Studies of regulation of pseudohyphae development and invasiveness in yeast have resulted in the identification of genes encoding kinases, phosphatases, and transcription factors (Blacketer et al., 1993
; Liu et al., 1993
; Gimeno and Fink, 1994
; Ward et al., 1995
; Gavrias et al., 1996
; Roberts et al., 1997
). Ultimately, changes must occur at the cell surface to enable cells to invade substrate or to adhere to form a filamentous chain of cells. Flo11p has been previously identified as a flocculin, a cell surface molecule responsible for the calcium-dependent nonsexual aggregation known as flocculation (Lo and Dranginis, 1996
). The work described here demonstrates that this flocculin is required for invasion and pseudohyphae development. Flo11p is the first cell wall protein shown to be involved in these processes. Recently, a screen for mutants defective in filamentous growth led to the identification of genes encoding structural proteins that interact with the cytoskeleton and genes responsible for bud site selection as well as previously identified components of the signal transduction pathway, but did not recover FLO11
(Mosch and Fink, 1997
). However, this screen also did not lead to the identification of several other genes known to be required for filamentous growth, suggesting that the screen was not saturated.
The transcriptional activator Ste12p is a downstream target of the pheromone-responsive MAPK cascade, elements of which are also required for the filamentous response (Liu et al., 1993
). Ste12p activates two different sets of genes, the mating genes and the filamentation genes, in response to different signals. A transcriptional reporter containing promoter sequences from the Ty1 element, FG(TyA)::lacZ, has been shown to be activated by Ste12p during filamentous growth, but not during the mating response (Mosch et al., 1996
). This filamentation-specific transcriptional induction was recently shown to be a consequence of the cooperative binding of Ste12p and Tec1p to a filamentation and invasion response element (FRE), which is comprised of a binding site for Ste12p adjacent to a binding site for Tec1p (Madhani and Fink, 1997
). Two genes have thus far been described which have FRE elements: Ty1
and the transcriptional factor TEC1
itself, which seems to participate in a positive feedback loop (Madhani and Fink, 1997
, however, represents the first reported functionally important downstream gene in the filamentous response pathway which contains a FRE in its promoter.
We present evidence that FLO11 is regulated by the transcriptional activator Ste12p. First, strains with a deletion of STE12 do not express FLO11 RNA. Second, the upstream region of FLO11 contains a consensus FRE sequence. Finally, expression of FLO11 on a high-copy plasmid overcomes the inability of the ste12-deletion strain to invade agar, indicating that activation of FLO11 is the primary mechanism by which the transcriptional activator Ste12p promotes invasion.
It is possible that the adhesion, invasion, and filamentation functions of FLO11
are related. One hypothesis is that FLO11
is responsible for the adhesion of mother and daughter cells in the filamentous chain of cells of pseudohyphae. The adhesion properties of Flo11p might also account for its critical role in invasiveness. Adhesion to substrate may play a role in the mechanism of invasion or cell–cell adhesion may provide a stable filament of cells, allowing the force of cell division to drive invasion. A relationship between flocculation and pseudohyphae formation was recently suggested by the discovery that PHD10
, an activator of pseudohyphae development, is the same gene as FLO8
(Liu et al., 1996
), a putative transcriptional activator of the flocculin FLO1
(Kobayashi et al., 1996
). A nonsense mutation in FLO8
accounts for the inability of the common laboratory yeast strain S288C to form pseudohyphae (Liu et al., 1996
). It is possible that Flo8p also activates transcription of FLO11
, and that this could account for the role of FLO8
in pseudohyphae formation. Other flocculins such as Flo1p must not play the same role as Flo11p in Σ1278b strains of yeast, since deletion of FLO11
abolishes the ability to invade or to form pseudohyphae. Flocculation per se does not cause invasiveness or filamentation, since we and others (Gimeno et al., 1992
) have observed flocculent strains that do not invade or form filaments.
We show that FLO11
transcripts are induced in diploid cells upon starvation for nitrogen, but not in haploids. Conversely, in rich media FLO11
transcripts persist in haploids but are not expressed in diploids. It may be that the diploid-specific repression of FLO11
in rich media is accomplished through the activity of the FRE, since a lacZ
reporter construct regulated by a FRE was shown to be expressed in diploids grown on rich media at less than one-tenth the level in haploids grown in rich media (Madhani and Fink, 1997
). Interestingly, overexpression of FLO11
in diploids on rich media enables them to invade, suggesting that the differential expression of FLO11
accounts for the differences in the abilities of haploid and diploid cells to invade. The expression of the FLO11
flocculin on the cell surface may represent a final common pathway of the transcription activators and other regulators of invasiveness and pseudohyphae development.