Leaf Plasmodesmata Respond Differently to TMV, ToBRFV and TYLCV Infection

Macromolecule and cytosolic signal distribution throughout the plant employs a unique cellular and intracellular mechanism called plasmodesmata (PD). Plant viruses spread throughout plants via PD using their movement proteins (MPs). Viral MPs induce changes in plasmodesmata’s structure and alter their ability to move macromolecule and cytosolic signals. The developmental distribution of a family member of proteins termed plasmodesmata located proteins number 5 (PDLP5) conjugated to GFP (PDLP5-GFP) is described here. The GFP enables the visual localization of PDLP5 in the cell via confocal microscopy. We observed that PDLP5-GFP protein is present in seed protein bodies and immediately after seed imbibition in the plasma membrane. The effect of three different plant viruses, the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV, tobamoviruses), and tomato yellow leaf curl virus (TYLCV, begomoviruses), on PDLP5-GFP accumulation at the plasmodesmata was tested. In tobacco leaf, TMV and ToBRFV increased PDLP5-GFP amount at the plasmodesmata of cell types compared to control. However, there was no statistically significant difference in tomato leaf. On the other hand, TYLCV decreased PDLP5-GFP quantity in plasmodesmata in all tomato leaf cells compared to control, without any significant effect on plasmodesmata in tobacco leaf cells

Shoot Regeneration Is Not a Single Cell Event

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4–5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days

A Novel Route Controlling Begomovirus Resistance by the Messenger RNA Surveillance Factor Pelota.

Tomato yellow leaf curl virus (TYLCV) is a devastating disease of tomato (Solanum lycopersicum) that can be effectively controlled by the deployment of resistant cultivars. The TYLCV-resistant line TY172 carries a major recessive locus for TYLCV resistance, designated ty-5, on chromosome 4. In this study, the association between 27 polymorphic DNA markers, spanning the ty-5 locus, and the resistance characteristics of individual plants inoculated with TYLCV in 51 segregating recombinant populations were analyzed. These analyses localized ty-5 into a 425 bp region containing two transversions: one in the first exon of a gene encoding the tomato homolog of the messenger RNA surveillance factor Pelota (Pelo), and a second in its proximal promoter. Analyses of susceptible and resistant lines revealed that the relative transcript level of the gene remained unchanged, regardless of whether the plants were infected with TYLCV or not. This suggests that the polymorphism discovered in the coding region of the gene controls the resistance. Silencing of Pelo in a susceptible line rendered the transgenic plants highly resistant, while in the resistant line TY172 had no effect on symptom development. In addition, over-expression of the susceptible allele of the gene in the resistant TY172 line rendered it susceptible, while over-expression of the resistant allele in susceptible plants had no effect. These results confirm that Pelo is the gene controlling resistance at the ty-5 locus. Pelo, implicated in the ribosome recycling-phase of protein synthesis, offers an alternative route to promote resistance to TYLCV and other viruses

Association between DNA markers spanning the <i>ty-5</i> locus and disease severity in representative segregating populations.

<p>In the ruler presented to the left of each analysis: yellow-shaded markers are homozygous for the allele originated from the susceptible line M-82 (SS), red-shaded markers are homozygous for the allele originated from the resistant line TY172 (RR) and gray-shaded markers are heterozygous (SR); the analysis of variance presented at the bottom of each population was carried out with different markers: the marker in red asterisks is the one that was used as an independent variable in each analysis; different superscript letters above means indicate statistically significant difference, <i>P</i><0.05, between genotypes for each population separately; A-D: four different representative segregating populations; DSI (disease severity index) was determined at 28 DPI (days post inoculation) and at 42 DPI, the DSI values presented is an average of both readings.</p

Effect of over-expressing the <i>Pelo</i> allele from M-82 plants in transgenic TY172 plants.

<p>^a NT = Not transgenic; T = Transgenic</p><p>^b Fold increase of <i>Pelo</i> transcription depicts the level of <i>Pelo</i> transcript level in the transgenic plants divided by the transcript level in the non-transgenic plants.</p><p>^c DSI = TYLCV disease severity index. Plants were scored and sampled 28 days post inoculation.</p><p>^d VCN = Virus copy number</p><p>Different superscript letters indicate statistically significant differences between transgenic and non-transgenic plants for each trait separately.</p><p>Effect of over-expressing the <i>Pelo</i> allele from M-82 plants in transgenic TY172 plants.</p

Average relative transcript levels of the <i>Pelo</i> gene in resistant TY172 and susceptible M-82 and R13 plants.

<p>The two experiments, A and B, were performed in two different years and analyzed independently. Ten samples, each sample composed of three different plants, were analyzed for each line, treatment and date. Identical superscript letters indicate that there is no statistical difference, <i>P</i>>0.05, between means for each date separately within each experiment. ± SEM.</p><p>Average relative transcript levels of the <i>Pelo</i> gene in resistant TY172 and susceptible M-82 and R13 plants.</p

Effect of <i>Pelo</i> silencing in transgenic susceptible (R13) and resistant (TY172) plants.

<p>^a NT = Not transgenic; T = Transgenic</p><p>^b Fold reduction of <i>Pelo</i> transcription depicts the level of <i>Pelo</i> transcription in the non-transgenic plants divided by the transcription level in the transgenic plants.</p><p>^c DSI = TYLCV disease severity index. Plants were scored and sampled 28 days post inoculation.</p><p>^d VCN = Virus copy number</p><p>Different superscript letters indicate statistically significant differences between transgenic and non-transgenic plants for each trait separately.</p><p>Effect of <i>Pelo</i> silencing in transgenic susceptible (R13) and resistant (TY172) plants.</p

Amino-acid sequence of the <i>Pelo</i> gene in the resistant TY172 line compared to the susceptible line M-82.

<p>The substitution of Valine¹⁶ (susceptible lines) to a Glycine (resistant TY172 line) is highlighted with magenta; GenBank accession numbers for TY172 and M-82 are KC447285 and KC447286, respectively.</p