A combined microscopy approach to study plant-phytoplasma interaction using Arabidopsis thaliana.

Phytoplasmas, obligate parasites of plants and phloem-feeding insects, belong to Mollicutes (Lee et al., 2004) and are associated with several hundreds of diseases affecting over one thousand plant species, including many economically important crops (Marcone, 2014). There is no effective curative strategy available so far, so the sole ways to limit the infection outbreaks are the use of insecticides and the removal of symptomatic plants (Bertaccini et al., 2014). Even if not all infections are necessarily deleterious, symptoms in infected plants suggest heavy disorders of phloem functions and growth-regulator balancing (Lee et al., 2000). Upon their discovery (Doi et al., 1967), the study of phytoplasmas has been hindered by the extreme difficulty to culture them in vitro, due to their lack of fundamental metabolic pathways (Bai et al., 2006). Moreover, the study in natural plant hosts is often limited by environmental conditions, long plant life cycle and poor knowledge of host-plant biology. Therefore, in the last decade some authors suggested to use Arabidopsis thaliana as model plant for studying phytoplasma-plant interactions. This choice was supported by the correspondence between the macroscopic symptoms developed in infected A. thaliana and those observed in natural host plants (Bressan and Purcell, 2005; Hoshi et al., 2009; Cettul and Firrao, 2011; MacLean et al., 2011). Nevertheless, morphological and ultrastructural modifications occurring in infected A. thaliana tissues have never been described in detail. In this work, we adopted a combined microscopy approach to verify if this plant is a reliable model for the study of phytoplasma-plant interactions at microscopical level. Using DAPI and fluorescence microscopy (FM), phytoplasma presence and localization were demonstrated in every infected plant. Transmission electron microscopy (TEM) observations confirmed phytoplasma massive presence into the sieve elements (SEs) (Figure 1). Phytoplasma appeared well preserved, with typical pleomorphic shape, free-floating and dividing in the lumen or adhered to SE membrane, probably connecting to the host (Marcone et al., 2014; Buxa et al., 2015). Phytoplasmas also established relationships with sieve element reticulum (SER). Pathogen presence, probably linked to nutrient uptake (Celli et al., 2015; Musetti et al., 2016), caused SER hyperproliferation, as observed in many other plant-phytoplasma interaction (Rudzinska-Langwald and Kaminska, 2001; Buxa et al., 2015) (Figure 1). Pathogen spread was documented by the passage through sieve pores. As remarked above, phytoplasma presence affected host plant development (Lee et al., 2000). In infected A. thaliana plants, light microscopy (LM) evidenced a profound disturbance in phloem morphology at histological level, mainly consisting in collapse, necrosis and hyperplasia of the phloem components. The relationship between necrosis and hyperplasia could be explained as a plant response to the impaired phloem functionality (Oshima et al., 2001) or due to pathogen effectors (Bai et al., 2009; Sugio et al., 2011). At ultrastructural level, as previously observed in other phytoplasma hosts (Musetti et al., 2000; 2013; Kaminska et al., 2001; Santi et al., 2013), phloem components showed plasmolysis or were collapsed or necrotized. Even in vital SEs, abnormalities of cell membrane profile and cell wall thickness were visible. TEM observations showed two typical plant responses to phytoplasma infection: phloem-protein agglutination and callose deposition at the sieve plates, which limited sieve-pore diameter (Figure 1). These phenomena have been interpreted as a plant reaction to physically limit pathogen spread (Lherminier et al., 2003; Gamalero et al., 2010; Luna et al., 2011; Musetti et al., 2010; 2013). Phloem functionality experiments using CFDA and confocal laser scanner microscopy (CLSM) suggested that sieve-pore obstruction leads to phloem impairment (Figure 2 A, C). This phenomenon is also associated to the accumulation of photo-assimilates, visible as chloroplast starch deposits under LM and TEM (Figure 2 B, D), as previously reported in other host plants (Maust et al., 2003; Junqueira et al., 2004; Musetti et al., 2013). This study proved that phloem tissue of infected A. thaliana presented the main morphological and ultrastructural response to phytoplasma infection as reported in natural hosts. Moreover, analyses carried on A. thaliana were not affected by troubles linked to low phytoplasma titre and uneven distribution, typical of woody plants. Therefore, we can state that A. thaliana revealed a reliable model plant for phytoplasma-plant interactions, concerning both macroscopic symptoms and morphological and ultrastructural changes

Epifluorescence microscopy imaging of phytoplasmas in embedded leaf tissues using DAPI and SYTO13 fluorochromes

The use of DNA-specific dyes, i. e. DAPI, is extensively reported for phytoplasma detection in fresh plant materials. However, fluorescence-based microscopy and imaging of fresh tissues often evidences technical limitations which are more significant in infected tissues, because phenolic and other defense-related compounds accumulate in the cell wall and in the vacuole making difficult sample preparation. In this paper we describe a method based on the use of epifluorescence microscopy and the DNA probes DAPI and SYTO13\uae for phytoplasma visualization in resin-embedded plant tissues. The method allows detection of phytoplasmas and it is recommended for tissues that are recalcitrant to conventional imaging

Changes in Physiological and Agronomical Parameters of Barley (Hordeum vulgare) Exposed to Cerium and Titanium Dioxide Nanoparticles

The aims of our experiment were to evaluate the uptake and translocation of cerium and titaniumoxide nanoparticles and to verify their effects on the growth cycle of barley (Hordeum vulgare L.). Barley plants were grown to physiological maturity in soil enriched with either 0, 500 or 1000 mg kg1 cerium oxide nanoparticles (nCeO2) or titanium oxide nanoparticles (nTiO2) and their combination. The growth cycle of nCeO2 and nTiO2 treated plants was about 10 days longer than the controls. In nCeO2 treated plants the number of tillers, leaf area and the number of spikes per plant were reduced respectively by 35.5%, 28.3% and 30% (p \ua4 0.05). nTiO2 stimulated plant growth and compensated for the adverse effects of nCeO2. Concentrations of Ce and Ti in aboveground plant fractions were minute. The fate of nanomaterials within the plant tissues was different. Crystalline nTiO2 aggregates were detected within the leaf tissues of barley, whereas nCeO2 was not present in the form of nanoclusters

Phloem cytochemical modification and gene expression following the recovery of apple plants from apple proliferation

Recovery of apple trees from apple proliferation was studied by combining ultrastructural, cytochemical, and gene expression analyses to possibly reveal changes linked to recovery-associated resistance. When compared with either healthy or visibly diseased plants, recovered apple trees showed abnormal callose and phloem-protein accumulation in their leaf phloem. Although cytochemical localization detected Ca2+ ions in the phloem of all the three plant groups, Ca2+ concentration was remarkably higher in the phloem cytosol of recovered trees. The expression patterns of five genes encoding callose synthase and of four genes encoding phloem proteins were analyzed by quantitative real-time reverse transcription- polymerase chain reaction. In comparison to both healthy and diseased plants, four of the above nine genes were remarkably upregulated in recovered trees. As in infected apple trees, phytoplasma disappear from the crown during winter, but persist in the roots, and it is suggested that callose synthesis/deposition and phloem-protein plugging of the sieve tubes would form physical barriers preventing the recolonization of the crown during the following spring. Since callose deposition and phloem-protein aggregation are both Ca2+-dependent processes, the present results suggest that an inward flux of Ca2+ across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees.L'articolo é disponibile sul sito dell'editore: http://www.apsjournals.apsnet.or

With or Without You: Altered Plant Response to Boron-Deficiency in Hydroponically Grown Grapevines Infected by Grapevine Pinot Gris Virus Suggests a Relation Between Grapevine Leaf Mottling and Deformation Symptom Occurrence and Boron Plant Availability

Despite the increasing spread of Grapevine Leaf Mottling and Deformation (GLMD) worldwide, little is known about its etiology. After identification of grapevine Pinot gris virus (GPGV) as the presumptive causal agent of the disease in 2015, various publications have evaluated GPGV involvement in GLMD. Nevertheless, there are only partial clues to explain the presence of GPGV in both symptomatic and asymptomatic grapevines and the mechanisms that trigger symptom development, and so a consideration of new factors is required. Given the similarities between GLMD and boron (B)-deficiency symptoms in grapevine plants, we posited that GPGV interferes in B homeostasis. By using a hydroponic system to control B availability, we investigated the effects of different B supplies on grapevine phenotype and those of GPGV infection on B acquisition and translocation machinery, by means of microscopy, ionomic and gene expression analyses in both roots and leaves. The transcription of the genes regulating B homeostasis was unaffected by the presence of GPGV alone, but was severely altered in plants exposed to both GPGV infection and B-deficiency, allowing us to speculate that the capricious and patchy occurrence of GLMD symptoms in the field may not be related solely to GPGV, but to GPGV interference in plant responses to different B availabilities. This hypothesis found preliminary positive confirmations in analyses on field-grown plants

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease. \ua9 2017 The Author(s

Filamentous sieve element proteins are able to limit phloem mass flow, but not phytoplasma spread

In Fabaceae, dispersion of forisomes\u2014highly ordered aggregates of sieve element proteins\u2014in response to phytoplasma infection was proposed to limit phloem mass flow and, hence, prevent pathogen spread. In this study, the involvement of filamentous sieve element proteins in the containment of phytoplasmas was investigated in non-Fabaceae plants. Healthy and infected Arabidopsis plants lacking one or two genes related to sieve element filament formation\u2014AtSEOR1 (At3g01680), AtSEOR2 (At3g01670), and AtPP2-A1 (At4g19840)\u2014were analysed. TEM images revealed that phytoplasma infection induces phloem protein filament formation in both the wild-type and mutant lines. This result suggests that, in contrast to previous hypotheses, sieve element filaments can be produced independently of AtSEOR1 and AtSEOR2 genes. Filament presence was accompanied by a compensatory overexpression of sieve element protein genes in infected mutant lines in comparison with wild-type lines. No correlation was found between phloem mass flow limitation and phytoplasma titre, which suggests that sieve element proteins are involved in defence mechanisms other than mechanical limitation of the pathogen

Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements

Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by ‘Candidatus Phytoplasma solani’, the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Expression analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes

Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements

Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by ‘Candidatus Phytoplasma solani,’ the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes

Agroinoculation of Grapevine Pinot Gris Virus in tobacco and grapevine provides insights on viral pathogenesis

The Grapevine Pinot Gris disease (GPG-d) is a novel disease characterized by symptoms such as leaf mottling and deformation, which has been recently reported in grapevines, and mostly in Pinot gris. Plants show obvious symptoms at the beginning of the growing season, while during summer symptom recovery frequently occurs, manifesting as symptomless leaves. A new Trichovirus, named Grapevine Pinot gris virus (GPGV), which belongs to the family Betaflexiviridae was found in association with infected plants. The detection of the virus in asymptomatic grapevines raised doubts about disease aetiology. Therefore, the primary target of this work was to set up a reliable system for the study of the disease in controlled conditions, avoiding interfering factor(s) that could affect symptom development. To this end, two clones of the virus, pRI::GPGV-vir and pRI::GPGV-lat, were generated from total RNA collected from one symptomatic and one asymptomatic Pinot gris grapevine, respectively. The clones, which encompassed the entire genome of the virus, were used in Agrobacterium-mediated inoculation of Vitis vinifera and Nicotiana benthamiana plants. All inoculated plants developed symptoms regardless of their inoculum source, demonstrating a correlation between the presence of GPGV and symptomatic manifestations. Four months post inoculum, the grapevines inoculated with the pRI::GPGV-lat clone developed asymptomatic leaves that were still positive to GPGV detection. Three to four weeks later (i.e. ca. 5 months post inoculum), the same phenomenon was observed in the grapevines inoculated with pRI::GPGV-vir. This observation perfectly matches symptom progression in infected field-grown grapevines, suggesting a possible role for plant antiviral mechanisms, such as RNA silencing, in the recovery process.</div