Silencing of ATP Synthase ß Impairs Egg Development in the Leafhopper Scaphoideus titanus, Vector of the Phytoplasma Associated with Grapevine Flavescence Dorée.

Scaphoideus titanus (Hemiptera: Cicadellidae) is the natural vector of Flavescence dorée phytoplasma, a quarantine pest of grapevine with severe impact on European viticulture. RNA interference (RNAi) machinery components are present in S. titanus transcriptome and injection of ATP synthase ß dsRNAs into adults caused gene silencing, starting three days post injection (dpi) up to 20 dpi, leading to decrease cognate protein. Silencing of this gene in the closely related leafhopper Euscelidiusvariegatus previously showed female sterility and lack of mature eggs in ovaries. Here, alteration of developing egg morphology in S. titanus ovaries as well as overexpression of hexamerin transcript (amino acid storage protein) and cathepsin L protein (lysosome proteinase) were observed in dsATP-injected females. To evaluate RNAi-specificity, E.variegatus was used as dsRNA-receiving model-species. Different doses of two sets of dsRNA-constructs targeting distinct portions of ATP synthase ß gene of both species induced silencing, lack of egg development, and female sterility in E. variegatus, indicating that off-target effects must be evaluated case by case. The effectiveness of RNAi in S. titanus provides a powerful tool for functional genomics of this non-model species and paves the way toward RNAi-based strategies to limit vector population, despite several technical and regulatory constraints that still need to be overcome to allow open field application.

Silencing of ATP synthase ß reduces phytoplasma multiplication in a leafhopper vector.

The leafhopper Euscelidius variegatus is a natural vector of the chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of the Flavescence dorée phytoplasma (FDp). Previous studies indicated a crucial role for insect ATP synthase α and ß subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase ß was obtained by injection of specific dsRNAs in E. variegatus. Here we present the long-lasting nature of such silencing, its effects on the small RNA profile, the significant reduction of the corresponding protein expression, and the impact on phytoplasma acquisition capability. The specific transcript expression was silenced at least up to 37 days post injection with an average reduction of 100 times in insects injected with dsRNAs targeting ATP synthase ß (dsATP) compared with those injected with dsRNAs targeting green fluorescent protein (dsGFP), used as negative controls. Specific silencing of this gene was also confirmed at protein level at 15 days after the injection. Total sRNA reads mapping to dsATP and dsGFP sequences in analysed libraries showed in both cases a peak of 21 nt, a length consistent with the generation of dsRNA-derived siRNAs by RNAi pathway. Reads mapped exclusively to the fragment corresponding to the injected dsATPs, probably indicating the absence of a secondary machinery for siRNA synthesis. Insects injected either with dsATP or dsGFP successfully acquired CYp and FDp during feeding on infected plants. However, the average phytoplasma amount in dsATP insects was significantly lower than that measured in dsGFP specimens, indicating a probable reduction of the pathogen multiplication when ATP synthase ß was silenced. The role of the insect ATP synthase ß during phytoplasma infection process is discussed.

Molecular memory of Flavescence dorée phytoplasma in recovering grapevines.

Flavescence dorée (FD) is a destructive phytoplasma disease of European grapevines. Spontaneous and cultivar-dependent recovery (REC) may occur in the field in FD-infected vines starting the year following the first symptoms. However, the biological underpinnings of this process are still largely unexplored. In this study, transcriptome sequencing (RNAseq), whole-genome bisulphite sequencing (WGBS) and metabolite analysis were combined to dissect molecular and metabolic changes associated to FD and REC in leaf veins collected in the field from healthy (H), FD and REC plants of the highly susceptible Vitis vinifera 'Barbera'. Genes involved in flavonoid biosynthesis, carbohydrate metabolism and stress responses were overexpressed in FD conditions, whereas transcripts linked to hormone and stilbene metabolisms were upregulated in REC vines. Accumulation patterns of abscisic acid and stilbenoid compounds analysed in the same samples confirmed the RNAseq data. In recovery conditions, we also observed the persistence of some FD-induced expression changes concerning inhibition of photosynthetic processes and stress responses. Several differentially expressed genes tied to those pathways also underwent post-transcriptional regulation by microRNAs, as outlined by merging our transcriptomic data set with a previously conducted smallRNAseq analysis. Investigations by WGBS analysis also revealed different DNA methylation marks between REC and H leaves, occurring within the promoters of genes tied to photosynthesis and secondary metabolism. The results allowed us to advance the existence of a "molecular memory" of FDp infection, involving alterations in the DNA methylation status of REC plants potentially related to transcriptional reprogramming events, in turn triggering changes in hormonal and secondary metabolite profiles.

Biological characterization of Euscelidius variegatus iflavirus 1.

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.

Differential gene expression in two grapevine cultivars recovered from "flavescence dorée".

The biological bases of recovery of two grapevine cultivars, Nebbiolo and Barbera, showing different susceptibility and recovery ability to "flavescence dorée" (FD) phytoplasma infection were investigated. The expression over one vegetative season, in FD-recovered and healthy grapevines, of 18 genes involved in defence, hydrogen peroxide and hormone production was verified at two time points. Difference (Δ) between the relative expressions of August and July were calculated for each target gene of both cultivars. The significance of differences among groups assessed by univariate and multivariate statistical methods, and sPLS-DA analyses of the Δ gene expression values, showed that control and recovered grapevines of both cultivars were clearly separated. The Barbera-specific deregulation of defence genes supports a stronger response of this variety, within a general frame of interactions among H2O2, jasmonate and ethylene metabolisms, common to both varieties. This may strengthen the hypothesis that FD-recovered Barbera grapevines modulate transcription of their genes to cope with potential damages associated to the alteration of their oxidative status. Nebbiolo variety would fit into this picture, although with a less intense response, in line with its lower degree of susceptibility and recovery incidence to FD, compared to Barbera. The results evidenced a scenario where plant response to phytoplasma infection is highly affected by climatic and edaphic conditions. Nevertheless, even after several years from the original FD infection, it was still possible to distinguish, at molecular level, control and recovered grapevines of both cultivars by analyzing their overall-season response, rather than that of a single time point.

Transcriptomic Analyses of Phytoplasmas.

Transcriptomic analyses addressed to study phytoplasma gene expression may present few difficulties due to the uncultivable nature of these intracellular, obligate pathogens. While RNA extraction from insect vectors does not imply any particular adaptation of the protocols used in most commercial kits, RNA isolation from phytoplasma-infected plants can be a challenging task, given the high levels of polyphenol contents and accumulation of sucrose and starch in the different plant tissues. Here, we describe two different transcriptomic approaches, one focused on RNA phytoplasma sequencing and the other on phytoplasma quantitative gene expression in relation to pathogen load.

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.

Dissecting interplays between Vitis vinifera L. and grapevine virus B (GVB) under field conditions.

Plant virus infections are often difficult to characterize as they result from a complex molecular and physiological interplay between a pathogen and its host. In this study, the impact of the phloem-limited grapevine virus B (GVB) on the Vitis vinifera L. wine-red cultivar Albarossa was analysed under field conditions. Trials were carried out over two growing seasons by combining agronomic, molecular, biochemical and ecophysiological approaches. The data showed that GVB did not induce macroscopic symptoms on 'Albarossa', but affected the ecophysiological performances of vines in terms of assimilation rates, particularly at the end of the season, without compromising yield and vigour. In GVB-infected plants, the accumulation of soluble carbohydrates in the leaves and transcriptional changes in sugar- and photosynthetic-related genes seemed to trigger defence responses similar to those observed in plants infected by phytoplasmas, although to a lesser extent. In addition, GVB activated berry secondary metabolism. In particular, total anthocyanins and their acetylated forms accumulated at higher levels in GVB-infected than in GVB-free berries, consistent with the expression profiles of the related biosynthetic genes. These results contribute to improve our understanding of the multifaceted grapevine-virus interaction.

Transmission of Penicillium aurantiogriseum partiti-like virus 1 to a new fungal host (Cryphonectria parasitica) confers higher resistance to salinity and reveals adaptive genomic changes.

We attempted to transfect six recently characterized virus species to protoplasts of Penicillium janczewskii and Chryphonectria parasitica. None of the recovered P. janczewskii colonies was positive for the transfected viruses, but Penicillium aurantiogriseum partiti-like virus 1 (PaPLV1) was detected in three distinct regenerated C. parasitica colonies. We screened the phenotype of the infected strains in up to 45 different conditions combining different media, salinity and temperatures: our results show that the infected strains grow slower than the virus- free in most of the tested conditions with the exception of halophilic stress in a specific nutrient combination media. We proceeded to characterize molecularly the population of distinct isolates of PaPLV1 infected C. parasitica through RNAseq: comparison to the viral population present in the original host - P. auratiogriseum - showed that two isolates accumulated non-synonymous mutations suggesting adaptation to the new host. RNAseq analyses identified a second genomic RNA segment and northern blot of RNA extracted from purified virus suspensions allowed establishing that PaPLV1 is at least bipartite in nature and that it forms isometric virions of circa 36-38 nm in diameter. In light of these new acquisitions, we discuss the taxonomic placement of PaPLV1 inside the Partitiviridae.

Structural modification of cuminaldehyde thiosemicarbazone increases inhibition specificity toward aflatoxin biosynthesis and sclerotia development in Aspergillus flavus.

Aspergillus flavus is an opportunistic mold that represents a serious threat for human and animal health due to its ability to synthesize and release, on food and feed commodities, different toxic secondary metabolites. Among them, aflatoxin B1 is one of the most dangerous since it is provided with a strong cancerogenic and mutagenic activity. Controlling fungal contamination on the different crops that may host A. flavus is considered a priority by sanitary authorities of an increasing number of countries due also to the fact that, owing to global temperature increase, the geographic areas that are expected to be prone to experience sudden A. flavus outbreaks are widening. Among the different pre- and post-harvest strategies that may be put forward in order to prevent fungal and/or mycotoxin contamination, fungicides are still considered a prominent weapon. We have here analyzed different structural modifications of a natural-derived compound (cuminaldehyde thiosemicarbazone) for their fungistatic and anti-aflatoxigenic activity. In particular, we have focused our attention on one of the compound that presented a prominent anti-aflatoxin specificity, and performed a set of physiological and molecular analyses, taking also advantage of yeast (Saccharomyces cerevisiae) cell as an experimental model.

Identification of putative effector genes and their transcripts in three strains related to 'Candidatus Phytoplasma aurantifolia'.

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.

Space-Time Point Pattern Analysis of Flavescence Dorée Epidemic in a Grapevine Field: Disease Progression and Recovery.

Analyses of space-time statistical features of a flavescence dorée (FD) epidemic in Vitis vinifera plants are presented. FD spread was surveyed from 2011 to 2015 in a vineyard of 17,500 m2 surface area in the Piemonte region, Italy; count and position of symptomatic plants were used to test the hypothesis of epidemic Complete Spatial Randomness and isotropicity in the space-time static (year-by-year) point pattern measure. Space-time dynamic (year-to-year) point pattern analyses were applied to newly infected and recovered plants to highlight statistics of FD progression and regression over time. Results highlighted point patterns ranging from disperse (at small scales) to aggregated (at large scales) over the years, suggesting that the FD epidemic is characterized by multiscale properties that may depend on infection incidence, vector population, and flight behavior. Dynamic analyses showed moderate preferential progression and regression along rows. Nearly uniform distributions of direction and negative exponential distributions of distance of newly symptomatic and recovered plants relative to existing symptomatic plants highlighted features of vector mobility similar to Brownian motion. These evidences indicate that space-time epidemics modeling should include environmental setting (e.g., vineyard geometry and topography) to capture anisotropicity as well as statistical features of vector flight behavior, plant recovery and susceptibility, and plant mortality.

Diagnosis of Phytoplasmas by Real-Time PCR Using Locked Nucleic Acid (LNA) Probes.

Phytoplasma infections are regularly reported worldwide, and concerns about their threats on agricultural production, especially in relation to global climate change, are increasing. Sensitive and reliable detection methods are important to ensure that propagation material is free of phytoplasma infection and for epidemiological studies that may provide information to limit the extent of phytoplasma diseases and to prevent large-scale crop losses. The detection method described here uses LNA chemistry in real-time PCR. It has been developed and validated for use on potatoes, and its sensitivity and specificity make it suitable for use in postentry potato quarantine and initiation of potato nuclear stocks to ensure that material is phytoplasma-free.

RNA-Seq profile of flavescence dorée phytoplasma in grapevine.

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.

Metabolic and transcript analysis of the flavonoid pathway in diseased and recovered Nebbiolo and Barbera grapevines (Vitis vinifera†L.) following infection by Flavescence dorée phytoplasma.

Flavescence dorée phytoplasma (FDp) infections seriously affect production and survival of grapevine. We analysed the changes in the flavonoid pathway occurring in two red cultivars, the highly susceptible Barbera and the less susceptible Nebbiolo, following FDp infection. A combination of metabolic and transcript analyses was used to quantify flavonoid compounds and expression of a set of genes involved in their biosynthesis. Quantification of anthocyanins, flavonols, proanthocyanidins and related biosynthetic enzymes was performed over the vegetative season, at four time points, on healthy, infected and recovered plants. A strong activation of anthocyanin accumulation was observed in infected Barbera leaves, while the response was less marked in Nebbiolo. Proanthocyanidins also accumulated mainly in infected Barbera leaves, even if basal proanthocyanidin concentration was higher in healthy and recovered Nebbiolo. Biochemical data were supported by transcript analysis: genes of the stem flavonoid pathway and of the anthocyanin and proanthocyanidin branches were expressed at a higher level in infected than in healthy plants, with a different magnitude between the two cultivars. Based on our results, we hypothesize that flavonoid accumulation is a physiological consequence of FD infection without affecting phytoplasma multiplication, although proanthocyanidin accumulation could help repel further infection by the insect vector.

A DNA origami nanorobot controlled by nucleic acid hybridization.

A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.

Genome wide sequence analysis grants unbiased definition of species boundaries in "Candidatus Phytoplasma".

The phytoplasmas are currently named using the Candidatus category, as the inability to grow them in vitro prevented (i) the performance of tests, such as DNA-DNA hybridization, that are regarded as necessary to establish species boundaries, and (ii) the deposition of type strains in culture collections. The recent accession to complete or nearly complete genome sequence information disclosed the opportunity to apply to the uncultivable phytoplasmas the same taxonomic approaches used for other bacteria. In this work, the genomes of 14 strains, belonging to the 16SrI, 16SrIII, 16SrV and 16SrX groups, including the species "Ca. P. asteris", "Ca. P. mali", "Ca. P. pyri", "Ca. P. pruni", and "Ca. P. australiense" were analyzed along with Acholeplasma laidlawi, to determine their taxonomic relatedness. Average nucleotide index (ANIm), tetranucleotide signature frequency correlation index (Tetra), and multilocus sequence analysis of 107 shared genes using both phylogenetic inference of concatenated (DNA and amino acid) sequences and consensus networks, were carried out. The results were in large agreement with the previously established 16S rDNA based classification schemes. Moreover, the taxonomic relationships within the 16SrI, 16SrIII and 16SrX groups, that represent clusters of strains whose relatedness could not be determined by 16SrDNA analysis, could be comparatively evaluated with non-subjective criteria. "Ca. P. mali" and "Ca. P. pyri" were found to meet the genome characteristics for the retention into two different, yet strictly related species; representatives of subgroups 16SrI-A and 16SrI-B were also found to meet the standards used in other bacteria to distinguish separate species; the genomes of the strains belonging to 16SrIII were found more closely related, suggesting that their subdivision into Candidatus species should be approached with caution.

Hydrogen peroxide accumulation and transcriptional changes in grapevines recovered from flavescence dorée disease.

Flavescence dorée (FD) is considered one of the most severe phytoplasma diseases affecting grapevine. The spontaneous, complete, and stable remission of the symptoms of FD (recovery) is a phenomenon that may occur in infected grapevines. The molecular bases of this phenomenon are still unclear, although some works suggest that recovery could be linked to the accumulation of hydrogen peroxide (H2O2). Several genes coding for enzymes involved in H2O2 metabolism, in the ascorbate-glutathione cycle, defense responses, and the biosynthesis of hormones were identified. The H2O2 content was biochemically determined and the expression levels of 44 genes were analyzed through quantitative real-time reverse-transcription polymerase chain reaction in healthy (H), infected by FD-associated phytoplasma (I), and 2-years-recovered (R) plants of Vitis vinifera 'Barbera'. In tissues of R plants, large amounts of H2O2 were detected, essentially linked to an upregulation of genes involved in the production of H2O2 (germin-like protein and glycolate oxidase); whereas, in I grapevines, the overexpression of some scavenging genes reduced the quantity of H2O2. The recovery state was characterized by the activation of ethylene biosynthesis and of defense genes not linked to salicylic acid (SA) signaling, such as the WRKY2 transcription factor. Conversely, I plants reacted to phytoplasma with SA-mediated signaling, even though this response does not appear to be effective against the pathogen.

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

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 < 0.05) were identified in infected compared to healthy plants, including proteins involved in photosynthesis, response to stress and the antioxidant system. Many were not differentially phosphorylated in recovered compared to healthy plants, pointing to their specific role in responding to infection, followed by a return to a steady-state phosphorylation level after remission of symptoms. Gene ontology (GO) enrichment and statistical analysis showed that the general main category "response to stimulus" was over-represented in both infected and recovered plants but, in the latter, the specific child category "response to biotic stimulus" was no longer found, suggesting a return to steady-state levels for those proteins specifically required for defence against pathogens. 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.