks, J.B., and Fink, G.R. (1978). Transformation of Yeast. Proc. Natl. Acad. Sci. USA 75, 1929-1933.

Roeder, G.S., Farabaugh, P.J., Chaleff, D.J., and Fink, G.R. (1980). The Origins of Gene Instability in Yeast. Science 209, 1375-1380.

Bender, J. and Fink, G.R. (1995). Epigenetic control of an endogenous gene family is revealed by a novel blue fluorescent mutant of Arabidopsis. Cell 83, 725-734.

Madhani, H.D., Galitski, T., Lander, E.S., and Fink, G.R. (1999). Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants. Proc. Natl Acad. Sci. 96, 12530-12535.

Updated February 20, 2003