Increasing temperatures due to changing global climate are interfering with plant-pollinator mutualism, an interaction facilitated mainly by floral colour and scent. Gas chromatography-mass spectroscopy analyses revealed that increasing ambient temperature leads to a decrease in phenylpropanoid-based floral scent production in two Petunia × hybrida varieties, P720 and Blue Spark, acclimated at 22/16 or 28/22 °C (day/night). This decrease could be attributed to down-regulation of scent-related structural gene expression from both phenylpropanoid and shikimate pathways, and up-regulation of a negative regulator of scent production, emission of benzenoids V (EOBV). To test whether the negative effect of increased temperature on scent production can be reduced in flowers with enhanced metabolic flow in the phenylpropanoid pathway, we analysed floral volatile production by transgenic 'Blue Spark' plants overexpressing CaMV 35S-driven Arabidopsis thaliana production of anthocyanin pigments 1 (PAP1) under elevated versus standard temperature conditions. Flowers of 35S:PAP1 transgenic plants produced the same or even higher levels of volatiles when exposed to a long-term high-temperature regime. This phenotype was also evident when analysing relevant gene expression as inferred from sequencing the transcriptome of 35S:PAP1 transgenic flowers under the two temperature regimes. Thus, up-regulation of transcription might negate the adverse effects of temperature on scent production.

Leaf morphogenesis and differentiation are highly flexible processes. The development of compound leaves is characterized by an extended morphogenesis stage compared with that of simple leaves. The tomato mutant clausa (clau) possesses extremely elaborate compound leaves. Here we show that this elaboration is generated by further extension of the morphogenetic window, partly via the activity of ectopic meristems present on clau leaves. Further, we propose that CLAU might negatively affect expression of the NAM/CUC gene GOBLET (GOB), an important modulator of compound-leaf development, as GOB expression is elevated in clau mutants and reducing GOB expression suppresses the clau phenotype. Expression of GOB is also elevated in the compound leaf mutant lyrate (lyr), and the remarkable enhancement of the clau phenotype by lyr suggests that clau and lyr affect leaf development and GOB in different pathways.

Plants exhibit high capacity to regenerate in three alternative pathways: tissue repair, somatic embryogenesis and de novo organogenesis. For most plants, de novo organ initiation can be easily achieved in tissue culture by exposing explants to auxin and/or cytokinin, yet the competence to regenerate varies among species and within tissues from the same plant. In Arabidopsis, root explants incubated directly on cytokinin-rich shoot inducing medium (SIM-direct), are incapable of regenerating shoots, and a pre-incubation step on auxin-rich callus inducing medium (CIM) is required to acquire competency to regenerate on the SIM. However the mechanism underlying competency acquisition still remains elusive. Here we show that the chromomethylase 3 (cmt3) mutant which exhibits significant reduction in CHG methylation, shows high capacity to regenerate on SIM-direct and that regeneration occurs via direct organogenesis. In WT, WUSCHEL (WUS) promoter, an essential gene for shoot formation, is highly methylated, and its expression on SIM requires pre-incubation on CIM. However, in cmt3, WUS expression induced by SIM-direct. We propose that pre-incubation on CIM is required for the re-activation of cell division. Following the transfer of roots to SIM, the intensive cell division activity continues, and in the presence of cytokinin leads to a dilution in DNA methylation that allows certain genes required for shoot regeneration to respond to SIM, thereby advancing shoot formation.

Establishment of seedlings is a key factor in achievement of uniform field stands and, consequently, stable yields. Under Mediterranean conditions, soil moisture in the upper layer is limited and seedlings may be exposed to frequent dehydration events. The presence of the Reduced height (Rht)-B1b and Rht-D1b semi-dominant dwarfing alleles results in insensitivity to gibberellin (GAI) and, hence, poor emergence from deep sowing. Introduction of alternative dwarfing genes and, thereby, preservation of the gibberellin response (GAR) and coleoptile length, contributes to better emergence from deep sowing. Initially 47 wheat cultivars carrying different Rht alleles were screened for their ability to emerge from deep sowing, and then 17 of them were selected for detailed physiological characterization in the field. The modern wheat lines containing GAI alleles showed significantly lower percentages of emergence from deep sowing than the GAR lines, i.e., 52 and 74%, respectively. Differences in early developmental stages were associated with grain yield, as indicated by a reduction of 37.3% in the modern GAI cultivars. Our results demonstrate the potential of alternative dwarfing genes for improving seedling establishment and grain yields in Mediterranean-like environments.

There is a debate concerning mono- or poly-phyletic origins of the Near Eastern crops. In parallel, some authors claim that domestication was not possible within the natural range of the wild progenitors due to wild alleles flow into the nascent crops. Here we address both, the mono- or poly-phyletic origins and the domestications within or without the natural range of the progenitor, debates in order to understand the relationship between domesticated chickpea (Cicer arietinum L.) and its wild progenitor (C. reticulatum Ladizinsky) with special emphasis on its domestication centre in southeastern Turkey. A set of 103 chickpea cultivars and landraces from the major growing regions alongside wild accessions (C. reticulatum, C. echinospermum P.H Davis and C. bijugum K.H. Rech) sampled across the natural distribution range in eastern Turkey were genotyped with 194 SNPs markers. The genetic affinities between and within the studied taxa were assessed. The analysis suggests a mono-phyletic origin of the cultigen, with several wild accession as likely members of the wild stock of the cultigen. Clear separation between the wild and domesticated germplasm was apparent, with negligible level of admixture. A single C. reticulatum accession shows morphological and allelic signatures of admixture, a likely result of introgression. No evidence of geneflow from the wild into domesticated germplasm was found. The traditional farming systems of southeaster Turkey are characterized by occurrence of sympatric wild progenitor-domesticated forms of chickpea (and likewise cereals and other grain legumes). Therefore, both the authentic crop landraces and the wild populations native to the area are a unique genetic resource. Our results grant support to the notion of domestication within the natural distribution range of the wild progenitor, suggesting that the Neolithic domesticators were fully capable of selecting the desired phenotypes even when facing rare wild-domesticated introgression events.

Molecular markers allowed breeders to mendelize quantitative trait loci (QTL) providing another demonstration that quantitative traits are governed by the same principles as single qualitative genes. This research extends the QTL analysis to two and three QTL and tests our ability to mendelize an oligogenic trait. In tomato, agricultural yield is determined by the weight of the fruits harvested per unit area and the total soluble solids (% Brix)-sugars and acids. The current study explores the segregation of multiple independent yield-related QTL that were identified and mapped using introgression lines (IL) of Solanum pennellii in cultivated processing tomato (S. lycopersicum). We screened 45 different double and triple IL-QTL combinations for agricultural yield, to identify QTL pyramids that behaved in an additive manner and were suitable substrate for mendelizing an oligogenic trait. A pyramid of three independent QTL that significantly improved Brix(∗)Yield (BXY - the soluble solids output per unit area) compared to M82 was selected. In the progenies of the tri-hybrid we bred using markers a nearly isogenic 'immortalized F2.' While the common mode of QTL-QTL interactions across the 45 IL-QTLs combinations was less than additive, the three QTLs in the selected triple-stack performed in an additive manner which made it an exceptional material for breeding. This study demonstrates that using the phenotypic effect of all 27 possible QTL-alleles combinations it is possible to make reliable predictions about the genotypes that will maximize the yield.

Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG (*)) of the shikimate pathway under a constitutive promoter. The presence of AroG(*) protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG (*) transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG (*). This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG(*) cells, and the relative frequencies of the different anthocyanins changed as well.

The role of molecular mechanisms in the regulation of leaf hydraulics (K(leaf)) is still not well understood. We hypothesized that aquaporins (AQPs) in the bundle sheath may regulate K(leaf). To examine this hypothesis, AQP genes were constitutively silenced using artificial microRNAs and recovery was achieved by targeting the expression of the tobacco AQP (NtAQP1) to bundle-sheath cells in the silenced plants. Constitutively silenced PIP1 plants exhibited decreased PIP1 transcript levels and decreased K(leaf). However, once the plants were recovered with NtAQP1, their K(leaf) values were almost the same as those of WT plants. We also demonstrate the important role of ABA, acting via AQP, in that recovery and K(leaf) regulation. These results support our previously raised hypothesis concerning the role of bundle-sheath AQPs in the regulation of leaf hydraulics.

Acylsugars are insecticidal specialized metabolites produced in the glandular trichomes of plants in the Solanaceae family. In the tomato clade of the Solanum genus, acylsugars consist of aliphatic acids of different chain lengths esterified to sucrose, or less frequently to glucose. Through liquid chromatography-mass spectrometry screening of introgression lines, we previously identified a region of chromosome 8 in the Solanum pennellii LA0716 genome (IL8-1/8-1-1) that causes the cultivated tomato Solanum lycopersicum to shift from producing acylsucroses with abundant 3-methylbutanoic acid acyl chains derived from leucine metabolism to 2-methylpropanoic acid acyl chains derived from valine metabolism. We describe multiple lines of evidence implicating a trichome-expressed gene from this region as playing a role in this shift. S. lycopersicum M82 SlIPMS3 (Solyc08g014230) encodes a functional end product inhibition-insensitive version of the committing enzyme of leucine biosynthesis, isopropylmalate synthase, missing the carboxyl-terminal 160 amino acids. In contrast, the S. pennellii LA0716 IPMS3 allele found in IL8-1/8-1-1 encodes a nonfunctional truncated IPMS protein. M82 transformed with an SlIPMS3 RNA interference construct exhibited an acylsugar profile similar to that of IL8-1-1, whereas the expression of SlIPMS3 in IL8-1-1 partially restored the M82 acylsugar phenotype. These IPMS3 alleles are polymorphic in 14 S. pennellii accessions spread throughout the geographical range of occurrence for this species and are associated with acylsugars containing varying amounts of 2-methylpropanoic acid and 3-methylbutanoic acid acyl chains.

Wheat is one of the Neolithic founder crops domesticated ~10 500 years ago. Following the domestication episode, its evolution under domestication has resulted in various genetic modifications. Grain weight, embryo weight, and the interaction between those factors were examined among domesticated durum wheat and its direct progenitor, wild emmer wheat. Experimental data show that grain weight has increased over the course of wheat evolution without any parallel change in embryo weight, resulting in a significantly reduced (30%) embryo weight/grain weight ratio in domesticated wheat. The genetic factors associated with these modifications were further investigated using a population of recombinant inbred substitution lines that segregated for chromosome 2A. A cluster of loci affecting grain weight and shape was identified on the long arm of chromosome 2AL. Interestingly, a novel locus controlling embryo weight was mapped on chromosome 2AS, on which the wild emmer allele promotes heavier embryos and greater seedling vigour. To the best of our knowledge, this is the first report of a QTL for embryo weight in wheat. The results suggest a differential selection of grain and embryo weight during the evolution of domesticated wheat. It is argued that conscious selection by early farmers favouring larger grains and smaller embryos appears to have resulted in a significant change in endosperm weight/embryo weight ratio in the domesticated wheat. Exposing the genetic factors associated with endosperm and embryo size improves our understanding of the evolutionary dynamics of wheat under domestication and is likely to be useful for future wheat-breeding efforts.

Understanding how different plants prioritize carbon gain and drought vulnerability under a variable water supply is important for predicting which trees will maximize woody biomass production under different environmental conditions. Here, Populus balsamifera (BS, isohydric genotype), P. simonii (SI, previously uncharacterized stomatal behaviour), and their cross, P. balsamifera x simonii (BSxSI, anisohydric genotype) were studied to assess the physiological basis for biomass accumulation and water-use efficiency across a range of water availabilities. Under ample water, whole plant stomatal conductance (gs), transpiration (E), and growth rates were higher in anisohydric genotypes (SI and BSxSI) than in isohydric poplars (BS). Under drought, all genotypes regulated the leaf to stem water potential gradient via changes in gs, synchronizing leaf hydraulic conductance (Kleaf) and E: isohydric plants reduced Kleaf, gs, and E, whereas anisohydric genotypes maintained high Kleaf and E, which reduced both leaf and stem water potentials. Nevertheless, SI poplars reduced their plant hydraulic conductance (Kplant) during water stress and, unlike, BSxSI plants, recovered rapidly from drought. Low gs of the isohydric BS under drought reduced CO2 assimilation rates and biomass potential under moderate water stress. While anisohydric genotypes had the fastest growth under ample water and higher photosynthetic rates under increasing water stress, isohydric poplars had higher water-use efficiency. Overall, the results indicate three strategies for how closely related biomass species deal with water stress: survival-isohydric (BS), sensitive-anisohydric (BSxSI), and resilience-anisohydric (SI). Implications for woody biomass growth, water-use efficiency, and survival under variable environmental conditions are discussed.

UNLABELLED: Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted exclusively by the whitefly Bemisia tabaci in a persistent, circulative manner. Replication of TYLCV in its vector remains controversial, and thus far, the virus has been considered to be nonpropagative. Following 8 h of acquisition on TYLCV-infected tomato plants or purified virions and then transfer to non-TYLCV-host cotton plants, the amounts of virus inside whitefly adults significantly increased (>2-fold) during the first few days and then continuously decreased, as measured by the amounts of genes on both virus DNA strands. Reported alterations in insect immune and defense responses upon virus retention led us to hypothesize a role for the immune response in suppressing virus replication. After virus acquisition, stress conditions were imposed on whiteflies, and the levels of three viral gene sequences were measured over time. When whiteflies were exposed to TYLCV and treatment with two different pesticides, the virus levels continuously increased. Upon exposure to heat stress, the virus levels gradually decreased, without any initial accumulation. Switching of whiteflies between pesticide, heat stress, and control treatments caused fluctuating increases and decreases in virus levels. Fluorescence in situ hybridization analysis confirmed these results and showed virus signals inside midgut epithelial cell nuclei. Combining the pesticide and heat treatments with virus acquisition had significant effects on fecundity. Altogether, our results demonstrate for the first time that a single-stranded DNA plant virus can replicate in its hemipteran vector. IMPORTANCE: Plant viruses in agricultural crops are of great concern worldwide. Many of them are transmitted from infected to healthy plants by insects. Persistently transmitted viruses often have a complex association with their vectors; however, most are believed not to replicate within these vectors. Such replication is important, as it contributes to the virus's spread and can impact vector biology. Tomato yellow leaf curl virus (TYLCV) is a devastating begomovirus that infects tomatoes. It is persistently transmitted by the whitefly Bemisia tabaci but is believed not to replicate in the insect. To demonstrate that TYLCV is, in fact, propagative (i.e., it replicates in its insect host), we hypothesized that insect defenses play a role in suppressing virus replication. We thus exposed whitefly to pesticide and heat stress conditions to manipulate its physiology, and we showed that under such conditions, the virus is able to replicate and significantly influence the insect's fecundity.

The plant vascular system serves as a conduit for delivery of both nutrients and signaling molecules to various distantly located organs. The anucleate sieve tube system of the angiosperm phloem delivers sugars and amino acids to developing organs, and has recently been shown to contain a unique population of RNA and proteins. Grafting studies have established that a number of these macromolecules are capable of moving long distances between tissues, thus providing support for operation of a phloem-mediated inter-organ communication network. Currently, our knowledge of the roles played by such phloem-borne macromolecules is in its infancy. Here, we show that, in tomato, translocation of a phloem-mobile cyclophilin, SlCyp1, from a wild-type scion into a mutant rootstock results in restoration of vascular development and lateral root initiation. This process occurs through reactivation of auxin response pathways and reprogramming of the root transcriptome. Moreover, we show that long-distance trafficking of SlCyp1 is associated with regulation of the shoot-to-root ratio in response to changing light intensities, by modulating root growth. We conclude that long-distance trafficking of SlCyp1 acts as a rheostat to control the shoot-to-root ratio, by mediating root development to integrate photosynthesis and light intensity with requirements for access to water and mineral nutrients.

The mechanism underlying the emission of phenylpropanoid volatiles is poorly understood. Here, we reveal the involvement of PH4, a petunia MYB-R2R3 transcription factor previously studied for its role in vacuolar acidification, in floral volatile emission. We used the virus-induced gene silencing (VIGS) approach to knock down PH4 expression in petunia, measured volatile emission and internal pool sizes by GC-MS, and analyzed transcript abundances of scent-related phenylpropanoid genes in flowers. Silencing of PH4 resulted in a marked decrease in floral phenylpropanoid volatile emission, with a concurrent increase in internal pool levels. Expression of scent-related phenylpropanoid genes was not affected. To identify putative scent-related targets of PH4, we silenced PH5, a tonoplast-localized H(+) -ATPase that maintains vacuolar pH homeostasis. Suppression of PH5 did not yield the reduced-emission phenotype, suggesting that PH4 does not operate in the context of floral scent through regulation of vacuolar pH. We conclude that PH4 is a key floral regulator that integrates volatile production and emission processes and interconnects two essential floral traits - color and scent.

Gibberellin (GA) regulates plant development primarily by triggering the degradation/deactivation of the DELLA proteins. However, it remains unclear whether all GA responses are regulated by DELLAs. Tomato (Solanum lycopersicum) has a single DELLA gene named PROCERA (PRO), and its recessive pro allele exhibits constitutive GA activity but retains responsiveness to external GA. In the loss-of-function mutant pro(ΔGRAS), all examined GA developmental responses were considerably enhanced relative to pro and a defect in seed desiccation tolerance was uncovered. As pro, but not pro(ΔGRAS), elongation was promoted by GA treatment, pro may retain residual DELLA activity. In agreement with homeostatic feedback regulation of the GA biosynthetic pathway, we found that GA20oxidase1 expression was suppressed in pro(ΔGRAS) and was not affected by exogenous GA3. In contrast, expression of GA2oxidase4 was not affected by the elevated GA signaling in pro(ΔGRAS) but was strongly induced by exogenous GA3. Since a similar response was found in Arabidopsis thaliana plants with impaired activity of all five DELLA genes, we suggest that homeostatic GA responses are regulated by both DELLA-dependent and -independent pathways. Transcriptome analysis of GA-treated pro(ΔGRAS) leaves suggests that 5% of all GA-regulated genes in tomato are DELLA independent.

Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE2 (COBL2), a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE gene family in Arabidopsis (Arabidopsis thaliana), as coexpressed with other genes involved in cellulose deposition in mucilage secretory cells. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised, as demonstrated by scanning electron microscopy and in situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole-seed crystalline cellulose content compared with the wild type. These data establish that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation.

Advances in understanding plant biology, novel genetic resources, genome modification, and omics technologies generate new solutions for food security and novel biomaterials production under changing environmental conditions. New gene and germplasm candidates that are anticipated to lead to improved crop yields and other plant traits under stress have to pass long development phases based on trial and error using large-scale field evaluation. Therefore, quantitative, objective, and automated screening methods combined with decision-making algorithms are likely to have many advantages, enabling rapid screening of the most promising crop lines at an early stage followed by final mandatory field experiments. The combination of novel molecular tools, screening technologies, and economic evaluation should become the main goal of the plant biotechnological revolution in agriculture.

BACKGROUND: Drought is the major environmental stress threatening crop-plant productivity worldwide. Identification of new genes and metabolic pathways involved in plant adaptation to progressive drought stress at the reproductive stage is of great interest for agricultural research. RESULTS: We developed a novel Cross-Species meta-Analysis of progressive Drought stress at the reproductive stage (CSA:Drought) to identify key drought adaptive genes and mechanisms and to test their evolutionary conservation. Empirically defined filtering criteria were used to facilitate a robust integration of 17 deposited microarray experiments (148 arrays) of Arabidopsis, rice, wheat and barley. By prioritizing consistency over intensity, our approach was able to identify 225 differentially expressed genes shared across studies and taxa. Gene ontology enrichment and pathway analyses classified the shared genes into functional categories involved predominantly in metabolic processes (e.g. amino acid and carbohydrate metabolism), regulatory function (e.g. protein degradation and transcription) and response to stimulus. We further investigated drought related cis-acting elements in the shared gene promoters, and the evolutionary conservation of shared genes. The universal nature of the identified drought-adaptive genes was further validated in a fifth species, Brachypodium distachyon that was not included in the meta-analysis. qPCR analysis of 27, randomly selected, shared orthologs showed similar expression pattern as was found by the CSA:Drought.In accordance, morpho-physiological characterization of progressive drought stress, in B. distachyon, highlighted the key role of osmotic adjustment as evolutionary conserved drought-adaptive mechanism. CONCLUSIONS: Our CSA:Drought strategy highlights major drought-adaptive genes and metabolic pathways that were only partially, if at all, reported in the original studies included in the meta-analysis. These genes include a group of unclassified genes that could be involved in novel drought adaptation mechanisms. The identified shared genes can provide a useful resource for subsequent research to better understand the mechanisms involved in drought adaptation across-species and can serve as a potential set of molecular biomarkers for progressive drought experiments.

During desiccation, homoiochlorophyllous resurrection plants retain most of their photosynthetic apparatus, allowing them to resume photosynthetic activity quickly upon water availability. These plants rely on various mechanisms to prevent the formation of reactive oxygen species and/or protect their tissues from the damage they inflict. In this work, we addressed the issue of how homoiochlorophyllous resurrection plants deal with the problem of excessive excitation/electron pressures during dehydration using Craterostigma pumilum as a model plant. To investigate the alterations in the supramolecular organization of photosynthetic protein complexes, we examined cryoimmobilized, freeze-fractured leaf tissues using (cryo)scanning electron microscopy. These examinations revealed rearrangements of photosystem II (PSII) complexes, including a lowered density during moderate dehydration, consistent with a lower level of PSII proteins, as shown by biochemical analyses. The latter also showed a considerable decrease in the level of cytochrome f early during dehydration, suggesting that initial regulation of the inhibition of electron transport is achieved via the cytochrome b6f complex. Upon further dehydration, PSII complexes are observed to arrange into rows and semicrystalline arrays, which correlates with the significant accumulation of sucrose and the appearance of inverted hexagonal lipid phases within the membranes. As opposed to PSII and cytochrome f, the light-harvesting antenna complexes of PSII remain stable throughout the course of dehydration. Altogether, these results, along with photosynthetic activity measurements, suggest that the protection of retained photosynthetic components is achieved, at least in part, via the structural rearrangements of PSII and (likely) light-harvesting antenna complexes into a photochemically quenched state.

The spread of Tomato yellow leaf curl virus (TYLCV) was accompanied by the formation of coat protein (CP) aggregates of increasing size in the cytoplasm and nucleus of infected tomato (Solanum lycopersicum) cells. In order to better understand the TYLCV-host interaction, we investigated the properties and the subcellular accumulation pattern of the non-structural viral protein V2. CP and V2 are the only sense-oriented genes on the virus circular single-stranded DNA genome. Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP. V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein. The formation and the 26S proteasome-mediated degradation of V2 aggregates were dependent on the integrity of the actin and microtubule cytoskeleton. We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.