Romina Giacometti

Catalytic isoforms Tpk1 and Tpk2 of Candida albicans PKA have non-redundant roles in stress response and glycogen storage.

Candida albicans cAMP‐dependent protein kinase (PKA) is coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1). In this organism the cAMP/PKA signalling pathway mediates basic cellular processes, such as the yeast‐to‐hyphae transition and cell cycle regulation. In the present study, we investigated the role of C. albicans PKA in response to saline, heat and oxidative stresses as well as in glycogen storage. To fine‐tune the analysis, we performed the studies on several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in a different BCY1 genetic background. We observed that tpk1Δ/tpk1Δ strains developed a lower tolerance to saline exposure, heat shock and oxidative stress, while wild‐type and tpk2Δ/tpk2Δ mutants were resistant to these stresses, indicating that both isoforms play different roles in the stress response pathway. We also found that regardless of the TPK background, heterozygous and homozygous BCY1 mutants were highly sensitive to heat treatment. Surprisingly, we observed that those strains devoid of one or both TPK1 alleles were defective in glycogen storage, while strains lacking Tpk2 accumulated higher levels of the polysaccharide, indicating that Tpk1 and Tpk2 have opposite roles in carbohydrate metabolism. Copyright

Candida albicans Tpk1p and Tpk2p isoforms differentially regulate pseudohyphal development, biofilm structure, cell aggregation and adhesins expression

Candida albicans undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments. In this organism, cAMP‐dependent protein kinase (PKA), coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1), mediates basic cellular processes, such as the yeast‐to‐hypha transition and cell cycle regulation. It is known that both Tpk isoforms play positive roles in vegetative growth and filamentation, although distinct roles have been found in virulence, stress response and glycogen storage. However, little is known regarding the participation of Tpk1p and/or Tpk2p in pseudohyphal development. This point was addressed using several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in different BCY1 genetic backgrounds. We observed that under hypha‐only inducing conditions, all BCY1 heterozygous strains shifted growth toward pseudohyphal morphology; however, the pseudohypha:hypha ratio was higher in strains devoid of TPK2. Under pseudohypha‐only inducing conditions, strains lacking TPK2 were prone to develop short and branched pseudohyphae. In tpk2Δ/tpk2Δ strains, biofilm architecture was markedly less dense, composed of short pseudohyphae and blastospores with reduced adhesion ability to abiotic material, suggesting a significant defect in cell adherence. Immunolabelling assays showed a decreased expression of adhesins Als1p and Als3p only in the tpk2Δ/tpk2Δ strain. Complementation of this mutant with a wild‐type copy of TPK2 restored all the altered functions: pseudohyphae elongation, biofilm composition, cell aggregation and adhesins expression. Our study suggests that the Tpk2p isoform may be part of a mechanism underlying not only polarized pseudohyphal morphogenesis but also cell adherence. Copyright

Arbuscular mycorrhiza detoxifying response against arsenic and pathogenic fungus in soybean.

UNLABELLED Uptake of Arsenic (As) in plant tissues can affect metabolism, causing physiological disorders, even death. As toxicity, but also pathogen infections trigger a generalised stress response called oxidative stress; however knowledge on the response of soybean (Glycine max L.) under multiple stressors is limited so far. Arbuscular mycorrhizal fungi (AMF) enhance the tolerance of host plants to abiotic and biotic stress. Thus, we investigated the effects of the AMF Rhizophagus intraradices on soybean grown in As-contaminated soils as well as in the presence of the pathogen Macrophomina phaseolina (charcoal rot of the stem). Plant parameters and degree of mycorrhizal colonization under the different assessed treatments were analyzed. Content of As in roots and leaves was quantified. Increasing As level in the soil stopped plant growth, but promoted plant As uptake. Inoculation of soybean plants with M. phaseolina accentuated As effect at all physiological levels. In the presence of mycorrhizal symbiosis biomass dramatically increased, and significantly reduced the As concentration in plant tissues. Mycorrhization decreased oxidative damage in the presence of both As and the pathogen. Furthermore, transcription analysis revealed that the high-affinity phosphate transporter from R. intraradices RiPT and the gene encoding a putative arsenic efflux pump RiArsA were up-regulated under higher As doses. These results suggest that R. intraradices is most likely to get involved in the defense response against M. phaseolina, but also in the reduction of arsenate to arsenite as a possible detoxification mechanism in AMF associations in soybean. CAPSULE ABSTRACT R. intraradices actively participates in the soybean antioxidant defense response against arsenic stress and M. phaseolina infection.

Early perception of stink bug damage in developing seeds of field-grown soybean induces chemical defences and reduces bug attack.

BACKGROUND Southern green stink bugs (Nezara viridula L.) invade field-grown soybean crops, where they feed on developing seeds and inject phytotoxic saliva, which causes yield reduction. Although leaf responses to herbivory are well studied, no information is available about the regulation of defences in seeds. RESULTS This study demonstrated that mitogen-activated protein kinases MPK3, MPK4 and MPK6 are expressed and activated in developing seeds of field-grown soybean and regulate a defensive response after stink bug damage. Although 10-20 min after stink bug feeding on seeds induced the expression of MPK3, MPK6 and MPK4, only MPK6 was phosphorylated after damage. Herbivory induced an early peak of jasmonic acid (JA) accumulation and ethylene (ET) emission after 3 h in developing seeds, whereas salicylic acid (SA) was also induced early, and at increasing levels up to 72 h after damage. Damaged seeds upregulated defensive genes typically modulated by JA/ET or SA, which in turn reduced the activity of digestive enzymes in the gut of stink bugs. Induced seeds were less preferred by stink bugs. CONCLUSION This study shows that stink bug damage induces seed defences, which is perceived early by MPKs that may activate defence metabolic pathways in developing seeds of field-grown soybean.

Expression of TPK1 and TPK2 genes encoding PKA catalytic subunits during growth and morphogenesis in Candida albicans

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 tpk1Δ and tpk2Δ 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 yeast‐to‐mycelial transition, a sharp increase in TPK1 mRNA levels and in PKA‐specific activity correlated with the onset of germ‐tube formation in strain tpk2Δ. We also showed that tpk1Δ strain exhibited a delayed morphogenetic shift in comparison with CAI4 and tpk2Δ strains in several liquid inducing media, reinforcing the idea that Tpk1p is important for faster germ‐tube appearance. Copyright

PIF-pocket as a target for C. albicans Pkh selective inhibitors.

The phosphoinositide-dependent protein kinase 1, PDK1, is a master kinase that phosphorylates the activation loop of up to 23 AGC kinases. S. cerevisiae has three PDK1 orthologues, Pkh1-3, which also phosphorylate AGC kinases (e.g., Ypk, Tpk, Pkc1, and Sch9). Pkh1 and 2 are redundant proteins involved in multiple essential cellular functions, including endocytosis and cell wall integrity. Based on similarities with the budding yeast, the Pkh of fungal infectious species was postulated as a novel target for antifungals. Here, we found that depletion of Pkh eventually induces oxidative stress and DNA double-strand breaks, leading to programmed cell death. This finding supports Pkh as an antifungal target since pharmacological inhibition of Pkh would lead to the death of yeast cells, the ultimate goal of antifungals. It was therefore of interest to further investigate the possibility to develop Pkh inhibitors with selectivity for Candida Pkh that would not inhibit the human ortholog. Here, we describe C. albicans Pkh2 biochemically, structurally and by using chemical probes in comparison to human PDK1. We found that a regulatory site on the C. albicans Pkh2 catalytic domain, the PIF-pocket, diverges from human PDK1. Indeed, we identified and characterized PS77, a new small allosteric inhibitor directed to the PIF-pocket, which has increased selectivity for C. albicans Pkh2. Together, our results describe novel features of the biology of Pkh and chemical biology approaches that support the validation of Pkh as a drug target for selective antifungals.

Cross regulation between Candida albicans catalytic and regulatory subunits of protein kinase A

In the pathogen Candida albicans protein kinase A (PKA) catalytic subunit is encoded by two genes TPK1 and TPK2 and the regulatory subunit by one gene, BCY1. PKA mediates several cellular processes such as cell cycle regulation and the yeast to hyphae transition, a key factor for C. albicans virulence. The catalytic isoforms Tpk1p and Tpk2p share redundant functions in vegetative growth and hyphal development, though they differentially regulate glycogen metabolism, the stress response pathway and pseudohyphal formation. In Saccharomyces cerevisiae it was earlier reported that BCY1 overexpression not only increased the amount of TPK3 mRNA but also its catalytic activity. In C. albicans a significant decrease in Bcy1p expression levels was already observed in tpk2Δ null strains. In this work we showed that the upregulation in Bcy1p expression was observed in a set of strains having a TPK1 or TPK2 allele reintegrated in its own locus, as well as in strains expressing the TPKs under the control of the constitutive ACT1 promoter. To confirm the cross regulation event between Bcy1p and Tpkp expression we generated a mutant strain with the lowest PKA activity carrying one TPK1 and a unique BCY1 allele with the aim to obtain two derived strains in which BCY1 or TPK1 were placed under their own promoters inserted in the RPS10 neutral locus. We found that placing one copy of BCY1 upregulated the levels of Tpk1p and its catalytic activity; while TPK1 insertion led to an increase in BCY1 mRNA, Bcy1p and in a high cAMP binding activity. Our results suggest that C. albicans cells were able to compensate for the increased levels of either Tpk1p or Tpk2p subunits with a corresponding elevation of Bcy1 protein levels and vice versa, implying a tightly regulated mechanism to balance holoenzyme formation.

Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein

cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.

Extracellular biosynthesis of bactericidal Ag/AgCl nanoparticles for crop protection using the fungus Macrophomina phaseolina

Synthesis of noble metal nanoparticles using natural products and living organisms has drawn a lot of interest owing to economic prospects and potential applicability in different fields. For this work we used the exudate of the soil fungus Macrophomina phaseolina for a low-cost method of green synthesis to obtain stable silver-silver chloride nanoparticles (Ag/AgCl-NPs). Reaction parameters including media and AgNO3 concentration were further optimized for NPs production. Spectral analysis revealed a peak at 420 nm that corresponds to the surface plasmon resonance of silver NPs. Scanning electron microscopy (SEM) analysis unveiled NPs spherical morphology with a size range of 5-30 nm. The crystalline nature of the synthesized NPs was examined by X-ray diffraction (XRD) analysis. The green synthesized NPs showed activity against gram-positive and gram-negative bacteria. No effect in fungi or yeast cells was detected, though a high inhibitory effect was observed on bacteria growth kinetics. Interaction of bacteria with Ag/AgCl-NPs led to cell membrane damage as observed by SEM, followed by an increase in oxidative stress, being this the possible mechanism behind the strong bactericidal activity depicted. In order to test its possible applicability as a seed protection agent the effect of Ag/AgCl-NPs dosage on soybean (Glycine max L.) seeds germination was also examined. Interestingly, not only the germination process was not affected by the NPs dosage or time of seeds incubation, but also no oxidative damage was detected in seeds after exposure to the biogenic nanoparticles.