Distribution and prevalence of viral genomes in Italian populations of the invasive brown marmorated stink bug Halyomorpha halys.

Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species that causes significant agricultural losses, especially to orchard fruits, vegetables, herbaceous and ornamental plants. It is also a nuisance pest that seeks shelter in indoor spaces during the winter months. Harnessing the H. halys virome can result in new environmentally sustainable approaches to contain its populations and its relatated agricultural damages. In this study, RNA-Seq data were used to explore the virome associated to ten field populations collected in the Lombardy region in Northern Italy. We identified six complete viral genomes, three of which were previously unknown, belonging to the orders Reovirales, Articulavirales, Ghabrivirales, Durnavirales, and Picornavirales. The prevalence of the six viruses was evaluated by Real-time reverse transcription-quantitative PCR on eighty individuals. Halyomorpha halys ifla-like virus 2 turned out to be the most geographically widespread virus, as it was found in more than 50% of the analyzed insects and in nine out of the ten sampling locations. Moreover, in some individuals, this iflavirus was found in association with each of the other viruses in various combinations that involved up to four viruses. Further studies on such virus-virus interactions and their relationships with the insect host may open the possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of H. halys.

Metatranscriptomic Assessment of the Microbial Community Associated With the Flavescence dorée Phytoplasma Insect Vector Scaphoideus titanus.

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 Scaphoideus titanus 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 de novo 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 S. titanus, 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 S. titanus: the obligate bacterial symbiont Candidatus Sulcia muelleri and an Ophiocordyceps-allied fungus distantly related to yeast-like symbionts described from other hemipterans. Bacteria of the genus Cardinium 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 S. titanus, this unbiased in silico characterization of its microbiome is paramount for identifying the naturally occurring targets for new biocontrol strategies to counteract Flavescence dorée spread in Europe.

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.

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.

Oleosin Cor a 15 is a novel allergen for Italian hazelnut allergic children.

BACKGROUND: Hazelnut allergy, which is characterized by symptoms that range from mild to severe, is one of the most common allergies in children throughout Europe, and an accurate diagnosis of this allergy is therefore essential. However, lipophilic allergens, such as oleosins, are generally underrepresented in diagnostic tests. We therefore sought to characterize the IgE reactivity of raw and roasted hazelnut oleosins, using the sera of hazelnut-allergic pediatric patients. METHODS: Raw and roasted hazelnut oil body-associated proteins were analyzed by means of 1D and 2D electrophoresis and MS. Oleosin IgE reactivity was assessed by immunoblotting with the sera of 27 children who have confirmed hazelnut allergies and from 10 tolerant subjects. A molecular characterization of the oleosins was performed by interrogating the C. avellana cv. Jefferson and cv. TGL genomes, and through expression and purification of the recombinant new allergen. RESULTS: A proteomic and genomic investigation allowed two new oleosins to be identified, in addition to Cor a 12 and Cor a 13, in hazelnut oil bodies. One of the new oleosins was registered as a new allergen, according to the WHO/IUIS Allergen Nomenclature Subcommittee criteria, and termed Cor a 15. Cor a 15 was the most frequently immunorecognized oleosin in our cohort. Oleosins resulted to be the only immunorecognized allergens in a subgroup of allergic patients who showed low ImmunoCAP assay IgE values and positive OFC and PbP. Hazelnut roasting resulted in an increase in oleosin immunoreactivity. CONCLUSION: A novel hazelnut oleosin, named Cor a 15, has been discovered. Cor a 15 could play a role in eliciting an allergic reaction in a subgroup of pediatric patients that exclusively immunorecognize oleosins. The high prevalence of hazelnut oleosin sensitization here reported further confirms the need to include oleosins in routine diagnostic procedures.

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.

The Molecular Priming of Defense Responses is Differently Regulated in Grapevine Genotypes Following Elicitor Application against Powdery Mildew.

Molecular changes associated with response to powdery mildew (PM) caused by Erysiphe necator have been largely explored in Vitis vinifera 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').

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.

New Viral Sequences Identified in the Flavescence Dorée Phytoplasma Vector Scaphoideus titanus.

The leafhopper Scaphoideus titanus is the primary vector of Flavescence dorée phytoplasma (FDp) in European vineyards. Flavescence dorée is one of the most severely damaging diseases of Vitis vinifera 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 S. titanus as highly specific and environmentally benign tools for biological control. (2)Methods: 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. (3)Results: 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. (4)Conclusions: 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 S. titanus could be the answer to increasing demand for a more sustainable viticulture.

Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale.

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, dnaK and malG, were developed for FD phytoplasma (FDp) characterization. The two genes and the vmpA locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region. Vitis vinifera, "gone-wild" V. vinifera and rootstocks, Clematis spp., and Scaphoideus titanus 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 malG 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 Vitis plants and associated S. titanus insects in the epidemiology of the disease. Genetic profiles of FDp found in Clematis 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 dnaK for discriminating between FD-C and FD-D strains and the high variability of malG sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale.IMPORTANCE 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.

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.

RNA interference of muscle actin and ATP synthase beta increases mortality of the phytoplasma vector Euscelidius variegatus.

BACKGROUND: RNA interference (RNAi) techniques have emerged as powerful tools to develop novel management strategies for the control of insect pests. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma and a laboratory vector of Flavescence dorée phytoplasma. Phytoplasmas are insect-borne bacterial plant pathogens that cause economically relevant crop losses worldwide. RESULTS: In this study, we demonstrated that microinjection of muscle actin and ATP synthase ß double-stranded (ds)RNAs into adult insects caused an exponential reduction in the expression of both genes, which began within 72 h of dsRNA administration and lasted for 14 days, leading to almost complete silencing of the target genes. Such silencing effects on muscle actin expression appeared to be both time- and dose-dependent. Our results also showed that the knockdown of both genes caused a significant decrease in survival rates in comparison with green fluorescent protein (GFP) dsRNA-injected control insects. CONCLUSION: The effectiveness of RNAi-based gene silencing in E. variegatus guarantees the availability of a powerful reverse genetic tool for the functional annotation of its genes and the identification of those potentially involved in the interaction with phytoplasmas. In addition, this study demonstrated that muscle actin and ATP synthase ß may represent candidate genes for RNAi-based control of E. variegatus. © 2018 Society of Chemical Industry.

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.

Two Phytoplasmas Elicit Different Responses in the Insect Vector Euscelidius variegatus Kirschbaum.

Phytoplasmas are plant-pathogenic bacteria transmitted by hemipteran insects. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of flavescence dorée phytoplasma (FDp). The two phytoplasmas induce different effects on this species: CYp slightly improves whereas FDp negatively affects insect fitness. To investigate the molecular bases of these different responses, transcriptome sequencing (RNA-seq) analysis of E. variegatus infected with either CYp or FDp was performed. The sequencing provided the first de novo transcriptome assembly for a phytoplasma vector and a starting point for further analyses on differentially regulated genes, mainly related to immune system and energy metabolism. Insect phenoloxidase activity, immunocompetence, and body pigmentation were measured to investigate the immune response, while respiration and movement rates were quantified to confirm the effects on energy metabolism. The activation of the insect immune response upon infection with FDp, which is not naturally transmitted by E. variegatus, confirmed that this bacterium is mostly perceived as a potential pathogen. Conversely, the acquisition of CYp, which is naturally transmitted by E. variegatus, seems to increase the insect fitness by inducing a prompt response to stress. This long-term relationship is likely to improve survival and dispersal of the infected insect, thus enhancing the opportunity of phytoplasma transmission.

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.

Genome sequence, prevalence and quantification of the first iflavirus identified in a phytoplasma insect vector.

The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CY) and an efficient vector of flavescence dorée phytoplasma (FD) under laboratory conditions. During a transcriptome sequencing (RNA-seq) project aimed at investigating the interactions between the insect and the two phytoplasmas, a 10,616-nucleotide-long contig with high sequence similarity to known picorna-like viruses was identified among the assembled insect transcripts. The discovery came totally unexpected, because insects from the laboratory colony did not show any evident symptom that could be related to the presence of a virus. The amino acid sequence, the shape and size of viral particles, and the results of phylogenetic analysis suggest that this virus, named Euscelidius variegatus virus 1 (EVV-1), can be considered a new member of a new species in the genus Iflavirus. EVV-1 was detected in all of the tested insects from the laboratory colony used for RNA-seq, both in phytoplasma-exposed and in non-exposed insects, but the viral load measured in FD-exposed samples was significantly lower than that in non-exposed insects. This result suggests the possible existence of an intriguing cross-talk among insects, endogenous bacteria, and viruses. The identification of two other E. variegatus laboratory colonies that were free of EVV-1 could represent the key to addressing some basic virological issues, e.g., viral replication and transmission mechanisms, and offer the opportunity to use infectious clones to express heterologous genes in the leafhopper and manipulate the expression of endogenous genes by promoting virus-induced gene silencing.

RiPEIP1, a gene from the arbuscular mycorrhizal fungus Rhizophagus irregularis, is preferentially expressed in planta and may be involved in root colonization.

Transcriptomics and genomics data recently obtained from the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis have offered new opportunities to decipher the contribution of the fungal partner to the establishment of the symbiotic association. The large number of genes which do not show similarity to known proteins witnesses the uniqueness of this group of plant-associated fungi. In this work, we characterize a gene that was called RiPEIP1 (Preferentially Expressed In Planta). Its expression is strongly induced in the intraradical phase, including arbuscules, and follows the expression profile of the Medicago truncatula phosphate transporter MtPT4, a molecular marker of a functional symbiosis. Indeed, mtpt4 mutant plants, which exhibit low mycorrhizal colonization and an accelerated arbuscule turnover, also show a reduced RiPEIP1 mRNA abundance. To further characterize RiPEIP1, in the absence of genetic transformation protocols for AM fungi, we took advantage of two different fungal heterologous systems. When expressed as a GFP fusion in yeast cells, RiPEIP1 localizes in the endomembrane system, in particular to the endoplasmic reticulum, which is consistent with the in silico prediction of four transmembrane domains. We then generated RiPEIP1-expressing strains of the fungus Oidiodendron maius, ericoid endomycorrhizal fungus for which transformation protocols are available. Roots of Vaccinium myrtillus colonized by RiPEIP1-expressing transgenic strains showed a higher mycorrhization level compared to roots colonized by the O. maius wild-type strain, suggesting that RiPEIP1 may regulate the root colonization process.

Complete Genome Sequences of the Obligate Symbionts "Candidatus Sulcia muelleri" and "Ca. Nasuia deltocephalinicola" from the Pestiferous Leafhopper Macrosteles quadripunctulatus (Hemiptera: Cicadellidae).

Two bacterial symbionts of the European pest leafhopper, Macrosteles quadripunctulatus (Hemiptera: Cicadellidae), were fully sequenced. "Candidatus Sulcia muelleri" and "Ca. Nasuia deltocephalinicola" represent two of the smallest known bacterial genomes at 190 kb and 112 kb, respectively. Genome sequences are nearly identical to strains reported from the closely related host species, M. quadrilineatus.

The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense.

The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV6710, with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CPwt, in situ immunolocalization experiments indicated that CP6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.