Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from a colonial form of growth to a filamentous pseudohyphal form. This dimorphism requires a polar budding pattern and elements of the MAP kinase signal transduction pathway essential for mating pheromone response in haploids. We report here that haploid strains exhibit an invasive growth behavior with many similarities to pseudohyphal development, including filament formation and agar penetration. Haploid filament formation depends on a switch from an axial to a bipolar mode of bud site selection. Filament formation is distinct from agar penetration in both haploids and diploids. We find that the same components of the MAP kinase cascade necessary for diploid pseudohyphal development (STE20, STEI1, STE7, and STE12) are also required for both filament formation and agar penetration in haploids. Thus, haploid yeast cells can enter either of two developmental pathways: mating or invasive growth, both of which depend on elements of a single MAP kinase cascade. Our results provide a novel developmental model to study the dynamics of signal transduction, with implications for higher eukaryotes.

RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology.

14-3-3 proteins are highly conserved ubiquitous proteins whose explicit functions have remained elusive. Here, we show that the S. cerevisiae 14-3-3 homologs BMH1 and BMH2 are not essential for viability or mating MAPK cascade signaling, but they are essential for pseudohyphal-development MAPK cascade signaling and other processes. Activated alleles of RAS2 and CDC42 induce pseudohyphal development and FG(TyA)-lacZ signaling in Bmh+ strains but not in ste20 (p65PAK) or bmh1 bmh2 mutant strains. Moreover, Bmh1p and Bmh2p associate with Ste20p in vivo. Three alleles of BMH1 encode proteins defective for FG(TyA)-lacZ signaling and association with Ste20p, yet these alleles complement other 14-3-3 functions. Therefore, the 14-3-3 proteins are specifically required for RAS/MAPK cascade signaling during pseudohyphal development in S. cerevisiae.

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The bacterium Agrobacterium tumefaciens transforms eukaryotic hosts by transferring DNA to the recipient cell where it is integrated and expressed. Bacterial factors involved in this interkingdom gene transfer have been described, but less is known about host-cell factors. Using the yeast Saccharomyces cerevisiae as a model host, we devised a genetic screen to identify yeast mutants with altered transformation sensitivities. Twenty-four adenine auxotrophs were identified that exhibited supersensitivity to A. tumefaciens-mediated transformation when deprived of adenine. We extended these results to plants by showing that purine synthesis inhibitors cause supersensitivity to A. tumefaciens transformation in three plant species. The magnitude of this effect is large and does not depend on prior genetic manipulations of host cells. These data indicate the utility of yeast as a model for the transformation process and identify purine biosynthesis as a key determinant of transformation efficiency. These findings should increase the utility of A. tumefaciens in genetic engineering.A grobacterium tumefaciens, a Gram-negative soil bacterium, genetically transforms plants by transferring DNA to the host cell where it is integrated into the host chromosome and expressed. Exogenous DNA sequences introduced into transferred DNA (T-DNA) vectors can be delivered to plants, making A. tumefaciens a cornerstone of plant genetic engineering. Under controlled conditions, A. tumefaciens can also transform mammalian cells and a variety of fungi, including the yeast Saccharomyces cerevisiae (1-6).Understanding the cellular factors influencing transformation will provide broader insights into the mechanisms underlying interkingdom DNA transfer and should increase the utility of A. tumefaciens in genetic engineering. Bacterial factors that control virulence gene induction as well as processing and delivery of the T-DNA have been studied extensively (7,8). Recently, a few host-cell factors have been identified that participate in A. tumefaciens-mediated transformation. Studies in Arabidopsis thaliana have implicated histone H2A in chromosomal integration of the T-DNA (9). Studies in S. cerevisiae have implicated a nuclear pore protein in T-DNA nuclear import (10) and nonhomologous end-joining proteins in T-DNA chromosomal integration (11). To date, however, the facile yeast system has not been used to perform a large-scale screen to identify host factors that influence transformation sensitivity. Consequently, we devised a genetic screen to isolate yeast mutants with altered sensitivity to A. tumefaciens-mediated transformation. This approach revealed an unexpected link between transformation efficiency and de novo biosynthesis of adenine, an essential purine precursor of DNA, RNA, and ATP. Materials and MethodsStrains and Plasmids. The supervirulent A. tumefaciens strain EHA105 harboring pKP506 served as the bacterial donor strain in yeast-transformation experiments (1). The pKP506 plasmid contains the yeast TRP1 marker and the ARS1 rep...

Display technologies link proteins with the genes that encode them, providing a means of selecting proteins with desired properties through the process of directed evolution. Here, we describe DNA/protein attachment and recovery tools (DARTs), a novel polypeptide display technology that utilizes the Agrobacterium tumefaciens protein VirD2 to generate DNA-protein hybrid molecules. The resulting DNA-protein hybrids are small, robust, and are not expected to be subject to the synthesis and selection biases associated with viral- and cell-based display systems. We demonstrated that these DNA-protein hybrids could be used to display a variety of peptides that bind to appropriate antibodies for immunodetection and immunopurification. Further, the DNA components of the hybrid molecules can hybridize to complementary DNA molecules in solution or on a solid substrate. Because full-length VirD2 self-associated, we constructed a truncation that did not self-associate but still exhibited DNA linking activity and efficiently displayed peptides. Finally, we purified DNA-protein hybrids using their displayed peptide epitopes and amplified their DNA components by polymerase chain reaction. We suggest that the DART polypeptide display system will be valuable for performing directed evolution and generating protein arrays.