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Phytoplasmas are phloem-limited plant pathogenic bacteria causing diseases in many plant species. They are transmitted by Hemipteran insect species in a persistent-propagative manner. Phytoplasmas are wall-less, and their membrane proteins are involved in pathogen internalization into host cells. We focused on the immunodominant membrane protein (Imp) of Flavescence dorée phytoplasma (FDp), a grapevine quarantine pest and a major threat to European viticulture. Scaphoideus titanus is the main natural vector of FDp to grapevine, whereas Euscelidius variegatus is commonly used as laboratory vector. Previous works indicated that recombinant Imp of two FDp strains (FD-C and FD-D) selectively interact with gut proteins from vector species rather than those from non-vectors. Here, similar patterns of interacting insect gut proteins were obtained from both vector species, following pull-down with His-tagged FDp Imps. After identification of several targets, four S. titanus and five E. variegatus proteins interacting with Imp were further characterized by measuring expression in different insect tissues and in healthy vs. infected insects. Specific RNAi silencing of two of these vector genes, namely natterin and legumain, resulted in a significant reduction of phytoplasma multiplication in insects upon pathogen acquisition, compared to control insects. Natterin displays a DM9 domain and legumain possesses a signature of G protein receptor, supporting their involvement as FDp Imp receptors. Outcomes of this work are discussed with particular attention devoted to the gain of knowledge on host/pathogen interaction as well as to the potential impact on improvement phytoplasma disease management. • Grapevine Flavescence dorée phytoplasma (FDp) is a severe quarantine plant disease. • Recombinant Imp proteins of FDp-C and D strains interact with insect gut elements. • RNAi silencing of insect natterin and legumain reduces phytoplasma load in vectors. • They have features of protein interaction mediating pathogen colonization of vector. • Natterin and legumain may work as pathogen receptors in gut of insect vectors. [ABSTRACT FROM AUTHOR]

The leafhopper Euscelidius variegatus Kirschbaum (Hemiptera: Cicadellidae) is a natural vector of the 'Candidatus Phytoplasma asteris' and a laboratory vector of the phytoplasma associated with grapevine Flavescence dorée. Previous studies indicated that RNA interference (RNAi) by dsRNA injection efficiently works in E. variegatus and that silencing of ATP synthase β increases mortality and impairs phytoplasma multiplication. Here, a nearly complete female sterility was observed when ATP synthase β was silenced. The sterility was associated with the absence of mature eggs in the ovaries. On the contrary, male genitalia morphology and sperm motility were similar to those observed in E. variegatus control specimens, treated with dsRNAs targeting green fluorescent protein (dsGFP). An over-expression of hexamerin (amino acid storage protein) and cathepsin L (lysosome proteinase) was observed at transcript and protein level in dsATP-injected females in comparison with dsGFP-injected insects, in both whole body and dissected ovary samples. Conversely, the expression of other genes known to be involved in oocyte development, namely vitellogenin, perilipin and digestive cystein protein, was not altered in dsATP-injected E. variegatus insects in comparison with dsGFP ones. Possible roles of ATP synthase β, hexamerin and cathepsin L in oocyte and egg development are discussed. Insecticide treatments against vectors are the main strategies to counteract phytoplasma diseases, with negative impact on environment and public health. RNAi is a promising sustainable approach against insect vectors and ATP synthase β could be a valid target gene, as its silencing, besides increasing mortality and reducing phytoplasma multiplication, induces female sterility. [ABSTRACT FROM AUTHOR]

Flavescence dorée (FD) is a threat for wine production in the vineyard landscape of Piemonte, Langhe-Roero and Monferrato, Italy. Spread of the disease is dependent on complex interactions between insect, plant and phytoplasma. In the Piemonte region, wine production is based on local cultivars. The role of six local grapevine varieties as a source of inoculum for the vector Scaphoideus titanus was investigated. FD phytoplasma (FDP) load was compared among red and white varieties with different susceptibility to FD. Laboratory-reared healthy S. titanus nymphs were caged for acquisition on infected plants to measure phytoplasma acquisition efficiency following feeding on different cultivars. FDP load for Arneis was significantly lower than for other varieties. Acquisition efficiency depended on grapevine variety and on FDP load in the source plants, and there was a positive interaction for acquisition between variety and phytoplasma load. S. titanus acquired FDP with high efficiency from the most susceptible varieties, suggesting that disease diffusion correlates more with vector acquisition efficiency than with FDP load in source grapevines. In conclusion, although acquisition efficiency depends on grapevine variety and on FDP load in the plant, even varieties supporting low FDP multiplication can be highly susceptible and good sources for vector infection, while poorly susceptible varieties may host high phytoplasma loads. [ABSTRACT FROM AUTHOR]

Background: The phytoplasma-borne disease flavescence dorée is still a threat to European viticulture, despite mandatory control measures and prophylaxis against the leafhopper vector. Given the economic importance of grapevine, it is essential to find alternative strategies to contain the spread, in order to possibly reduce the current use of harmful insecticides. Further studies of the pathogen, the vector and the mechanisms of phytoplasma-host interactions could improve our understanding of the disease. In this work, RNA-Seq technology followed by three de novo assembly strategies was used to provide the first comprehensive transcriptomics landscape of flavescence dorée phytoplasma (FD) infecting field-grown Vitis vinifera leaves. Results: With an average of 8300 FD-mapped reads per library, we assembled 347 sequences, corresponding to 215 annotated genes, and identified 10 previously unannotated genes, 15 polycistronic transcripts and three genes supposedly localized in the gaps of the FD92 draft genome. Furthermore, we improved the annotation of 44 genes with the addition of 5'/3' untranslated regions. Functional classification revealed that the most expressed genes were either related to translation and protein biosynthesis or hypothetical proteins with unknown function. Some of these hypothetical proteins were predicted to be secreted, so they could be bacterial effectors with a potential role in modulating the interaction with the host plant. Interestingly, qRT-PCR validation of the RNA-Seq expression values confirmed that a group II intron represented the FD genomic region with the highest expression during grapevine infection. This mobile element may contribute to the genomic plasticity that is necessary for the phytoplasma to increase its fitness and endorse host-adaptive strategies. Conclusions: The RNA-Seq technology was successfully applied for the first time to analyse the FD global transcriptome profile during grapevine infection. Our results provided new insights into the transcriptional organization and gene structure of FD. This may represent the starting point for the application of high-throughput sequencing technologies to study differential expression in FD and in other phytoplasmas with an unprecedented resolution. [ABSTRACT FROM AUTHOR]

Flavescence dorée (FD) is a quarantine grapevine disease caused by a phytoplasma transmitted by the leafhopper Scaphoideus titanus Ball. FD management relies on compulsory insecticide treatments, roguing of infected plants, and substitution with certified material. Some grapevine cultivars show a spontaneous remission of symptoms (recovery). To determine if recovery is a suitable strategy to co-exist with disease in areas of strong infestation, the qualitative aspects of grapes, musts, and wines obtained from recovered Barbera and Chardonnay grapevines were investigated in two productive vineyards. Following field observations, about 1500 plants in each vineyard were divided into healthy (asymptomatic and negative in phytoplasma molecular diagnosis) and recovered (asymptomatic the year of observation but infected the year before). Maturation curves and microvinification tests followed by oenological and sensory analyses showed that maturation trends of recovered grapes were in line with those from healthy plants and the final qualities of wines were comparable. The spread of FD has strongly increased in Piedmont (Italy) in recent decades. Management strategies to cope with the disease are necessary to preserve traditional wine production. Despite the yield from recovered grapevines is quantitatively lower than that from healthy ones, we showed here that the wine quality is, however, preserved. [ABSTRACT FROM AUTHOR]

Abstract Many aspects of plant diseases caused by phytoplasmas are still unknown, as these pathogens are phloem restricted, uncultivable wall-less bacteria and must be studied always in association with their host. Phytoplasma transcripts are strongly underrepresented within host tissues and this poses problems for gene expression analyses. In this study, a procedure was established to infect the model plant Arabidopsis thaliana with the phytoplasma Flavescence dorée, a serious threat to European viticulture. Rates of phytoplasma infective insects and transmission efficiency to A. thaliana as well as pathogen loads were measured in different tissues of infected A. thaliana plants, and modification of phloem cell ultrastructure was observed in infected plant tissues at microscopic level. Moreover, a protocol for the application of laser microdissection to analyze plant and phytoplasma gene expression profiles in the specific colonized tissue was designed. The procedure allowed a good preservation of the plant tissue anatomy. Results showed that the extracted RNA was suitable for qualitative and quantitative RT-PCR, since both plant and pathogen transcripts, either abundant or rare ones, could be detected without any pre-amplification step. The combined use of laser microdissection approach and A. thaliana to study phytoplasmas opens the way to exploit biological, molecular and bioinformatic tools available for the model plant and to elucidate key pathways of the infection mechanisms of these important plant pathogen. [ABSTRACT FROM AUTHOR]

Molecular mechanisms underlying phytoplasma interactions with host plants are largely unknown. In this study attempts were made to identify effectors of three phytoplasma strains related to ‘ Ca. P. aurantifolia’, crotalaria phyllody (CrP), faba bean phyllody (FBP), and witches’ broom disease of lime (WBDL), using information from draft genome of peanut witches’ broom phytoplasma. Seven putative effectors were identified in WBDL genome (SAP11, SAP21, Eff64, Eff115, Eff197, Eff211 and EffSAP67), five (SAP11, SAP21, Eff64, Eff99 and Eff197) in CrP and two (SAP11, Eff64) in FBP. No homologs to Eff64, Eff197 and Eff211 in phytoplasmas of other phylogenetic groups were found. SAP11 and Eff64 homologs of ‘ Ca. P. aurantifolia’ strains shared at least 95.9% identity and were detected in the three phytoplasmas, supporting their role within the group. Five of the putative effectors (SAP11, SAP21, Eff64, Eff115, and Eff99) were transcribed from total RNA extracts of periwinkle plants infected with these phytoplasmas. Transcription profiles of selected putative effectors of CrP, FBP and WBDL indicated that SAP11 transcripts were the most abundant in the three phytoplasmas. SAP21 transcript levels were comparable to those of SAP11 for CrP and not measurable for the other phytoplasmas. Eff64 had the lowest transcription level irrespective of sampling date and phytoplasma isolate. Eff115 transcript levels were the highest in WBDL infected plants. This work reports the first sequence information for 14 putative effectors in three strains related to ‘ Ca. P. aurantifolia’, and offers novel insight into the transcription profile of five of them during infection of periwinkle. [ABSTRACT FROM AUTHOR]

• The iflavirus EVV-1 covertly infects the phytoplasma vector Euscelidius variegatus. • The highest viral loads occurred in ovaries and in first to third instar nymphs. • Vertical transmission is efficient and horizontal transmission may occur via cuticle. Virus-based biocontrol technologies represent sustainable alternatives to pesticides and insecticides. Phytoplasmas are prokaryotic plant pathogens causing severe losses to crops worldwide. Novel approaches are needed since insecticides against their insect vectors and rogueing of infected plants are the only available strategies to counteract phytoplasma diseases. A new iflavirus, named EVV-1, has been described in the leafhopper phytoplasma vector Euscelidius variegatus , raising the potential to use virus-based application strategies against phytoplasma disease. Here transmission routes of EVV-1 are characterized, and localization within the host reveals the mechanism of insect tolerance to virus infection. Both vertical and horizontal transmission of EVV-1 occur and vertical transmission was more efficient. The virus is systemic and occurs in all life-stages, with the highest loads measured in ovaries and first to third instar nymphs. The basic knowledge gained here on the biology of the virus is crucial for possible future application of iflaviruses as biocontrol agents. [ABSTRACT FROM AUTHOR]