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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 ‘
Phytoplasmas are biotrophic plant-pathogenic wall-less prokaryotes (class
Plant–phytoplasma interactions have been poorly characterized due to a lack of techniques. Thus far, it has been impossible to transform or genetically modify phytoplasmas, or simply isolate different strains from mixtures present in nature (
Infection of plants by phytoplasmas leads to massive changes in phloem physiology associated with a severely impaired assimilate translocation (
By western blot analyses of protein extracts from midribs of healthy and ‘
The studies provided evidence that infection of
The preparation of plant material and the microscopy analyses have been performed according the methods previously reported by
Four
Phytoplasma presence was assessed in randomly collected leaf samples by real time RT-PCR analyses. Total RNA was extracted from 1 g of frozen leaf midribs using RNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany). RNAs were reverse-transcribed using a QuantiTect Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany) with random hexamers, following the manufacturer’s instructions. Real time RT-PCR analyses were performed using the primers 16S stol F2/R3 based on the 16SrRNA gene of ‘
Fifteen randomly chosen leaf midrib segments, sampled from the four either infected or healthy tomato plants, were cut into pieces 6–7 mm in length, fixed in a solution of 3 % glutaraldehyde in 0.1M phosphate buffer (PB), pH 7.2, for 2 h at 4°C, washed for 30 min at 4°C in PB and post-fixed for 2 h with 1% (w/v) OsO4 in PB at 4°C (
Fifteen randomly chosen leaf midrib segments were excised from infected or healthy tomato plants. Segments were cut into small portions (6–7 mm in length), fixed in 0.2% glutaraldehyde, rinsed in 0.1 M PB, pH 7.4 and dehydrated in graded ethanol series (25-, 50-, 75%, 30 min for each step) at 4°C. After 1 h of the final 100% ethanol step, the samples were infiltrated in a hard-grade London Resin White (LRW; Electron Microscopy Sciences, Fort Washington, PA, USA)-100% ethanol mixture in the proportion 1:2 for 30 min, followed by LRW:ethanol 2:1 for 30 min, and 100% LRW overnight at room temperature (with a change 1 h after the start of the infiltration). The samples were embedded in Eppendorf tubes using fresh LRW containing benzoyl peroxide 2% (w/w) according to manufacturer’s protocol and polymerized for 24 h at 60°C (
Several serial ultrathin sections (60–70 nm) of about 60 LR-White-embedded samples from each healthy or infected plant were cut using an ultramicrotome (Reichert Leica Ultracut E ultramicrotome, Leica Microsystems, Wetzlar, Germany) and collected on carbon/formvar coated 400 mesh nickel grids (Electron Microscopy Sciences, Fort Washington, PA, USA). To visualize the presence and distribution of actin in LR-White-embedded plant tissue, immunogold-labeling technique was performed (modified after
To assess the subcellular distribution of actin labeling, immunogold particle number was determined in healthy and infected sieve elements. Gold spots were manually counted and recorded on plasma membrane, cytoplasm (i.e., mictoplasm,
Total proteins were extracted from
An ANOVA procedure with SPSS software version 21.0 (IBM SPSS Statistics, Armonk, NY, USA) evaluated differences in the number of gold particles observed in healthy and infected sieve elements. Homogeneity of variance and distributional assumptions were assessed via the Levene test. A significance level of 0.05 was used for all comparisons.
A densitometric analysis was conducted on actin and BiP Western Blot signals with Quantity One 4.6.6. Bio-Rad Software (Bio-Rad Laboratories, Hercules, CA, USA). A total of six samples for each plant were analyzed. The statistical analysis of densitometric values was performed with the unpaired
Control plants were regularly grown, without disease symptoms. In stolbur-infected plants, typical symptoms, such as leaf yellowing, leaf-size reduction, witches’ brooms and stunting, emerged nearly 2 months after grafting (
Molecular detection of ‘
Well | Label | Primers | Cq |
---|---|---|---|
F1 | Stolbur-infected |
16SRT f2r3 | 17.98 |
F2 | Stolbur-infected |
16SRT f2r3 | 19.38 |
F3 | Stolbur-infected |
16SRT f2r3 | 17.54 |
F4 | Stolbur-infected |
16SRT f2r3 | 16.10 |
F5 | C. roseus Stol+ | 16SRT f2r3 | 17.55 |
F6 | Grapevine Stol+ | 16SRT f2r3 | 23.50 |
F7 | Tomato C– | 16SRT f2r3 | None |
F8 | H2O | 16SRT f2r3 | None |
In total, 60 sections from the 15 embedded blocks have been screened by TEM. TEM images revealed the sieve-element plasma membrane appressed to the cell wall in healthy leaves (
Control sections (from both healthy and infected samples), incubated with buffer alone, did not show labeling (not shown). In agreement with labeling with α-actin-gold-conjugated antibodies, actin occurred along the sieve-element membrane (
In infected samples, high spatial resolution images revealed a co-localization of sieve-element actin and phytoplasma cells (
Gold particles were counted to determine the labeling distribution in sieve-element membrane, mictoplasm and lumen. The countings were statistically analyzed (
Sieve elements of healthy and infected tomato were analyzed by immunogold labeling and electron microscopy, to assess actin subcellular distribution.
Sample | # Fields | Membrane | Lumen | Mictoplasm | Total gold particles |
---|---|---|---|---|---|
Healthy | 9 | 12.00 ± 2.35 a | 4.78 ± 5.09 b | 16.89 ± 6.17 a | 303 |
Infected | 9 | 0.00 ± 0.00 | 23.11 ± 1.62 c | 0.00 ± 0.00 | 210 |
TEM images of healthy samples showed SER stacks mostly orientated parallel to the sieve-element plasma membrane (
Western Blot analyses (
It has been advanced that cytological relationships between phytoplasmas and sieve elements are essential for the establishment of pathogenic activity in the host (
Among the diverse traits of
Here, TEM observations evidence major modifications of the plasma membrane in infected sieve elements. Parietally located phytoplasmas do not only adhere to the SER (
Both animal and plant pathogens actively interact with the host cytoskeleton to successfully enter in the host (
In our study, the connection between the invader phytoplasma and sieve-element actin has been described
To gain additional evidence for actin involvement in the sieve-element interaction with phytoplasmas, quantitative actin expression analyses were carried out. Western blotting and gold labeling demonstrated that the interaction between phytoplasmas and actin in infected sieve elements is associated with a decrease of the amount of actin. This interpretation should be made with care, as it departs from the assumption that the changes occur in sieve elements, the exclusive location of phytoplasmas. It is not excluded that part of the changes occurs in the surrounding (vascular) cells given the use of entire midribs. Nevertheless, a similar reduction of actin content in infected cells measured in expression studies (
Dynamic actin re-arrangement is regulated by a pool of actin-binding proteins, named actin depolarizing factors (ADFs), which sense stresses and environmental modifications and regulate the cytoskeleton through diverse biochemical activities (
In stolbur-diseased plants, ADF genes have been reported significantly overexpressed (
Furthermore, ADF activates actin-based motility of bacteria, as reported for
In conclusion, our results show that stolbur-phytoplasma infection results in a significant re-organization of the sieve-element ultrastructure in phloem tissue of
Despite the structural interconnections between phytoplasmas and the host sieve-element plasma membrane and actin and the massive impact of phytoplasma infection on the SER ultrastructure, the functional nature of the interactions remains largely unclear. The changes probably express a transformation that benefits growth, maintenance and transport of phytoplasmas. Phytoplasmas may effectively re-arrange the host ultrastructure to enable nutrient supply and systemic spread
RM and SB conceived the project under the supervision of AvB and KK, SB, and RM established the protocols for the electron microscopy and performed microscopical observations. AL prepared infected tomato plants. FDM and RP performed phytoplasma detection by real-time RT-PCR on tomato leaves. FD and LS established the protocols and performed western blot analyses. SB and RM wrote the manuscript with extensive support of AvB.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by the International Giessen Graduate Centre for the Life Sciences (GGL) founded by the Deutscher Akademischer Austauschdienst (DAAD) and by the University of Udine.