Development of MRM methods to quantify proteins isoforms of poliphenol oxidase

Comunicaciones a congreso

Phosphoprotein enrichment from soluble and microsomal fractions of grapevine (vitis vinifera cv. gamay) cell culture using metal oxide affinity chromatography (MOAC)

Comunicaciones a congreso

A tau class glutathione-S-transferase is involved in trans-resveratrol transport out of grapevine cells

Vitis vinifera cell cultures respond to pathogens and elicitors by synthesizing and extracellularly accumulating stilbenoid phytoalexins. Large amounts of trans-resveratrol (t-R) are produced when a cell culture is elicited with methylated cyclodextrins (MBCD), either alone or combined with methyl jasmonate (MeJA). t-R transport to the extracellular medium, which represents the apoplastic space, would place this antifungal defense right in the battlefield to efficiently fight against pathogen attack. Yet despite their physiological relevance, these transport pathways are mostly unknown. A broad hypothesis-free DIGE-based proteomic experiment of a temporal series of elicited grapevine cell cultures was performed to explore the expression profiles of t-R biosynthetic proteins and other co-expressing proteins potentially involved in such a cell response. A correlation between two tau class glutathione-S-transferases (GSTs) with several stilbene synthase and phenylalanine ammonia-lyase isoforms, and with the t-R metabolite itself, was found and further assessed by a qRT-PCR gene expression analysis. The best candidate, GSTU-2, was cloned from the cDNA of the MBCD + MeJA-elicited grapevine cells and used for Agrobacterium-mediated grapevine cell transformation. The non-elicited lines that overexpressed GSTU-2 displayed an extracellular t-R accumulating phenotype, but stabilization of t-R required the addition to culture medium of adsorbent compounds, e.g., PVP or β-cyclodextrin. The wild-type cell cultures accumulated no t-R, not even in the presence of adsorbents. The transient expression of the GSTU-2-GFP fusion proteins in grapevine cells showed localisation in the plasma membrane, and the immunoprecipitation of HA-tagged GSTU-2 revealed its interaction with HIR, a plasma membrane-bound protein. These findings are consistent with a functional role in transport. This is the first report providing several pieces of experimental evidence for the involvement of a specific tau class GST in t-R transport to the extracellular medium

Plant virome reconstruction and antiviral RNAi characterization by deep sequencing of small RNAs from dried leaves

In plants, RNA interference (RNAi) generates small interfering (si)RNAs from entire genomes of viruses, satellites and viroids. Therefore, deep small (s)RNA sequencing is a universal approach for virome reconstruction and RNAi characterization. We tested this approach on dried barley leaves from field surveys. Illumina sequencing of sRNAs from 2 plant samples identified in both plants Hordeum vulgare endornavirus (HvEV) and barley yellow mosaic bymovirus (BaYMV) and, additionally in one plant, a novel strain of Japanese soil-borne wheat mosaic furovirus (JSBWMV). De novo and reference-based sRNA assembly yielded complete or near-complete genomic RNAs of these viruses. While plant sRNAs showed broad size distribution, viral sRNAs were predominantly 21 and 22 nucleotides long with 5'-terminal uridine or adenine, and were derived from both genomic strands. These bona fide siRNAs are presumably processed from double-stranded RNA precursors by Dicer-like (DCL) 4 and DCL2, respectively, and associated with Argonaute 1 and 2 proteins. For BaYMV (but not HvEV, or JSBWMV), 24-nucleotide sRNAs represented the third most abundant class, suggesting DCL3 contribution to antibymovirus defence. Thus, viral siRNAs are well preserved in dried leaf tissues and not contaminated by non-RNAi degradation products, enabling both complete virome reconstruction and inference of RNAi components mediating antiviral defense.Diversification for Tobacco Growing Farms by the alternative crop Stevia rebaudiana Berton

Induced expression of selected plant defence related genes in pot azalea, Rhododendron simsii hybrid

A set of putative marker genes to study plant defense responses against Polyphagotarsonemus latus, a key pest in the production of Rhododendron simsii hybrids, was selected and validated. Genes belonged to the biosynthetic pathway of phytohormones jasmonic acid (JA) (RsLOX, RsAOS, RsAOC, RsOPR3 and RsJMT) and salicylic acid (SA) (RsPAL and RsICS). Furthermore, RsPPO, a putative marker gene for oxidative stress response was successfully cloned from R. simsii. A CTAB-based extraction protocol was optimized to assure excellent RNA quality for subsequent RT-qPCR analysis. The RT-qPCR protocol was extensively tested and RsRG7 and RsRG14 were selected as reference genes from a geNorm pilot study. Validation of the marker genes was done after application with elicitors [methyl jasmonate (MeJA), coronatine, beta-aminobutyric acid and acibenzolar-Smethyl] or wounding. Both 100 mu M MeJA and 0.1 lM coronatine had a significant effect on the expression of all marker genes. Foliar application of MeJA on the shoots resulted in a significantly earlier response when compared to root application and subsequent sampling of the shoots. Expression patterns after MeJA treatment were generally the same in six R. simsii genotypes: 'Nordlicht', 'Elien', 'Aiko Pink', ' Michelle Marie', 'Mevrouw Gerard Kint' and 'Sachsenstern'. Wounding resulted in the same expression patterns as MeJA treatment except for RsJMT. None of the genotypes showed a significant induction of the latter gene 6 h upon wounding. Findings of these experiments indicated that the tolerant genotype 'Elien' has low basal expression levels of RsPPO. This might be the first step towards the breeding of mite-tolerant genotypes