Activation of a calcium-dependent protein kinase involved in the Azospirillum growth promotion in rice.

Rice seedlings (Oryza sativa) inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense FT326 showed an enhanced development of the root system 3 days after inoculation. Later on, a remarkable enlargement of shoots was also evident. An increase in the Ca2+-dependent histone kinase activity was also detected as a result of inoculation. The biochemical characterization and Western-blot analysis of the kinase strongly supports the hypothesis that it belongs to a member of the rice CDPK family. The fact that the amount of the protein did not change upon inoculation seems to indicate that a posttranslational activation is responsible for the change in the enzymatic activity. An in-gel kinase experiment identified a 46 kDa CDPK like protein kinase as a putative component of the signal transduction pathway triggered by Azospirillum inoculation. To our knowledge, this is the first report on the possible involvement of a Ca2+-dependent protein kinase in promotion of rice plants growth by A. brasilense.

Candida albicans lacking the gene encoding the regulatory subunit of protein kinase A displays a defect in hyphal formation and an altered localization of the catalytic subunit.

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.

The two isoforms of the cAMP-dependent protein kinase catalytic subunit are involved in the control of dimorphism in the human fungal pathogen Candida albicans.

We have cloned the Candida albicans TPK2 gene encoding a cAMP-dependent protein kinase (PKA) catalytic subunit and generated a tpk2 homozygous null mutant to assess its ability to germinate in liquid media. N-acetylglucosamine (GlcNAc)-induced germ-tube formation was attenuated in the tpk2 strain and enhanced by compounds that are known to increase the PKA activity in situ. Germination was completely blocked in the presence of the myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI). These results indicate that TPK1 acts positively in regulating the morphogenetic transition in C. albicans in the absence of the TPK2 gene. We were able to identify an mRNA from this second form of PKA in both wild-type and tpk2 null mutant cells. We found that PKA activity measured in the mutant lacking the TPK2 gene was about 10% of that displayed by the wild-type. The finding that the germinative response of tpk2 null mutant to serum was severely diminished at low serum concentrations indicates that the level of PKA is an important determinant of filamentous growth at low serum concentrations. The extent of germination attained at higher serum concentrations (5%) was similar in the wild-type and in the tpk2 null mutant strains suggesting that under these conditions germination was triggered through a PKA-independent pathway.