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MicroRNAs (miRNAs) are short, single stranded RNA molecules that regulate the stability and translation of messenger RNAs in diverse eukaryotic groups. Several miRNA genes are of ancient origin and have been maintained in the genomes of animal and plant taxa for hundreds of millions of years, playing key roles in development and physiology. In the last decade, genome and small RNA (sRNA) sequencing of several plant species have helped unveil the evolutionary history of land plants. Among these, the fern group (monilophytes) occupies a key phylogenetic position, as it represents the closest extant cousin taxon of seed plants, i.e. gymno-and angiosperms. However, in spite of their evolutionary, economic and ecological importance, no fern genome has been sequenced yet and few genomic resources are available for this group. Here, we sequenced the small RNA fraction of an epiphytic South American fern, Pleopeltis minima (Polypodiaceae), and compared it to plant miRNA databases, allowing for the identification of miRNA families that are shared by all land plants, shared by all vascular plants (tracheophytes) or shared by euphyllophytes (ferns and seed plants) only. Using the recently described transcriptome of another fern, Lygodium japonicum, we also estimated the degree of conservation of fern miRNA targets in relation to other plant groups. Our results pinpoint the origin of several miRNA families in the land plant evolutionary tree with more precision and are a resource for future genomic and functional studies of fern miRNAs. Fil: Berruezo, Florencia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Silva Junqueira de Souza, Flavio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Picca, Pablo Ignacio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Nemirovsky, Sergio Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Martínez Tosar, Leandro Julián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Rivero, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; Argentina Fil: Mentaberry, Alejandro Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Zelada, Alicia Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina

A His-tagged truncated version of Toxoplasma gondii dense granule 4 protein (Gra4163-345) was transiently expressed in tobacco leaves. Two genetic constructions were used to accomplish this goal. In one of them, based in a Potato virus X (PVX) amplicon, the sequence encoding His-Gra4163-345 was placed under control of an additional PVX coat protein subgenomic promoter. In the other, the same sequence was fused to an apoplastic transport signal and placed under the direction of the cauliflower mosaic virus 35S promoter. His-Gra4163-345 accumulation in agroinfiltrated tobacco leaves was estimated by Western blot analysis using mouse anti-Gra4 antibody and a seropositive human serum. Here, we demonstrated the feasibility of producing a Gra4 antigen using transient expression methods in plants. Fil: Ferraro, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Laguía Becher, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Ángel, Sergio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Zelada, Alicia Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Mentaberry, Alejandro Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Clemente, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

We adapted a previously described Agrobacterium-mediated transient expression system to test the expression level of three constructs carrying the surface antigen 1 (SAG1) of Toxoplasma gondii. Two constructs were based in a Potato virus X (PVX) amplicon. In one of them, the PVX movement protein genes were replaced by the sag1 gene. In the other, the sag1 gene was placed under the control of an additional coat protein subgenomic promoter. In the third construct, the sag1 gene was fused to an apoplastic peptide signal under the CaMV 35S promoter. Western blot analysis of leaf extracts infiltrated with each construct revealed a protein of 35 kDa. SAG1 accumulation in leaves ranged from 0.1 to 0.06% of total soluble protein (equivalent to 10 µg and 6 µg of SAG1 per gram of fresh leaf tissue, respectively). Three of five human seropositive samples reacted with tobacco-expressed SAG1 in Western blot analysis. The C3H mice were immunized with SAG-expressing leaf extracts and perorally challenged with a nonlethal dose of the T. gondii Me49 strain. Mice vaccinated with SAG1 showed significantly lower brain cyst burdens compared to those from the control group. Immunization with SAG1-expressing leaves elicited a specific humoral response with predominant participation of type IgG2a. In conclusion, a functional SAG1 version could be transiently expressed in tobacco leaves.

In this paper we demonstrate that the Candida albicans 20S proteasome is in vivo phosphorylated and is a good in vitro substrate (S(0.5) 14nM) of homologous protein kinase CK2 (CK2). We identify alpha6/C2, alpha3/C9, and alpha5/Pup2 proteasome subunits as the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome components. In vitro phosphorylation by homologous CK2 holoenzyme occurs only in the presence of polylysine, a characteristic that distinguishes the yeast proteasomes from mammalian proteasomes which are phosphorylated by CK2 in the absence of polycations. The major in vivo phosphate acceptor is the alpha3/C9 subunit, being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by endoproteinase Glu-C digestion from in vivo labeled alpha3/C9 subunit, from in vitro phosphorylation by homologous CK2 holoenzyme, and from the recombinant alpha3/C9 subunit phosphorylated by recombinant human CK2-alpha subunit are identical, suggesting that CK2 is likely responsible for in vivo phosphorylation of this subunit. Direct mutational analysis shows that serine 248 is the residue of the alpha3/C9 subunit phosphorylated by CK2. The in vitro stoichiometry of phosphorylation of the alpha6/C2 and alpha3/C9 proteasome subunits by CK2 can be estimated as 0.7-0.8 and 0.4-0.5 mol of phosphate per mole of subunit, respectively. These results are consistent with the relative abundance of the unphosphorylated and phosphorylated isoforms of these subunits present in the purified 20S proteasome preparation. Our demonstration of phosphorylation of C. albicans proteasome suggests that phosphorylation might be a general mechanism of regulation of proteasome activity.

TThe Triple Gene Block 1 (TGBp1) protein encoded by the Potato virus X is a multifunctional protein that acts as a suppressor of RNA silencing or facilitates the passage of virus from cell to cell by promoting the plasmodesmata opening. We previously showed that the membrane raft protein StRemorin1.3 is able to impair PVX infection. Here, we show that overexpressed StRemorin1.3 does not impair the silencing suppressor activity of TGBp1, but affects its ability to increase plasmodesmata permeability. A similar effect on plasmodesmata permeability was observed with other movement proteins, suggesting that REM is a general regulator of plasmodesmal size exclusion limit. These results add to our knowledge of the mechanisms underlying the StREM1.3 role in virus infection. Fil: Perraki, Artemis. Universite de Bordeaux; Francia. Centre National de la Recherche Scientifique; Francia Fil: Binaghi, Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Mecchia, Martin Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Gronnier, Julien. Universite de Bordeaux; Francia. Centre National de la Recherche Scientifique; Francia Fil: German Retana, Sylvie. Institut National de la Recherche Agronomique; Francia Fil: Mongrand, Sébastien. Universite de Bordeaux; Francia. Centre National de la Recherche Scientifique; Francia Fil: Bayer, Emmanuelle. Universite de Bordeaux; Francia. Centre National de la Recherche Scientifique; Francia Fil: Zelada, Alicia Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Germain, Véronique. Centre National de la Recherche Scientifique; Francia. Universite de Bordeaux; Francia

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 Km for ATP and Kemptide were 30 microM and 60 microM 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-[32P]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.

Background: Soil is among the most diverse and complex environments in the world. Soil microorganisms play an essential role in biogeochemical cycles and affect plant growth and crop production. However, our knowledge of the relationship between species-assemblies and soil ecosystem processes is still very limited. The aim of this study was to generate a comprehensive metagenomic survey to evaluate the effect of high-input agricultural practices on soil microbial communities. Results: We collected soil samples from three different areas in the Argentinean Pampean region under three different types of land uses and two soil sources (bulk and rhizospheric). We extracted total DNA from all samples and also synthetized cDNA from rhizospheric samples. Using 454-FLX technology, we generated 112 16S ribosomal DNA and 14 16S ribosomal RNA amplicon libraries totaling 1.3 M reads and 36 shotgun metagenome libraries totaling 17.8 million reads (7.7 GB). Our preliminary results suggested that water availability could be the primary driver that defined microbial assemblages over land use and soil source. However, when water was not a limiting resource (annual precipitation >800 mm) land use was a primary driver. Conclusion: This was the first metagenomic study of soil conducted in Argentina and our datasets are among the few large soil datasets publicly available. The detailed analysis of these data will provide a step forward in our understanding of how soil microbiomes respond to high-input agricultural systems, and they will serve as a useful comparison with other soil metagenomic studies worldwide., Instituto de Biotecnologia y Biologia Molecular

We have previously shown that in the ascomycete Saccobolus platensis the presence of cyclic AMP can overcome the requirement for light to produce apothecia. With the aim to gain insight into the phototransduction mechanism the existence of G protein-like molecules was investigated. By using the GTP analog [ 35 S]GTP-γS we detected the existence of GTP-binding proteins; the dissociation constant for the binding of the nucleotide was calculated ( K d = 52 n M ); the binding was effectively competed by GTP and GDPPS and to a lesser extent by ATP. We also demonstrated by SDS-PAGE the existence of proteins of 48, 41, 26, and 15 kDa that are substrates of the cholera toxin-dependent ADP-ribosylation reaction. Proteins resolved by SDS-PAGE were identified by Western blotting with antisera specific for vertebrate G protein subunits; the antibody anti-α common revealed two main bands at 48 and 41 kDa; the AS/7 antibody (anti-α t , −α 1 , and −a i2 ) led to the identification of one band at 41 kDa; the antibody anti-β subunit revealed a polipeptide band at around 37 kDa. The antibody anti-α common also abolished the binding of [ 35 S]GTP-γS to crude membranes. The results presented in this paper provide evidence for the existence in S. platensis of proteins that proved to have a behavior similar to that of mammalian G proteins.

We have previously reported that Candida albicans protein kinase CK2 is composed of two distinct catalytic (α- and α′-) and two distinct regulatory (β- and β′-) subunits. We report here the isolation of two cDNAs clones, CaCKB1 and CaCKB2, encoding C. albicans β- and β′-subunits, respectively. The predicted β- and β′- proteins have calculated molecular masses of 34 kDa and 31 kDa and show all major features conserved in β-subunits of other organisms, including the N-terminal autophosphorylation site, the internal acidic region and a potential metal-binding motif. The deduced amino acid sequence of C. albicans β-subunit displays 48% identity with that of Saccharomyces cerevisiae and has an unusually long C-terminal acidic region containing a putative autophosphorylation site. C. albicans β′ shows 54% sequence identity with its S. cerevisiae homologue. Semi-quantitative RT-PCR analyses indicate that the mRNAs corresponding to both subunits are present in similar amounts in the yeast and hyphal forms of the fungus. To evaluate the biochemical properties of C. albicans β- and β′-subunits, both proteins were expressed in Escherichia coli and purified. Experiments performed in vitro indicate that both recombinant subunits reconstitute a fully functional holoenzyme when incubated with stoichiometric amounts of human recombinant α-subunit, as judged by their ability to abolish basal phosphorylation of calmodulin by human recombinant α-subunit and the reversion of the inhibitory effect by polylysine. In addition, both regulatory subunits can be phosphorylated by human recombinant α subunit. Phylogenetic analysis of β- and β′-proteins of C. albicans and other organisms shows that the CKB gene duplication occurred before the split of the ascomycete and basidiomycete lineages. cDNA sequences of C. albicans CKB1 (Accession No. AF0599060) and CKB2 (Accession No. AY172319) have been deposited in the GenBank database. Fil: Zelada, Alicia Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Silva Junqueira de Souza, Flavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Walz, Katherina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina Fil: Giasson, Luc. Laval University; Canadá Fil: Di Bernardo, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina

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. Fil: Cloutier, Monikca. GREB Laval University; Canadá Fil: Castilla Lozano, Maria del Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina Fil: Bolduc, Nathalie. GREB Laval University; Canadá Fil: Zelada, Alicia Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina Fil: Martineau, Philippe. GREB Laval University; Canadá Fil: Bouillon, Marlène. GREB Laval University; Canadá Fil: Magee, Beatrice B.. University of Minnesota; Estados Unidos Fil: Di Bernardo, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina Fil: Giasson, Luc. GREB Laval University; Canadá Fil: Cantore, Maria Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina

The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser180) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser180 is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [32P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.