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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Phytoplasmas are insect-borne pathogenic bacteria that cause major economic losses to several crops worldwide. The dynamic microbial community associated with insect vectors influences several ...aspects of their biology, including their vector competence for pathogens. Unraveling the diversity of the microbiome of phytoplasma insect vectors is gaining increasing importance in the quest to develop novel microbe-based pest control strategies that can minimize the use of insecticides for better environmental quality. The leafhopper
is the primary vector of the Flavescence dorée phytoplasma, a quarantine pest which is dramatically affecting the main grape-growing European countries. In this study, the RNA-Seq data, which were previously used for insect virus discovery, were further explored to assess the composition of the whole microbial community associated with insects caught in the wild in both its native (the United States) and invasive (Europe) areas. The first
assembly of the insect transcriptome was used to filter the host sequencing reads. The remaining ones were assembled into contigs and analyzed by blastx to provide the taxonomic identification of the microorganisms associated with
, including the non-bacterial components. By comparing the transcriptomic libraries, we could differentiate the stable and consistent associations from the more ephemeral and flexible ones. Two species appeared to be universal to the core microbiome of
the obligate bacterial symbiont
Sulcia muelleri and an
-allied fungus distantly related to yeast-like symbionts described from other hemipterans. Bacteria of the genus
have been identified as another dominant member of the microbiome, but only in the European specimens. Although we are yet to witness how the interplay among the microorganisms influences the vector competence of
, this unbiased
characterization of its microbiome is paramount for identifying the naturally occurring targets for new biocontrol strategies to counteract Flavescence dorée spread in Europe.
(Hemiptera:
) 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
transcriptome and injection of ATP synthase β 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
previously showed female sterility and lack of mature eggs in ovaries. Here, alteration of developing egg morphology in
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,
was used as dsRNA-receiving model-species. Different doses of two sets of dsRNA-constructs targeting distinct portions of ATP synthase β gene of both species induced silencing, lack of egg development, and female sterility in
, indicating that off-target effects must be evaluated case by case. The effectiveness of RNAi in
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.
Molecular changes associated with response to powdery mildew (PM) caused by
have been largely explored in
cultivars, but little is known on transcriptional and metabolic modifications following ...application of resistance elicitors against this disease. In this study, the whole transcriptome sequencing, and hormone and metabolite analyses were combined to dissect long-term defense mechanisms induced by molecular reprogramming events in PM-infected 'Moscato' and 'Nebbiolo' leaves treated with three resistance inducers: acibenzolar-S-methyl, potassium phosphonate, and laminarin. Although all compounds were effective in counteracting the disease, acibenzolar-S-methyl caused the most intense transcriptional modifications in both cultivars. These involved a strong down-regulation of photosynthesis and energy metabolism and changes in carbohydrate accumulation and partitioning that most likely shifted the plant growth-defense trade-off towards the establishment of disease resistance processes. It was also shown that genotype-associated metabolic signals significantly affected the cultivar defense machinery. Indeed, 'Nebbiolo' and 'Moscato' built up different defense strategies, often enhanced by the application of a specific elicitor, which resulted in either reinforcement of early defense mechanisms (e.g., epicuticular wax deposition and overexpression of pathogenesis-related genes in 'Nebbiolo'), or accumulation of endogenous hormones and antimicrobial compounds (e.g., high content of abscisic acid, jasmonic acid, and viniferin in 'Moscato').
The leafhopper
is the primary vector of Flavescence dorée phytoplasma (FDp) in European vineyards. Flavescence dorée is one of the most severely damaging diseases of
and, consequently, a major threat ...to grape and wine production in several European countries. Control measures are compulsory, but they mainly involve large-scale insecticide treatments, with detrimental impacts on the environment. One possible solution is to exploit the largely unexplored genetic diversity of viruses infecting
as highly specific and environmentally benign tools for biological control.
A metatranscriptomic approach was adopted to identify viruses that may infect individuals caught in the wild in both its native (United States) and invasive (Europe) areas. Reverse transcription PCR was used to confirm their presence in RNA pools and explore their prevalence.
We described nine new RNA viruses, including members of "Picorna-Calici", "Permutotetra", "Bunya-Arena", "Reo", "Partiti-Picobirna", "Luteo-Sobemo" and "Toti-Chryso" clades. A marked difference in the diversity and abundance of the viral species was observed between the US population and the European ones.
This work represents the first survey to assess the viral community of a phytoplasma insect vector. The possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of
could be the answer to increasing demand for a more sustainable viticulture.
To study the role of wild areas around the vineyards in the epidemiology of flavescence dorée (FD) and track the origin of new foci, two phytoplasma genetic markers,
and
, were developed for FD ...phytoplasma (FDp) characterization. The two genes and the
locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region.
, "gone-wild"
and rootstocks,
spp., and
adults were sampled within and outside the vineyards. A range of genotypes infecting the different hosts of the FDp epidemiological cycle was found. Type FD-C isolates were fairly homogeneous compared to type FD-D ones. Most of the FD-D variability was correlated with the
sequence, and a duplication of this locus was demonstrated for this strain. Coinfection with FD-C and FD-D strains was rare, suggesting possible competition between the two. Similar levels of FDp genetic variation recorded for grapevines or leafhoppers of cultivated and wild areas and co-occurrence of many FDp genotypes inside and outside the vineyards supported the idea of the importance of wild or abandoned
plants and associated
insects in the epidemiology of the disease. Genetic profiles of FDp found in
were never found in the other hosts, indicating that this species does not take part in the disease cycle in the area. Due to the robustness of analyses using
for discriminating between FD-C and FD-D strains and the high variability of
sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale.
Flavescence dorée, a threatening disease of grapevine caused by FD phytoplasma (FDp), is distributed within the most important wine-producing areas of Europe and has severe effects on both vineyard productivity and landscape management. FDp is a quarantine pest in Europe, and despite the efforts to contain the pathogen, the disease is still spreading. In this work, new genetic markers for the fine genetic characterization of FDp at local scale are presented. Our findings improve the knowledge of FDp epidemiological cycle and offer the possibility of tracking the route of the FDp infection. In particular, due to its high genetic variability, one of the newly developed markers could be sufficient to track the origin of new infection foci, either from the wild areas or from nurseries.
Flavescence dorée is an economically important vector-borne disease of grapevine in Europe caused by phytoplasmas belonging to the 16SrV ribosomal group. Expression profiles of 11 genes of the ...Flavescence dorée phytoplasma (FDp) were analysed over time following infection of natural (
Vitis vinifera
and the leafhopper vector
Scaphoideus titanus
) and experimental (
Vicia faba
and
Euscelidius variegatus
) hosts. Infected and symptomatic grapevine plants (Chardonnay) were sampled under natural field conditions in a productive vineyard in north-western Italy. Broad bean samples were assayed after the experimental inoculation with infectious
E. variegatus
. Adults of both vector species were analyzed following FDp acquisition from infected broad bean plants. The selected FDp genes were grouped according to their putative functions within different categories, namely ‘Membrane proteins’ (
imp
), ‘Regulative elements’ (
spo
VG,
rpo
D), ‘Protein metabolism, transport and secretion’ (
tld
D,
ysd
C,
fts
Y), ‘Stress response’ (
comp
83,
osm
C), ‘ABC transporters’ (
Co
ABC) and ‘Unknown’ function (
contig
12,
comp
115). All analyzed genes were expressed in the four different host species suggesting their crucial role during the FDp infection cycle. Moreover, some of them (
contig
12,
Co
ABC,
comp
83, and
imp
) might be considered essential for phytoplasma survival irrespective of the host, while
comp
115 seems to be required for insect infection. We showed that FDp is metabolically more active in insects than in plants, at least according to the pool of selected genes for this study, indicating that FDp behaves differently in the two hosts compared to other phytoplasma species/strains.
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•RNAi in the phytoplasma vector Euscelidius variegatus is long-lasting.•Silencing of ATP synthase β transcript reduces expression of the cognate protein.•dsRNA injection generates ...siRNAs mapping only on the fragment used to design dsRNAs.•Silenced insects acquire chrysanthemum yellows and Flavescence dorée phytoplasmas.•Silencing of ATP synthase β reduces the multiplication of both phytoplasmas.
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 β subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase β 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 β (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 β was silenced. The role of the insect ATP synthase β during phytoplasma infection process is discussed.
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