A comparison of different phytoplasma DNA extraction methods using competitive PCR

The primer pair 16endF-Tmod, specific for the16S/23S rDNA spacer region, amplifies different length fragments when the target DNA belongs to phytoplasmas of the X-clade and AP-clade in compared with phytoplasmas of the AY-clade. The cloned 16S/23S spacer of the tagete witches’-broom phytoplasma (TWB, AY-clade) was used in a competitive PCR assay to quantify the DNA of the apple proliferation phytoplasma (AP, AP-clade) and the clover phyllody phytoplasma (CP, X-clade) present in the nucleic acids extracted from infected periwinkle plants. Thus, four methods, normally used for the extraction of phytoplasma DNA from infected plant tissue, were compared to determine which was the most efficient in recovering phytoplasma nucleic acids. The different methods detected relatively minor differences in the yields obtained. It was concluded that the sampling and detection protocols are more critical than the DNA extraction method as far as sensitivity is concerned

miRVIT: A Novel miRNA Database and Its Application to Uncover Vitis Responses to Flavescence dorée Infection

Micro(mi)RNAs play crucial roles in plant developmental processes and in defense responses to biotic and abiotic stresses. In the last years, many works on small RNAs in grapevine (Vitis spp.) were published, and several conserved and putative novel grapevine-specific miRNAs were identified. In order to reorganize the high quantity of available data, we produced “miRVIT,” the first database of all novel grapevine miRNA candidates characterized so far, and still not deposited in miRBase. To this aim, each miRNA accession was renamed, repositioned in the last version of the grapevine genome, and compared with all the novel and conserved miRNAs detected in grapevine. Conserved and novel miRNAs cataloged in miRVIT were then used for analyzing Vitis vinifera plants infected by Flavescence dorée (FD), one of the most severe phytoplasma diseases affecting grapevine. The analysis of small RNAs from healthy, recovered (plants showing spontaneous and stable remission of symptoms), and FD-infected “Barbera” grapevines showed that FD altered the expression profiles of several miRNAs, including those involved in cell development and photosynthesis, jasmonate signaling, and disease resistance response. The application of miRVIT in a biological context confirmed the effectiveness of the followed approach, especially for the identification of novel miRNA candidates in grapevine. miRVIT database is available at http://mirvit.ipsp.cnr.it.Highlights: The application of the newly produced database of grapevine novel miRNAs to the analysis of plants infected by Flavescence dorée reveals key roles of miRNAs in photosynthesis and jasmonate signaling

Proteome responses of Vitis vinifera L. to 'flavescence dorée' phytoplasma infection

Abstract 'Flavescence dorée' is a serious phytoplasma disease affecting grapevine in several European countries. We are currently studying the interaction of 'flavescence dorée' phytoplasma with its natural plant host, by monitoring the effects of infection on the protein expression profile. The red-berried V. vinifera cv. Nebbiolo and Barbera were the source of plant material, as they are widely grown in Piedmont and produce wines of high quality. Moreover, the two varieties show a different sensitivity to the disease: 'Nebbiolo' is generally considered more tolerant to 'flavescence dorée' infection and shows milder symptoms than 'Barbera'. Total proteins from midrib tissues were separated by two dimensional gel electrophoresis, and differentially expressed spots (p ≤ 0.05, | ratio | > 2) were identified by mass spectrometry (MALDI-TOF-TOF) analysis. The proteins were further analyzed by Blat2GO software, in order to study their function and involvement in biological processes. By this way, we could develop interaction maps, showing the possible involvement of the proteins in several biological pathways

Novel aspects of grapevine response to phytoplasma infection investigated by a proteomic and phospho-proteomic approach with data integration into functional networks

Abstract Background Translational and post-translational protein modifications play a key role in the response of plants to pathogen infection. Among the latter, phosphorylation is critical in modulating protein structure, localization and interaction with other partners. In this work, we used a multiplex staining approach with 2D gels to study quantitative changes in the proteome and phosphoproteome of Flavescence dorée-affected and recovered ‘Barbera’ grapevines, compared to healthy plants. Results We identified 48 proteins that differentially changed in abundance, phosphorylation, or both in response to Flavescence dorée phytoplasma infection. Most of them did not show any significant difference in recovered plants, which, by contrast, were characterized by changes in abundance, phosphorylation, or both for 17 proteins not detected in infected plants. Some enzymes involved in the antioxidant response that were up-regulated in infected plants, such as isocitrate dehydrogenase and glutathione S-transferase, returned to healthy-state levels in recovered plants. Others belonging to the same functional category were even down-regulated in recovered plants (oxidoreductase GLYR1 and ascorbate peroxidase). Our proteomic approach thus agreed with previously published biochemical and RT-qPCR data which reported down-regulation of scavenging enzymes and accumulation of H2O2 in recovered plants, possibly suggesting a role for this molecule in remission from infection. Fifteen differentially phosphorylated proteins (| ratio | > 2, p  Conclusions Proteomic data were integrated into biological networks and their interactions were represented through a hypothetical model, showing the effects of protein modulation on primary metabolic ways and related secondary pathways. By following a multiplex-staining approach, we obtained new data on grapevine proteome pathways that specifically change at the phosphorylation level during phytoplasma infection and following recovery, focusing for the first time on phosphoproteome changes during pathogen infection in this host.</p