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The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.Candida albicans is an opportunistic human fungal pathogen of great medical significance in immunocompromised patients (25). This fungus has the capability of switching its mode of growth between budding yeast and hypha or pseudohypha in response to environmental signals. Genetic evidence indicates that the morphogenetic switch to the hyphal mode of growth, though associated with pathogenicity and virulence (20), is necessary but not sufficient to trigger disease (5). The relationship between morphology and pathogenicity has been the focus of intensive research devoted to the study of the developmental programs involved in the dimorphic transition.The remarkable conservation of signal transduction pathways in fungi allowed the identification of components of these pathways in several fungal species based on the insight gained from studying pseudohyphal differentiation in Saccharomyces cerevisiae. In C. albicans, two major pathways implicated in dimorphism could be established: the mitogen-activated protein kinase and the cyclic AMP (cAMP)/protein kinase A transduction pathways (for a review, see reference 19).Initial biochemical studies indicate that high cAMP levels promote the yeast-to-hypha transition in C. albicans (23,31). In addition, we have shown that in vivo inhibition of protein kinase A blocks hyphal growth induced by N-acetylglucosamine (GlcNAc) (6). Recent genetic studies allowed the identification of the genes involved in the cAMP/protein kinase A pathway. A transduction cascade similar to that of S. cerevisiae, with regard to location and function of the homologous components, has been established. Thus, CaRa...

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Tomato seeds were inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense FT326, and changes in parameters associated with plant growth were evaluated 15 days after inoculation. Azospirilla were localized on roots and within xylematic tissue. An increase in shoot and root fresh weight, main root hair length, and root surface indicated that inoculation with A. brasilense FT 326 resulted in plant growth improvement. The levels of indole-3-acetic acid (IAA) and ethylene, two of the phytohormones related to plant growth, were higher in inoculated plants. Exogenously supplied ethylene mimicked the effect of inoculation, and the addition of an inhibitor of its synthesis or of its physiological activity completely blocked A. brasilense growth promotion. Based on our results, we propose that the process of growth promotion triggered by A. brasilense inoculation involves a signaling pathway that has ethylene as a central, positive regulator.

We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.

cAMP-dependent protein kinase (PKA) from Candida albicans yeast cells was isolated and characterised. Structural parameters of the holoenzyme and those of its subunits suggested that C. albicans PKA is a tetramer of 287 kDa composed of two regulatory (R) subunits of 64 kDa and two catalytic (C) subunits of unusually large molecular mass of 78 kDa. The apparent K m for ATP and Kemptide were 30 µM and 60 µM respectively. The [A] 0.5 for cAMP activation was 150 nM with a Hill coefficient of 1.6. The holoenzyme undergoes autophosphorylation on the R subunit, a characteristic of the type-II R subunits. Photoaffinity labeling with 8-azido-[ 32 P]cAMP of crude extracts from yeast and mycelial cells strongly suggests that only one type of R subunit is present in the fungus. The R subunit was purified to apparent homogeneity as a protein of 64 kDa. A highly specific polyclonal antiserum raised against the purified protein immunoprecipitated a 64-kDa protein from crude extracts, indicating that the purified R subunit very probably represents the native form of the protein. The 78-kDa form of the C subunit was detected in crude extracts and in Mono Q Sepharose column fractions with heterologous anti-C Ig. It could be isolated by cAMP treatment of the holoenzyme immunoprecipitated from crude extracts with anti-R serum, but this form could not be purified further. Instead, a 60-kDa protein with the main characteristics of C subunit was purified to near homogeneity from soluble extracts of yeast cells. Evidence is presented that this protein very probably derives from the 78-kDa form by proteolytic degradation.

The transcript levels of Candida albicans TPK1 and TPK2 genes, encoding PKA catalytic subunits, as well as phosphotransferase activity, were measured in the parental strain CAI4 and in homozygous tpk1 and tpk2 mutants during vegetative growth and during yeast-to-mycelial transition in N-acetylglucosamine liquid inducing medium at 37• C. We observed two TPK2 transcripts, a major one of 1.8 kb and a minor one of 1.4 kb, and established by 3 -RACE that they originate from the recognition of the three polyadenylation signals present in the 3 untranslated region of the gene. During vegetative growth of CAI4 strain, the expression profiles of TPK1 and TPK2 varied similarly, reaching maximal expression at the late logarithmic phase. TPK1 mRNA levels were lower than those of TPK2 at all stages measured. In the corresponding homozygous tpk mutants, mRNA levels and the expression patterns of TPK1 and TPK2 were similar to those of CAI4, suggesting that the loss of one catalytic isoform is not compensated by overexpression of the other. Changes in PKA specific activity roughly correlated with fluctuations of mRNA expression levels. During yeastto-mycelial transition, a sharp increase in TPK1 mRNA levels and in PKA-specific activity correlated with the onset of germ-tube formation in strain tpk2 . We also showed that tpk1 strain exhibited a delayed morphogenetic shift in comparison with CAI4 and tpk2 strains in several liquid inducing media, reinforcing the idea that Tpk1p is important for faster germ-tube appearance.

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