Propiedades bioquímicas de los microtúbulos : Asociación de componentes lipídicos y actividades enzimáticas relacionadas

En este trabajo se muestra que los microtúbulos de diversas fuentes y obtenidos por distintos métodos tienen asociados lípidos fosforilados, identificados como fosfolípidos, entre los cuales el más abundante es la lecitina. Además de fosfolípidos, los microtúbulos tienen asociada una actividad de diglicérido quinasa, capaz de fosforilar, a partir de ATP, diglicéridos también asociados a las preparaciones microtubulares o bien exógenos, para dar ácido fosfatídico. Esta diglicérido quinasa no es una parte de la actividad del sobrenadante, y sus propiedades son muy semejantes a las de otras diglicérido quinasas. La diglicérido quinasa asociada a microtúbulos no presenta propiedades diferentes a la proteína quinasa de la misma fuente, pero los sitios activos de ambas actividades serían diferentes. La alteración en la estructura o contenido en fosfolípidos asociados a microtúbulos por diversos tratamientos producen alteraciones que no se deben a desnaturalización en el desarrollo de turbidez o viscosidad de las preparaciones microtubulares, indicando que estos fosfolípidos pueden tener algún papel en la reacción de polimerización.Fil:Daleo, Gustavo Raúl. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Propiedades bioquímicas de los microtúbulos : Asociación de componentes lipídicos y actividades enzimáticas relacionadas

En este trabajo se muestra que los microtúbulos de diversas fuentes y obtenidos por distintos métodos tienen asociados lípidos fosforilados, identificados como fosfolípidos, entre los cuales el más abundante es la lecitina. Además de fosfolípidos, los microtúbulos tienen asociada una actividad de diglicérido quinasa, capaz de fosforilar, a partir de ATP, diglicéridos también asociados a las preparaciones microtubulares o bien exógenos, para dar ácido fosfatídico. Esta diglicérido quinasa no es una parte de la actividad del sobrenadante, y sus propiedades son muy semejantes a las de otras diglicérido quinasas. La diglicérido quinasa asociada a microtúbulos no presenta propiedades diferentes a la proteína quinasa de la misma fuente, pero los sitios activos de ambas actividades serían diferentes. La alteración en la estructura o contenido en fosfolípidos asociados a microtúbulos por diversos tratamientos producen alteraciones que no se deben a desnaturalización en el desarrollo de turbidez o viscosidad de las preparaciones microtubulares, indicando que estos fosfolípidos pueden tener algún papel en la reacción de polimerización

Analysing the expression of genes associated with induced resistance in potato plants treated with phosphites

Phosphites (Phi) have the ability to protect plants against different pathogens, both through a direct effect in oomycete metabolism and by an indirect effect stimulating the plant´s natural defence responses. We have previously shown that KPhi foliar application to potato plants resulted in different protection levels against Phytophthora infestans depending on dose and plant age at application time. In order to identify genes that are involved in induced resistant in plants treated with KPhi, we analyzed by RT PCR, the time course of transcript levels of two genes which encode predicted transcription factors involved in pathogen perception and defence gene expression. Preliminary results showed that WRKY and NPR1 were differentially induced in plants both treated with Phi and infected with Phytophthora infestans, showing an earlier and highest induction than infected plants non treated with Phi. These results may allow us to hypothetize that Phi treatment might trigger a fast mechanism to protect potato plants to pathogen infections

Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase- 3 activity (StSBTc-3)

Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant–pathogen interaction.Fil: Fernández, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Daleo, Gustavo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Guevara, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentin

Identification of potassium phosphite responsive miRNAs and their targets in potato

Micro RNAs (miRNAs) are small single strand non-coding RNAs that regulate geneexpression at the post-transcriptional level, either by translational inhibition or mRNA degradation based on the extent of complementarity between the miRNA and its target mRNAs. Potato (Solanum tuberosum L.) is the most important horticultural crop in Argentina. Achieving an integrated control of diseases is crucial for this crop, where frequent agrochemical applications, particularly fungicides, are carried out. A promising strategy is based on promoting induced resistance through the application ofenvironmentally friendly compounds such as phosphites, inorganic salts of phosphorous acid. The use of phosphites in disease control management has provento be effective. Although the mechanisms underlying their effect remain unclear, we postulated that miRNAs could be involved. Therefore we performed next generation sequencing (NGS) in potato leaves treated and non treated with potassium phosphite (KPhi). We identified 25 miRNAs that were expressed differentially, 14 already annotated in miRBase and 11 mapped to the potato genome as potential new miRNAs. A prediction of miRNA targets showed genes related to pathogen resistance,transcription factors, and oxidative stress. We also analyzed in silico stress and phytohormone responsive cis-acting elements on differentially expressed pre miRNAs. Despite the fact that some of the differentially expressed miRNAs have been already identified, this is to our knowledge the first report identifying miRNAs responsive to abiocompatible stress resistance inducer such as potassium phosphite, in plants.Further characterization of these miRNAs and their target genes, might help toelucidate the molecular mechanisms underlying KPhi-induced resistance.Fil: Rey Burusco, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Daleo, Gustavo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Feldman, Mariana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Expression of plant specific domain of potato aspartic proteases (StAP-PSI) restricts P. infestans spread in potato leaves.

Plant specific insert (PSI) is a domain present in the precursors and mature atypical plant aspartic proteases (APs). Several plant APs have been associated with the plant mechanism of defence against pathogens. However, only two (StAP1 and StAP3, for Solanum tuberosum APs) of these proteases, contain the PSI domain into the mature form. We have previously reported the cytotoxic activity of the recombinant StAP-PSI towards plant pathogens. However the role of PSI domain of StAPs in the plant mechanism defense is still unknown. The aim of this work was to analyze the effect of transient expression of StAP-PSI in potato leaves infected by P. infestans. Results obtained show that StAP-PSI expression reduces the P. infestans affected area in a 60 % compared with the control ones. Analysis by qPCR shows an increase of the transcript level of the hypersensitive response marker (hsr203J) in potato leaves that express StAP-PSI, independent of the P. infestans infection; however the highest increase of this gene was detected in leaves at 6 h. after infection. Additionally, an increase of the WRKI1 transcript level was detected in potato leaves that express StAP-PSI. Results obtained here indicate that, PSI domain of StAPs could have a direct (as antimicrobial compound) and indirect (as an inductor molecule) role in the plant mechanism to restrict the pathogen spread

DEVDase activity is induced in potato leaves during Phytophthora infestans infection.

Programmed cell death (PCD) occurs in plants, animals and several branches of unicellular eukaryotes as a part of developmental and/or defense processes. Caspase proteases are universal mediators of animal apoptosis, a type of PCD. In plants, there are not animal caspase homologs; therefore, the characterization of caspase-like activities is of considerable importance to our understanding of PCD in plants. Here we report for the first time the involvement of caspase-3-like activity in the resistance mechanism of potato to Phytophthora infestans infection. We showed that disease development in infected potato leaves is dependent of caspase-3-like activity. Unlike plant DEVDases previously reported, this DEVDase activity was sensitive to the serine protease inhibitor PMSF. As reported for other subtilisin- like proteases with caspase activity, potato DEVDase activity was mainly localized in the apoplast. We demonstrated that in total protein extract DEVDase activity accounts for a 60% of serine pro

Roles of glycosylation on the antifungal activity and apoplast accumulation of StAPs (Solanum tuberosum aspartic proteases)

Specific roles of glycosylation appear to be protein-dependent. Plant aspartic proteases (APs) contain two or more consensus N-glycosylation sites; however, the importance of them is not well understood. StAPs (Solanum tuberosum aspartic proteases) are bifunctional proteins with both proteolytic and antimicrobial activities. These proteins are accumulated into the intercellular washing fluid of potato tubers and leaves after wounding or infection. In this paper we investigated the importance of glycosylation on the StAPs apoplast accumulation, biochemical parameters, and fungicidal activity. Assays to evaluate the importance of StAPs glycosylation groups by using glycosylation inhibitors demonstrate that carbohydrate portions are essential to StAPs accumulation into the apoplast of tubers and leaves after wounding or detachment, respectively. Bifunctional activity of StAPs is differentially affected by this post-translational modification. Results obtained show that not significant changes were produced in the physicochemical properties after StAPs deglycosylation (pH and thermal-optimum activity and index of protein surface hydrophobicity). Otherwise, StAPs antifungal activity is affected by deglycosylation. Deglycosylated StAPs (dgStAPs) fungicidal activity is lower than native StAPs at all concentrations and times assayed. In summary, glycosylation has not a significant role on the StAPs conformational structure. However, it is involved in the StAPs subcellular accumulation and antifungal activity suggesting that it could be necessary for StAPs membrane and/or protein interactions and subsequently its biological function(s).Fil: Pagano, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Mendieta, Julieta Renee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Muñoz, Fernando Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Daleo, Gustavo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Guevara, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Apoplastic hydrophobic proteins involved in tuber defense response to P. Infestans

During infection, oomycetes secrete effectors into the plant apoplast where they interact with host resistance proteins. In response, large amounts of protease and protease inhibitors (PIs), are accumulated. We analyzed differentially expressed Apoplastic Hydrophobic Proteins (AHPs) in potato tubers from Innovator (resistant) and Spunta (susceptible) cvs, after wounding and P. infestans infection. Intercellular washing fluid was extracted from control, wounded or infected tubers at 0, 24 and 48 h and chromatographed into a PepRPCtmHR5/5 in FPLC. After elution with acetonitrile, fractions were analyzed by SDS-PAGE and proteins identified by MALDITOF- MS. Innovator cv. showed a higher basal AHP content and hydrophobicity than Spunta cv. In the latter, infection induced accumulation of patatins and PIs, whereas in Innovator cv. no changes in PIs accumulation were observed. In response to P. infestans infection, lypoxigenase, enolase, annexin p34 and glutarredoxin/cyclophilin were accumulated in both cvs. Hydrophobicity of AHPs was higher after 24 h of wounding and infection in both cultivars. These results suggest that an increase in AHPs concentration would be related with the protection against the oomycete and with the degree of resistance to pathogens. Finally, changes in hydrophobicity of Pis may induce changes in proteaseinhibitor interaction affecting the defense response