Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species

Optimization of abdominal microinjection parameters. Description of parameter optimizations for abdominal microinjection experiments. (PDF 56 kb

Bacteriophage-Host Association in the Phytoplasma Insect Vector Euscelidius variegatus

Insect vectors transmit viruses and bacteria that can cause severe diseases in plants and economic losses due to a decrease in crop production. Insect vectors, like all other organisms, are colonized by a community of various microorganisms, which can influence their physiology, ecology, evolution, and also their competence as vectors. The important ecological meaning of bacteriophages in various ecosystems and their role in microbial communities has emerged in the past decade. However, only a few phages have been described so far in insect microbiomes. The leafhopper Euscelidius variegatus is a laboratory vector of the phytoplasma causing Flavescence dorée, a severe grapevine disease that threatens viticulture in Europe. Here, the presence of a temperate bacteriophage in E. variegatus (named Euscelidius variegatus phage 1, EVP-1) was revealed through both insect transcriptome analyses and electron microscopic observations. The bacterial host was isolated in axenic culture and identified as the bacterial endosymbiont of E. variegatus (BEV), recently assigned to the genus Candidatus Symbiopectobacterium. BEV harbors multiple prophages that become active in culture, suggesting that different environments can trigger different mechanisms, finely regulating the interactions among phages. Understanding the complex relationships within insect vector microbiomes may help in revealing possible microbe influences on pathogen transmission, and it is a crucial step toward innovative sustainable strategies for disease management in agriculture

Phytoplasma infefction of tomato in central Italy

Tomato plants (Lycopersicon esculentum Mill.) showing leaf yellowing or reddening, proliferation of lateral shoots, hypertrophied calyces, and greening of petals were found in four tomato-growing areas of central Italy during surveys carried out in 1999–2000. The highest disease incidence was recorded in 1999 (78%) and 2000 (82%) near Ronciglione (Viterbo province). The phytoplasmas associated with the disease were detected and characterised using PCR and RFLP. They belonged to 16S rRNA groups I, III, V and XII (sensu Lee et al., 1998. Int. J. Syst. Bacteriol., 48, 1153). Forty-five percent of tomato plants were infected with group I (max. 52% near Frascati and Ronciglione), 31% with group XII (max. 39% near Ronciglione) and 24% with other groups (max. 33% near Tarquinia and 23% near Fondi). Seven percent of tomato plants were infected with both group I and group XII. This is the first report of a group-III and a group-V phytoplasma found in tomato

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

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity

FLAVOSCREEN_MEDVI

Susceptibility to Flavescence dorée of different Vitis vinifera genotypes from North-Western Ital

Genetic diversity of Italian and French “bois noir” phytoplasma isolates

International audienceGenetic diversity of "bois noir" phytoplasma (BNp) isolates was determined by PCR-RFLP of the stolbur specific stol-1H10 gene encoding a putative membrane protein. BN-infected grapevines were collected from the most representative Italian and French wine producing regions. Weeds and vectors collected in infected vineyards from different Italian regions were also analysed for BN characterization. Size polymorphism of stol-1H10 gene was obtained from PCR assays. Twelve different RFLP patterns were detected in BNp grapevine isolates, one in Urtica dioca and four in Convolvolus arvensis isolates. Hyalesthes obsoletus BNp isolates with two prevalent RFLP profiles were detected in grapevines and in herbaceous hosts from the same vineyards. Stol-1H10 represents a useful non-ribosomal marker to type BNp in plant and insect hosts

Genetic diversity of Italian and French “bois noir” phytoplasma isolates

International audienceGenetic diversity of "bois noir" phytoplasma (BNp) isolates was determined by PCR-RFLP of the stolbur specific stol-1H10 gene encoding a putative membrane protein. BN-infected grapevines were collected from the most representative Italian and French wine producing regions. Weeds and vectors collected in infected vineyards from different Italian regions were also analysed for BN characterization. Size polymorphism of stol-1H10 gene was obtained from PCR assays. Twelve different RFLP patterns were detected in BNp grapevine isolates, one in Urtica dioca and four in Convolvolus arvensis isolates. Hyalesthes obsoletus BNp isolates with two prevalent RFLP profiles were detected in grapevines and in herbaceous hosts from the same vineyards. Stol-1H10 represents a useful non-ribosomal marker to type BNp in plant and insect hosts