A Scanning Electron Micrograph-based Resource for Identification of Loci Involved in Epidermal Development in Tomato: Elucidation of a New Function for the Mixta-like Transcription Factor in Leaves.

The aerial epidermis of plants plays a major role in environmental interactions, yet the development of the cellular components of the aerial epidermis-trichomes, stomata, and pavement cells-is still not fully understood. We have performed a detailed screen of the leaf epidermis in two generations of the well-established Solanum lycopersicum cv M82 × Solanum pennellii ac. LA716 introgression line (IL) population using a combination of scanning electron microscopy (SEM) techniques. Quantification of trichome and stomatal densities in the ILs revealed four genomic regions with a consistently low trichome density. This study also found ILs with abnormal proportions of different trichome types and aberrant trichome morphologies. This work has led to the identification of new, unexplored genomic regions with roles in trichome formation in tomato. This study investigated one interval in IL2-6 in more detail and identified a new function for the transcription factor SlMixta-like in determining trichome patterning in leaves. This illustrates how these SEM images, publicly available to the research community, provide an important dataset for further studies on epidermal development in tomato and other species of the Solanaceae family.

Distinct Differentiation Characteristics of Endothelium Determine Its Ability to Form Pseudo-Embryos in Tomato Ovules.

The endothelium is an additional cell layer, differentiating from the inner epidermis of the ovule integument. In tomato (Solanum lycopersicum L.), after fertilization, the endothelium separates from integument and becomes an independent tissue developing next to the growing embryo sac. In the absence of fertilization, the endothelium may proliferate and form pseudo-embryo. However, the course of the reorganization of endothelium into pseudo-embryo in tomato ovules is poorly understood. We aimed to investigate specific features of endothelium differentiation and the role of the endothelium in the development of fertilized and unfertilized tomato ovules. The ovules of tomato plants ("YaLF" line), produced by vegetative growth plants of transgenic tomato line expressing the ac gene, encoding chitin-binding protein from Amaranthus caudatus L., were investigated using light and transmission electron microscopy. We showed that in the fertilized ovule of normally developing fruit and in the unfertilized ovule of parthenocarpic fruit, separation of the endothelium from integument occurs via programmed death of cells of the integumental parenchyma, adjacent to the endothelium. Endothelial cells in normally developing ovules change their structural and functional specialization from meristematic to secretory and back to meristematic, and proliferate until seeds fully mature. The secretory activity of the endothelium is necessary for the lysis of dying cells of the integument and provides the space for the growth of the new sporophyte. However, in ovules of parthenocarpic fruits, pseudo-embryo cells do not change their structural and functional organization and remain meristematic, no zone of lysis is formed, and pseudo-embryo cells undergo programmed cell death. Our data shows the key role of the endothelium as a protective and secretory tissue, needed for the normal development of ovules.

Quantifying Plant Growth and Cell Proliferation with MorphoGraphX.

Confocal microscopy is widely used to live-image plant tissue. Cell outlines can be visualized using fluorescent probes that mark the cell wall or plasma membrane, enabling the confocal microscope to be used as a 3D scanner with submicron precision. After imaging, the data needs to be analyzed by specialized software to quantify the features of interest, such as cell size and shape, growth rates and anisotropy, and gene expression. Here we present a protocol for the 3D image processing software MorphoGraphX ( www.MorphoGraphX.org ) using time-lapse images of an Arabidopsis thaliana sepal and the shoot apex of tomato.

ABA signaling rather than ABA metabolism is involved in trehalose-induced drought tolerance in tomato plants.

MAIN CONCLUSION: Trehalose increased drought tolerance of tomato plants, accompanied by reduced water loss and closed stomata, which was associated with the upregulated ABA signaling-related genes expression, but not in ABA accumulation. Drought is one of the principal abiotic stresses that negatively influence the growth of plant and yield. Trehalose has great agronomic potential to improve the stress tolerance of plants. However, little information is available on the role of ABA and its signaling components in trehalose-induced drought tolerance. The aim of this study is to elucidate the potential mechanism by which trehalose regulates ABA in response to drought stress. In this study, 6-week-old tomato (Solanum lycopersicum cv. Ailsa Craig) plants were treated with 0 or 15.0 mM trehalose solution. Results showed that trehalose treatment significantly enhanced drought tolerance of tomato plants, accompanied by encouraged stomatal closure and protected chloroplast ultrastructure. Compared with controls, trehalose-treated plants showed lower hydrogen peroxide content and higher antioxidant enzymes activities, which contributed to alleviate oxidative damage caused by drought. Moreover, trehalose treatment decreased ABA content, which was followed by the downregulation of ABA biosynthesis genes expression and the upregulation of ABA catabolism genes expression. In contrast, exogenous trehalose upregulated transcript levels of ABA signaling-related genes, including SlPYL1/3/4/5/6/7/9, SlSnRK2.3/4, SlAREB1/2, and SlDREB1. These results suggested that trehalose treatment enhanced drought tolerance of tomato plants, and it's ABA signaling rather than ABA metabolism that was involved in trehalose-induced drought tolerance in tomato plants. These findings provide evidence for the physiological role of trehalose and bring about a new understanding of the possible relationship between trehalose and ABA.

Nitrogen-improved photosynthesis quantum yield is driven by increased thylakoid density, enhancing green light absorption.

A nitrogen supply is necessary for all plants. The multifaceted reasons why this nutrient stimulates plant dry weight accumulation are assessed herein. We compared tomato plants grown in full sunlight and in low light environments under four N doses and evaluated plant growth, photosynthetic and calorimetric parameters, leaf anatomy, chloroplast transmission electron microscopy (TEM) and a high resolution profile of optical leaf properties. Increases in N supplies allow tomato plants to grow faster in low light environments (91.5% shading), displaying a robust light harvesting machinery and, consequently, improved light harvesting efficiency. Ultrastructurally, high N doses were associated to a high number of grana per chloroplast and greater thylakoid stacking, as well as high electrodensity by TEM. Robust photosynthetic machinery improves green light absorption, but not blue or red. In addition, low construction and dark respiration costs were related to improved total dry weight accumulation in shade conditions. By applying multivariate analyses, we conclude that improved green light absorbance, improved quantum yield and greater palisade parenchyma cell area are the primary components that drive increased plant growth under natural light-limited photosynthesis.

Comparative study of the bioaccessibility of the colorless carotenoids phytoene and phytofluene in powders and pulps of tomato: microstructural analysis and effect of addition of sunflower oil.

The objective was to assess the potential bioavailability of phytoene (PT) and phytofluene (PTF) from tomato powders used as raw materials for supplements as compared to the pulp of a common tomato and a cherry tomato. PT and PTF are attracting much interest nowadays as they can provide health and cosmetic benefits. PT and PTF levels in the more concentrated powder were up to 1000 times higher than in the tomatoes. The bioaccessibility from the powders was lower as compared to the tomato fruits and increased markedly when sunflower oil was added. However, the best source of potentially absorbable PT and PTF (0.5 and 2 mg g-1 respectively) was by far the powder with higher levels of them. This result could be due to the higher carotenoid concentration in the powder, the reduction of the particle sizes, and the rupture of cell structures compared to the pulps.

Abnormal development of floral meristem triggers defective morphogenesis of generative system in transgenic tomatoes.

Parthenocarpy and fruit malformations are common among independent transgenic tomato lines, expressing genes encoding different pathogenesis-related (PR) protein and antimicrobal peptides. Abnormal phenotype developed independently of the expression and type of target genes, but distinctive features during flower and fruit development were detected in each transgenic line. We analyzed the morphology, anatomy, and cytoembryology of abnormal flowers and fruits from these transgenic tomato lines and compared them with flowers and fruits of wild tomatoes, line YaLF used for transformation, and transgenic plants with normal phenotype. We confirmed that the main cause of abnormal flower and fruit development was the alterations of determinate growth of generative meristem. These alterations triggered different types of anomalous growth, affecting the number of growing ectopic shoots and formation of new flowers. Investigation of the ovule ontogenesis did not show anomalies in embryo sac development, but fertilization did not occur and embryo sac degenerated. Nevertheless, the ovule continued to differentiate due to proliferation of endothelium cells. The latter substituted embryo sac and formed pseudoembryonic tissue. This process imitated embryogenesis and stimulated ovary growth, leading to the development of parthenocarpic fruit. We demonstrated that failed fertilization occurred due to defective male gametophyte formation, which was manifested in blocked division of the nucleus in the microspore and arrest of vegetative and generative cell formation. Maturing pollen grains were overgrown microspores, not competent for fertilization but capable to induce proliferation of endothelium and development of parthenocarpic ovary. Thus, our study provided new data on the structural transformations of reproductive organs during development of parthenocarpic fruits in transgenic tomato.

Exogenous Melatonin Mitigates Acid Rain Stress to Tomato Plants through Modulation of Leaf Ultrastructure, Photosynthesis and Antioxidant Potential.

Acid rain (AR) is a serious global environmental issue causing physio-morphological changes in plants. Melatonin, as an indoleamine molecule, has been known to mediate many physiological processes in plants under different kinds of environmental stress. However, the role of melatonin in acid rain stress tolerance remains inexpressible. This study investigated the possible role of melatonin on different physiological responses involving reactive oxygen species (ROS) metabolism in tomato plants under simulated acid rain (SAR) stress. SAR stress caused the inhibition of growth, damaged the grana lamella of the chloroplast, photosynthesis, and increased accumulation of ROS and lipid peroxidation in tomato plants. To cope the detrimental effect of SAR stress, plants under SAR condition had increased both enzymatic and nonenzymatic antioxidant substances compared with control plants. But such an increase in the antioxidant activities were incapable of inhibiting the destructive effect of SAR stress. Meanwhile, melatonin treatment increased SAR-stress tolerance by repairing the grana lamella of the chloroplast, improving photosynthesis and antioxidant activities compared with those in SAR-stressed plants. However, these possible effects of melatonin are dependent on concentration. Moreover, our study suggests that 100-µM melatonin treatment improved the SAR-stress tolerance by increasing photosynthesis and ROS scavenging antioxidant activities in tomato plants.

Studying microstructure and microstructural changes in plant tissues by advanced diffusion magnetic resonance imaging techniques.

As sessile organisms, plants must respond to the environment by adjusting their growth and development. Most of the plant body is formed post-embryonically by continuous activity of apical and lateral meristems. The development of lateral adventitious roots is a complex process, and therefore the development of methods that can visualize, non-invasively, the plant microstructure and organ initiation that occur during growth and development is of paramount importance. In this study, relaxation-based and advanced diffusion magnetic resonance imaging (MRI) methods including diffusion tensor (DTI), q-space diffusion imaging (QSI), and double-pulsed-field-gradient (d-PFG) MRI, at 14.1 T, were used to characterize the hypocotyl microstructure and the microstructural changes that occurred during the development of lateral adventitious roots in tomato. Better contrast was observed in relaxation-based MRI using higher in-plane resolution but this also resulted in a significant reduction in the signal-to-noise ratio of the T2-weighted MR images. Diffusion MRI revealed that water diffusion is highly anisotropic in the vascular cylinder. QSI and d-PGSE MRI showed that in the vascular cylinder some of the cells have sizes in the range of 6-10 µm. The MR images captured cell reorganization during adventitious root formation in the periphery of the primary vascular bundles, adjacent to the xylem pole that broke through the cortex and epidermis layers. This study demonstrates that MRI and diffusion MRI methods allow the non-invasive study of microstructural features of plants, and enable microstructural changes associated with adventitious root formation to be followed.

The parthenocarpic hydra mutant reveals a new function for a SPOROCYTELESS-like gene in the control of fruit set in tomato.

Fruit set is an essential process to ensure successful sexual plant reproduction. The development of the flower into a fruit is actively repressed in the absence of pollination. However, some cultivars from a few species are able to develop seedless fruits overcoming the standard restriction of unpollinated ovaries to growth. We report here the identification of the tomato hydra mutant that produces seedless (parthenocarpic) fruits. Seedless fruit production in hydra plants is linked to the absence of both male and female sporocyte development. The HYDRA gene is therefore essential for the initiation of sporogenesis in tomato. Using positional cloning, virus-induced gene silencing and expression analysis experiments, we identified the HYDRA gene and demonstrated that it encodes the tomato orthologue of SPOROCYTELESS/NOZZLE (SPL/NZZ) of Arabidopsis. We found that the precocious growth of the ovary is associated with changes in the expression of genes involved in gibberellin (GA) metabolism. Our results support the conservation of the function of SPL-like genes in the control of sporogenesis in plants. Moreover, this study uncovers a new function for the tomato SlSPL/HYDRA gene in the control of fruit initiation.

Ralstonia solanacearum Differentially Colonizes Roots of Resistant and Susceptible Tomato Plants.

Ralstonia solanacearum is the causal agent of bacterial wilt and infects over 200 plant species in 50 families. The soilborne bacterium is lethal to many solanaceous species, including tomato. Although resistant plants can carry high pathogen loads (between 105 and 108 CFU/g fresh weight), the disease is best controlled by the use of resistant cultivars, particularly resistant rootstocks. How these plants have latent infections yet maintain resistance is not clear. R. solanacearum first infects the plant through the root system and, thus, early root colonization events may be key to understanding resistance. We hypothesized that the distribution and timing of bacterial invasion differed in roots of resistant and susceptible tomato cultivars. Here, we use a combination of scanning electron microscopy and light microscopy to investigate R. solanacearum colonization in roots of soil-grown resistant and susceptible tomato cultivars at multiple time points after inoculation. Our results show that colonization of the root vascular cylinder is delayed in resistant 'Hawaii7996' and that, once bacteria enter the root vascular tissues, colonization in the vasculature is spatially restricted. Our data suggest that resistance is due, in part, to the ability of the resistant cultivar to restrict bacterial root colonization in space and time.

High-Temperature-Induced Defects in Tomato (Solanum lycopersicum) Anther and Pollen Development Are Associated with Reduced Expression of B-Class Floral Patterning Genes.

Sexual reproduction is a critical process in the life-cycle of plants and very sensitive to environmental perturbations. To better understand the effect of high temperature on plant reproduction, we cultivated tomato (Solanum lycopersicum) plants in continuous mild heat. Under this condition we observed a simultaneous reduction in pollen viability and appearance of anthers with pistil-like structures, while in a more thermotolerant genotype, both traits were improved. Ectopic expression of two pistil-specific genes, TRANSMITTING TISSUE SPECIFIC and TOMATO AGAMOUS LIKE11, in the anthers confirmed that the anthers had gained partial pistil identity. Concomitantly, expression of the B-class genes TOMATO APETALA3, TOMATO MADS BOX GENE6 (TM6) and LePISTILLATA was reduced in anthers under continuous mild heat. Plants in which TM6 was partially silenced reacted hypersensitively to temperature elevation with regard to the frequency of pistilloid anthers, pollen viability and pollen quantity. Taken together, these results suggest that high-temperature-induced down-regulation of tomato B-class genes contributes to anther deformations and reduced male fertility. Improving our understanding of how temperature perturbs the molecular mechanisms of anther and pollen development will be important in the view of maintaining agricultural output under current climate changes.

Tomato HAIRY MERISTEM genes are involved in meristem maintenance and compound leaf morphogenesis.

The HAIRY MERISTEM (HAM) genes function in meristem maintenance but play minor roles in the morphogenesis of a simple leaf that is determinate. Here, we functionally analyzed HAM genes in tomato and uncovered their involvement in compound leaf morphogenesis. Tomato encodes three HAM homologs, of which SlHAM and SlHAM2 (SlHAMs) are guided for cleavage by microRNA171 and are abundant in the shoot and floral meristems as well as in the compound leaf primordia. We found that SlHAMs silencing led to overproliferation of cells in the periphery of the meristems where SlHAM is localized. As in meristems, leaf-specific silencing of SlHAMs provoked overproliferation of meristematic cells in the organogenic compound leaf rachis. We further demonstrate that the meristematic cell overproliferation in both meristems and leaves was in part due to the misexpression of the stem cell regulator WUSCHEL, previously shown to be induced by cytokinin. Strikingly, reduction of cytokinin levels in SlHAMs-silenced leaves completely suppressed the overproliferation phenotype, suggesting a regulatory link between SlHAMs and cytokinin, a key hormone found to promote indeterminacy in meristems and leaves. Taken together, our data provide evidence that in addition to their conserved function in meristem maintenance, SlHAMs are also required for the proper morphogenesis of the compound leaf.

Interactions between 2-Cys peroxiredoxins and ascorbate in autophagosome formation during the heat stress response in Solanum lycopersicum.

2-Cys peroxiredoxins (2-CPs) function in the removal of hydrogen peroxide and lipid peroxides but their precise roles in the induction of autophagy have not been characterized. Here we show that heat stress, which is known to induce oxidative stress, leads to the simultaneous accumulation of transcripts encoding 2-CPs and autophagy proteins, as well as autophagosomes, in tomato (Solanum lycopersicum) plants. Virus-induced gene silencing of the tomato peroxiredoxin genes 2-CP1, 2-CP2, and 2-CP1/2 resulted in an increased sensitivity of tomato plants to heat stress. Silencing 2-CP2 or 2-CP1/2 increased the levels of transcripts associated with ascorbate biosynthesis but had no effect on the glutathione pool in the absence of stress. However, the heat-induced accumulation of transcripts associated with the water-water cycle was compromised by the loss of 2-CP1/2 functions. The transcript levels of autophagy-related genes ATG5 and ATG7 were higher in plants with impaired 2-CP1/2 functions, and the formation of autophagosomes increased, together with an accumulation of oxidized and insoluble proteins. Silencing of ATG5 or ATG7 increased the levels of 2-CP transcripts and protein but decreased heat stress tolerance. These results demonstrate that 2-CPs fulfil a pivotal role in heat stress tolerance in tomato, via interactions with ascorbate-dependent pathways and autophagy.

OHMS**: Phytoplasmas dictate changes in sieve-element ultrastructure to accommodate their requirements for nutrition, multiplication and translocation.

Phytoplasmas are among the most recently discovered plant pathogenic microorganisms so, many traits of the interactions with host plants and insect vectors are still unclear and need to be investigated. At now, it is impossible to determine the precise sequences leading to the onset of the relationship with the plant host cell. It is still unclear how phytoplasmas, located in the phloem sieve elements, exploit host cell to draw nutrition for their metabolism, growth and multiplication. In this work, basing on microscopical observations, we give insight about the structural interactions established by phytoplasmas and the sieve element plasma membrane, cytoskeleton, sieve endoplasmic reticulum, speculating about a possible functional role.

Morphological and transcriptional responses of Lycopersicon esculentum to hexavalent chromium in agricultural soil.

The carcinogenic, teratogenic, and mutagenic effects of hexavalent chromium (Cr[VI]) on living organisms through the food chain raise the immediate need to assess the potential toxicological impacts of Cr(VI) on human health. Therefore, the concentration-dependent responses of 12 Cr(VI)-responsive genes selected from a high-throughput Lycopersicon esculentum complementary DNA microarray were examined at different Cr concentrations. The results indicated that most of the genes were differentially expressed from 0.1 mg Cr/kg soil, whereas the lowest-observable-adverse-effect concentrations of Cr(VI) were 1.6 mg Cr/kg soil, 6.4 mg Cr/kg soil, 3.2 mg Cr/kg soil, and 0.4 mg Cr/kg soil for seed germination, root elongation, root biomass, and root morphology, respectively, implying that the transcriptional method was more sensitive than the traditional method in detecting Cr(VI) toxicity. Dose-dependent responses were observed for the relative expression of expansin (p = 0.778), probable chalcone-flavonone isomerase 3 (p = -0.496), and 12S seed storage protein CRD (p = -0.614); therefore, the authors propose the 3 genes as putative biomarkers in Cr(VI)-contaminated soil. Environ Toxicol Chem 2016;35:1751-1758. © 2015 SETAC.

The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces.

The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants remains unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to biofilm formation by OE1-1, which is required for OE1-1 virulence.

Pepino mosaic virus RNA-Dependent RNA Polymerase POL Domain Is a Hypersensitive Response-Like Elicitor Shared by Necrotic and Mild Isolates.

Pepino mosaic virus (PepMV) is an emerging pathogen that represents a serious threat to tomato production worldwide. PepMV-induced diseases manifest with a wide range of symptoms, including systemic necrosis. Our results showed that PepMV accumulation depends on the virus isolate, tomato cultivar, and environmental conditions, and associates with the development of necrosis. Substitution of lysine for glutamic acid at position 67 in the triple gene block 3 (TGB3) protein, previously described as a necrosis determinant, led to increased virus accumulation and was necessary but not sufficient to induce systemic necrosis. Systemic necrosis both in tomato and Nicotiana benthamiana shared hypersensitive response (HR) features, allowing the assessment of the role of different genomic regions on necrosis induction. Overexpression of both TGB3 and the polymerase domain (POL) of the RNA-dependent RNA polymerase (RdRp) resulted in necrosis, although only local expression of POL triggered HR-like symptoms. Our results also indicated that the necrosis-eliciting activity of POL resides in its highly conserved "palm" domain, and that necrosis was jasmonic acid-dependent but not salicylic acid-dependent. Altogether, our data suggest that the RdRp-POL domain plays an important role in PepMV necrosis induction, with necrosis development depending on the virus accumulation level, which can be modulated by the nature of TGB3, host genotype and environmental conditions.

Ultrastructure of the Epidermal Cell Wall and Cuticle of Tomato Fruit (Solanum lycopersicum L.) during Development.

The epidermis plays a pivotal role in plant development and interaction with the environment. However, it is still poorly understood, especially its outer epidermal wall: a singular wall covered by a cuticle. Changes in the cuticle and cell wall structures are important to fully understand their functions. In this work, an ultrastructure and immunocytochemical approach was taken to identify changes in the cuticle and the main components of the epidermal cell wall during tomato fruit development. A thin and uniform procuticle was already present before fruit set. During cell division, the inner side of the procuticle showed a globular structure with vesicle-like particles in the cell wall close to the cuticle. Transition between cell division and elongation was accompanied by a dramatic increase in cuticle thickness, which represented more than half of the outer epidermal wall, and the lamellate arrangement of the non-cutinized cell wall. Changes in this non-cutinized outer wall during development showed specific features not shared with other cell walls. The coordinated nature of the changes observed in the cuticle and the epidermal cell wall indicate a deep interaction between these two supramolecular structures. Hence, the cuticle should be interpreted within the context of the outer epidermal wall.

Ultrastructural insights into tomato infections caused by three different pathotypes of Pepino mosaic virus and immunolocalization of viral coat proteins.

This paper presents studies on an ultrastructural analysis of plant tissue infected with different pathotypes of Pepino mosaic virus (PepMV) and the immunolocalization of viral coat proteins. Because the PepMV virus replicates with a high mutation rate and exhibits significant genetic diversity, therefore, isolates of PepMV display a wide range of symptoms on infected plants. In this work, tomato plants of the Beta Lux cultivar were inoculated mechanically with three pathotypes representing the Chilean 2 (CH2) genotype: mild (PepMV-P22), necrotic (PepMV-P19) and yellowing (PepMV-P5-IY). The presence of viral particles in all infected plants in the different compartments of various cell types (i.e. spongy and palisade mesophyll, sieve elements and xylem vessels) was revealed via ultrastructural analyses. For the first time, it was possible to demonstrate the presence of crystalline inclusions, composed of virus-like particles. In the later stage of PepMV infection (14 dpi) various pathotype-dependent changes in the structure of the individual organelles (i.e. mitochondria, chloroplasts) were found. The strongest immunogold labeling of the viral coat proteins was also observed in plants infected by necrotic isolates. A large number of viral coat proteins were marked in the plant conductive elements, both xylem and phloem.