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Mathioudakis M.M.,Mediterranean Agronomic Institute of Chania | Mathioudakis M.M.,Aristotle University of Thessaloniki | Veiga R.S.L.,Mediterranean Agronomic Institute of Chania | Canto T.,CSIC - Biological Research Center | And 5 more authors.
Molecular Plant Pathology | Year: 2013

Various plant factors are co-opted by virus elements (RNA, proteins) and have been shown to act in pathways affecting virus accumulation and plant defence. Here, an interaction between Pepino mosaic virus (PepMV) triple gene block protein 1 (TGBp1; p26) and tomato catalase 1 (CAT1), a crucial enzyme in the decomposition of toxic hydrogen peroxide (H2O2), was identified using the yeast two-hybrid assay, and confirmed via an invitro pull-down assay and bimolecular fluorescent complementation (BiFC) inplanta. Each protein was independently localized within loci in the cytoplasm and nuclei, sites at which their interaction had been visualized by BiFC. Following PepMV inoculation, CAT mRNA and protein levels in leaves were unaltered at 0, 3 and 6 days (locally) and 8 days (systemically) post-inoculation; however, leaf extracts from the last two time points contained increased CAT activity and lower H2O2 levels. Overexpression of PepMV p26 invitro and inplanta conferred the same effect, suggesting an additional involvement of TGBp1 in potexvirus pathogenesis. The accumulation of PepMV genomic and subgenomic RNAs and the expression of viral coat protein in noninoculated (systemic) leaves were reduced significantly in CAT-silenced plants. It is postulated that, during PepMV infection, a p26-CAT1 interaction increases H2O2 scavenging, thus acting as a negative regulator of plant defence mechanisms to promote PepMV infections. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Kalivas A.,Aristotle University of Thessaloniki | Pasentsis K.,Institute of Agrobiotechnology CERTH | Argiriou A.,Institute of Agrobiotechnology CERTH | Darzentas N.,Institute of Agrobiotechnology CERTH | And 2 more authors.
Preparative Biochemistry and Biotechnology | Year: 2010

We describe an improvement of the RCA-RACE (rolling circle amplification-rapid amplification of cDNA ends) method, called family RCA-RACE (famRCA-RACE). The method is based on the generation of circular cDNA fragments, followed by rolling circle amplification of the circular cDNA using φ29 DNA polymerase and the application of PCR using degenerate outworking primers, designed for a conserved region of homologous genes, that allows the isolation of homologous cDNA sequences expressed in the mRNA preparation in a single polymerase chain reaction (PCR). As an example we present the isolation of seven NAC-like transcription factors cDNA sequences expressed in Crocus sativus flower, used for saffron production. Sequence alignment revealed that CsatNAC proteins contain the typical domain structure of plant NAC proteins, consisting of the conserved N-terminal NAC domain used to design the primers and the five subdomains. Phylogenetic analysis revealed that CsatNAC proteins fall in subgroup I of the NAC family of proteins. © Taylor & Francis Group, LLC.

Ignea C.,Mediterranean Agronomic Institute of Chania | Ignea C.,University of Crete | Cvetkovic I.,Mediterranean Agronomic Institute of Chania | Loupassaki S.,Mediterranean Agronomic Institute of Chania | And 5 more authors.
Microbial Cell Factories | Year: 2011

Background: Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway.Results: S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production.Conclusions: Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields. © 2011 Ignea et al; licensee BioMed Central Ltd.

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