A successful defence strategy in grapevine cultivar 'Tocai friulano' provides compartmentation of grapevine Flavescence dorée phytoplasma.

BACKGROUND: Flavescence dorée (FD) is a grapevine disease caused by phytoplasma and it is one of the most destructive pathologies in Europe. Nowadays, the only strategies used to control the epidemics are insecticides against vector, but more sustainable techniques are required. Completely resistant Vitis vinifera varieties have not been uncovered yet, but differences in susceptibility among cultivars and spontaneous recovery from FD symptoms have been observed. The grapevine cultivar 'Tocai friulano' shows very low susceptibility to FD but its defence strategy to counteract the phytoplasma spread has not been deciphered yet. In this work, the mechanisms occurring within 'Tocai friulano' FD-infected plants were examined in depth to identify the phytoplasma distribution and the defence pathways involved. RESULTS: In 'Tocai friulano' symptoms of FD-infection remained confined near the area where they appeared during all the vegetative season. Analyses of secondary phloem showed a total absence of FD phytoplasma (FDp) in the trunk and its disappearance in 2-year-old arms from July to November, which was different from 'Pinot gris', a highly susceptible variety. Diverse modulations of defence genes and accumulation of metabolites were revealed in 1-year-old canes of 'Tocai friulano' FD-infected plants, depending on the sanitary status. Symptomatic portions showed high activation of both jasmonate- and salicylate-mediated responses, together with a great accumulation of resveratrol. Whereas activation of jasmonate-mediated response and high content of ε-viniferin were identified in asymptomatic 1-year-old cane portions close to the symptomatic ones. CONCLUSION: Successful defence mechanisms activated near the symptomatic areas allowed the compartmentation of FD symptoms and phytoplasmas within the infected 'Tocai friulano' plants. These results could suggest specific agronomical practices to be adopted during FD management of this variety, and drive research of resistance genes against FD.

Screening potential insect vectors in a museum biorepository reveals undiscovered diversity of plant pathogens in natural areas.

Phytoplasmas (Mollicutes, Acholeplasmataceae), vector-borne obligate bacterial plant parasites, infect nearly 1,000 plant species and unknown numbers of insects, mainly leafhoppers (Hemiptera, Deltocephalinae), which play a key role in transmission and epidemiology. Although the plant-phytoplasma-insect association has been evolving for >300 million years, nearly all known phytoplasmas have been discovered as a result of the damage inflicted by phytoplasma diseases on crops. Few efforts have been made to study phytoplasmas occurring in noneconomically important plants in natural habitats. In this study, a subsample of leafhopper specimens preserved in a large museum biorepository was analyzed to unveil potential new associations. PCR screening for phytoplasmas performed on 227 phloem-feeding leafhoppers collected worldwide from natural habitats revealed the presence of 6 different previously unknown phytoplasma strains. This indicates that museum collections of herbivorous insects represent a rich and largely untapped resource for discovery of new plant pathogens, that natural areas worldwide harbor a diverse but largely undiscovered diversity of phytoplasmas and potential insect vectors, and that independent epidemiological cycles occur in such habitats, posing a potential threat of disease spillover into agricultural systems. Larger-scale future investigations will contribute to a better understanding of phytoplasma genetic diversity, insect host range, and insect-borne phytoplasma transmission and provide an early warning for the emergence of new phytoplasma diseases across global agroecosystems.

When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe.

Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties.

Grapevine comparative early transcriptomic profiling suggests that Flavescence dorée phytoplasma represses plant responses induced by vector feeding in susceptible varieties.

BACKGROUND: Flavescence dorée is the most serious grapevine yellows disease in Europe. It is caused by phytoplasmas which are transmitted from grapevine to grapevine by the leafhopper Scaphoideus titanus. Differences in susceptibility among grapevine varieties suggest the existence of specific genetic features associated with resistance to the phytoplasma and/or possibly with its vector. In this work, RNA-Seq was used to compare early transcriptional changes occurring during the three-trophic interaction between the phytoplasma, its vector and the grapevine, represented by two different cultivars, one very susceptible to the disease and the other scarcely susceptible. RESULTS: The comparative analysis of the constitutive transcriptomic profiles suggests the existence of passive defense strategies against the insect and/or the phytoplasma in the scarcely-susceptible cultivar. Moreover, the attack by the infective vector on the scarcely-susceptible variety prompted immediate and substantial transcriptomic changes that led to the rapid erection of further active defenses. On the other hand, in the most susceptible variety the response was delayed and mainly consisted of the induction of phytoalexin synthesis. Surprisingly, the jasmonic acid- and ethylene-mediated defense reactions, activated by the susceptible cultivar following FD-free insect feeding, were not detected in the presence of the phytoplasma-infected vector. CONCLUSIONS: The comparison of the transcriptomic response in two grapevine varieties with different levels of susceptibility to Flavescence dorèe highlighted both passive and active defense mechanisms against the vector and/or the pathogen in the scarcely-susceptible variety, as well as the capacity of the phytoplasmas to repress the defense reaction against the insect in the susceptible variety.

Grapevine Pinot gris virus seems to have recently been introduced to vineyards in Veneto, Italy.

Grapevine Pinot gris virus (GPGV), a member of the genus Trichovirus in the family Betaflexiviridae, was recently discovered in Italy and subsequently in other European countries and in Korea. In this study, we assessed the occurrence of GPGV in 441 samples from Western and Eastern Europe collected over the period 2002-2014. The results suggest that the virus had recently appeared in the Veneto region (Northeast Italy) and had been present in some Eastern European countries for at least 10 years. The molecular characterization of the 5'-terminal genomic region of several GPGV isolates from Italy and other European countries showed low polymorphism, with a maximum nucleotide sequence divergence of 3.2%.

Genetic diversity of European phytoplasmas of the 16SrV taxonomic group and proposal of 'Candidatus Phytoplasma rubi'.

In addition to the grapevine flavescence dorée phytoplasmas, other members of taxonomic group 16SrV phytoplasmas infect grapevines, alders and species of the genera Clematis and Rubus in Europe. In order to investigate which phytoplasmas constitute discrete, species-level taxa, several strains were analysed by comparing their 16S rRNA gene sequences and a set of five housekeeping genes. Whereas 16S rRNA gene sequence similarity values were >97.5 %, the proposed threshold to distinguish two 'Candidatus Phytoplasma' taxa, phylogenetic analysis of the combined sequences of the tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genetic loci showed that two discrete phylogenetic clusters could be clearly distinguished. The first cluster grouped flavescence dorée (FD) phytoplasmas, alder yellows (AldY) phytoplasmas, Clematis (CL) phytoplasmas and the Palatinate grapevine yellows (PGY) phytoplasmas. The second cluster comprised Rubus stunt (RS) phytoplasmas. In addition to the specificity of the insect vector, the Rubus stunt phytoplasma contained specific sequences in the 16S rRNA gene. Hence, the Rubus stunt phytoplasma 16S rRNA gene was sufficiently differentiated to represent a novel putative taxon: 'Candidatus Phytoplasma rubi'.

A new TaqMan method for the identification of phytoplasmas associated with grapevine yellows by real-time PCR assay.

Three real-time PCR systems for direct detection of phytoplasmas associated to Flavescence dorée (FD), Bois noir (BN) and aster yellows (AY) diseases were developed. TaqMan probes and primers were designed on the 16S ribosomal RNA sequences of phytoplasma genome. A further TaqMan assay, targeting a grapevine gene encoding for the chloroplast chaperonin 21, was developed in order to check the DNA quality and to verify the absence of PCR inhibition. A comparison between real-time PCR and conventional nested-PCR methods for phytoplasma detection was carried out on several reference samples from grapevine, periwinkle, other host plants and insect species. Detection of FD, BN and AY phytoplasma DNA on infected specimens was rapid, specific and reproducible. Sensitivity was as high as nested-PCR assay. The two procedures were then used on about 450 samples collected from grapevines showing yellows symptoms. The results showed that real-time PCR approach for phytodiagnostic purposes was more advantageous than nested-PCR method with regard to rapidity of the assay and reduced risk of sample cross contamination. These new protocols represent an improvement of existing analytical methods and could be used as a reliable diagnostic procedure in certification and control programs.

Improved strategies for the delivery of GFP-based Ca2+ sensors into the mitochondrial matrix.

The role of mitochondria in Ca2+ handling has acquired renewed interest in recent years in the field of cell signaling. Detailed studies of Ca2+ dynamics in this organelle at the single cell level have been hampered by technical problems in the available Ca2+ probes. Some of the latest generation GFP-based Ca2+ probes (Camgaroos, Cameleons and Pericams) show great potential to address this issue. Our data show that the choice of targeting sequence influences not only the overall efficiency of subcellular localization of the probes, but also their functional characteristics within the matrix. In particular, we here show that the use of a tandemly duplicated mitochondrial targeting sequence is capable of improving the delivery efficacy of all tested probes into the organelle's matrix, in particular that of Cameleon, a GFP-based Ca2+ probe that is otherwise largely mistargeted to the cytosol. The devised strategy should be generally applicable to other proteins that are characterized by poor targeting. Last, but not least, we also demonstrate that if the targeting sequence is not removed from the imported protein, the fluorescent properties and the Ca2+ affinity of the probe can be grossly affected.

Mitochondrial pH monitored by a new engineered green fluorescent protein mutant.

We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK(a) (pK(a)') around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca(2+) ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca(2+) accumulation by this organelle. Finally, we demonstrate that Ca(2+) mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.

Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a subpopulation of mitochondria.

To better understand the functional role of the mitochondrial network in shaping the Ca2+ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca2+ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca(2+)-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca2+ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca2+ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca2+ release channels (and thus responsible for the fast uptake of Ca2+ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum.