Figure 1.
Scheme of a Samsun tobacco plant showing position of leaves used in this study.
Figure 2.
Samsun tobacco plants recover from disease symptoms induced by AILV-V.
In A, local symptoms in inoculated leaves appeared by 7 dpi and consisted in chlorotic spots, small necrotic rings and line patterns etched in leaf epidermis. In B, systemic symptoms are shown in the 3rd and 4th leaf and consist in chlorotic or necrotic ringspots surrounding the veins and peripheral vein clearing. In C, young leaves emerged between 21 and 28 dpi showing the recovery phenotype from disease symptoms.
Figure 3.
AILV-V RNA-2 accumulation varies in the same leaf, progressively decreasing moving to the successive leaf.
Load of viral RNA (lines) was estimated by quantitative dot blot hybridization. RNA data are expressed as means of two independent experiments, were derived from spot intensity values of the target RNAs and were calculated on the basis of a standard curve generated by serial dilutions of a plasmid preparation containing the fragment of the RNA-2 of AILV-V targeted by the probe. Samples were collected from the 3rd, 4th, 5th, and 6th leaves at 24 h intervals from 10 to 23 dpi and then at 28 and 60 dpi. Each point in the line chart represents average of three biological replicates for each of the two experiments and error bars on lines represent the standard error among replicates. Figure shows also the relative quantity (RQ) of RDR6 and DCL4 transcripts (columns chart) in samples of tobacco plants collected at selected time points between 10 and 60 dpi with AILV-V. The values were first normalized on the accumulation level of the GAPDH mRNA (Δ cycle threshold [Ct] = CtGAPDH–Cttarget RNA) and then used to determine the relative quantification of each target RNA with a calibrator, according to the formula ΔΔCt = ΔCtcalibrator–ΔCttarget RNA. Each target mRNA in an individual mock-inoculated plant served as calibrator (RQ set to 1) for the respective gene. RQ for RDR6 and DCL4 transcripts was deduced by the formula expression 2−ΔΔCt. Columns represent mean RQ values from three biological replicates for each of the two experiments and different letters represent statistically significant differences values according to separate one-way ANOVA analysis for each target mRNA, using Tukey's test (P<0 05). Vertical bars on columns represent standard deviations among replicates.
Figure 4.
From the inoculated leaf, AILV-V moves first into shoot topmost leaf and then to lower leaves.
Dot blot hybridization of samples collected from leaves of tobacco plants from 1 to 7-V. Plants at the 1103 growth stage according to the scale for coding growth stages in tobacco – coresta (i.e. with the 3rd leaf unfolded) were inoculated mechanically on the 1st and 2nd leaf. Columns represent mean values of the intensity of dot blot hybridization signal from two technical replicates of samples collected from two plants at each time point. The intensity of hybridization signal with antisense probe to detect viral genome (+)RNA was estimated from serial dilutions of a plasmid preparation, containing the fragment of the RNA-2 of AILV-V targeted by the probe. Vertical bars represent the standard error. I = inoculated leaf; A = apical leaf of the shoot tip. 3rd = third leaf i.e. the first leaf above the two basal leaves used for rub inoculation; Mock = leaf mock-inoculated with buffer.
Table 1.
Comparison of accumulation of viral RNAs and expression level of RDR6 and DCL4 in time course experiments with tobacco plants challenged with AILV-V, PVY-SON41 or both viruses.
Figure 5.
AILV-V would not replicate in leaves recovered from disease symptoms but retains infectivity.
In A. local and systemic symptoms consisting, respectively, in chlorotic spots and line patterns and, chlorotic/necrotic ringspots surrounding the veins and peripheral vein clearing induced by AILV-V in tobacco at 12 dpi with sap extracted from tobacco leaves, which had recovered from disease symptoms at 40 dpi. In B. Northern blot hybridization for detection of (+)RNA (1) and (−)RNA (2) on RNA preparations extracted from the following sources: A = apical leaves at 40 dpi; 5thR and 5thL = samples collected from opposite sites (Right and Left) from the 5th leaf of two tobacco plants at 40 dpi with recovery phenotype; 4th = sample collected from the 4th leaf of a tobacco plant with severe symptoms of systemic infection; V = RNA from a purified preparation of AILV-V particles, used as positive control; H = sample collected from an healthy tobacco plant, used as negative control. The picture shows the presence of a weak signal of hybridization with the plus-sense RNA probe (which detects the replication specific minus-sense RNA) only in correspondence of the sample collected from the 4th leaf with severe symptoms of systemic infection. In C. Accumulation of AILV-V RNA2 determined by dot blot hybridization in leaf samples collected from two recovered plants (P1 and P2) at 28 dpi before and 14 days after secondary inoculation. + and – indicate positive (pAILV769 plasmid DNA) and negative (mock-inoculated plant leaf) controls, respectively.
Figure 6.
AILV-V enters SAM of Samsun tobacco at a very early stage of infection and persists there up to 40 dpi.
One of the two time-course analyses of AILV-V distribution in the SAM of Samsun tobacco showing longitudinal sections of meristem tissues at 7, 14, 21, 28 and 40 dpi. AILV was detected using polyclonal antibodies raised against AILV-V coat protein and signals were developed with alkaline phosphatase diluted 1∶500 in PBS/BSA buffer and stained in NBT/BCIP solution. Immunolocalization is demonstrated in cells with dark stain. Pictures show that between 7 and 28 dpi the virus was present in all meristem tissues and in leaf primordia while by 40 dpi, i.e. concomitantly with the recovery phase from disease symptoms, the virus appeared confined between the corpus and the rib meristem. Mock = SAM of healthy tobacco treated with AILV-V antiserum at 21 days after mock- inoculation and used as negative control. Bars = 100 µm.
Figure 7.
Small interfering RNAs (siRNA) produced in response to AILV-V infection remain below a detectable level.
In A and B, panels represent total RNA preparations enriched in siRNA obtained from samples collected from apical (Ap), rub-inoculated (I) and 3rd (3rd) leaf at 7, 14 and 21 dpi with AILV-V. RNA preparations were first separated in by polyacrylamide gel electrophoresis and stained with ethidium bromide (EtBr)), then transferred to nylon membrane by electroblotting and hybridized with an AILV-V -specific RNA probe (AILV-V) for the last 760 3′-terminal sequences, labeled with digoxigenin and hydrolyzed by alkaline treatment. H = total RNA extracted from an healthy tobacco leaf. P = 23 bp primer. Arrows point the position expected for the 23 bp primer, after hybridization. In C, detection of siRNAs in samples collected from leaves of a tobacco plant at 10 dpi with PVY-SON41. Panels show ethidium bromide staining (EtBr) after PAGE analysis and signals produced after hybridization with a PVY-specific RNA probe (PVY) labeled with digoxigenin and hydrolyzed by alkaline treatment. Arrows point position of 21 and 23 bp primers used as size markers.
Figure 8.
Plants expressing HC-Pro VSR do not enter the recovery phase during AILV-V infection.
In A, variation in the load of PVY HC-Pro protein detected by western blot in non-transgenic tobacco plants and tobacco plants transformed to express HC-Pro, upon infection with PVY-SON41. WT plants show increasing levels of HC-Pro protein from 14 to 40 dpi while HC-transgenic plants express: i) a steady-state level of the protein after mock-inoculation; ii) an increasing protein load at 14 dpi resulting from the sum of HC-Pro translated from the transgene and from viral transcript and iii) a strong downregulation of the accumulation of the HC-pro protein at 40 dpi caused by the activation of homology-dependent RNA silencing. In B, levels of viral RNAs in plants of tobacco wild-type (WT) and transformed with HC-Pro (HC), upon single and mixed infection of AILV-V and PVY-SON41. Columns represent mean values of the number of copies of viral RNAsx109 per µg of total RNA estimated from three biological replicates. Vertical bars represent the standard error. Quantitative dot blot was obtained from the intensity of hybridization signal estimated on the basis of a standard curve generated by serial dilutions of a plasmid preparation containing the RNA fragments targeted by the specific probes. Translation of HC-Pro from either transgenic or authentic virus transcripts favors the infection of AILV-V in WT plants so the plants do not show the recovery phenotype. A+P and P+A indicate the order of inoculation (A = AILV-V and P = PVY-SON41) in mixed infection. Samples were collected 14 days after the second inoculation, corresponding to 40 days from the first inoculation, from the newest fully developed leaves.
Figure 9.
Mixed infections of PVY-SON41 and AILV-V in tobacco exacerbate disease symptoms.
In A, mild mosaic and moderate leaf blade malformation induced in tobacco at 30 dpi with PVY-SON41. In B, Chlorotic/necrotic ringspots, severe reduction of leaf lamina and plant growth induced at 30 dpi by a mixed infection of PVY-SON41 and AILV-V.
Figure 10.
PVY-SON41 infection correlates with viral RNA continuous accumulation and suppression of RNA silencing.
Accumulation levels of viral RNA (lines) and transcription profiles of RDR6 and DCL4 were estimated by quantitative dot blot hybridization and qPCR, respectively. Symbols and protocols as in Figure 3.
Figure 11.
AILV-V is unable to revert GFP silencing while interferes with cell-to-cell movement of silencing signal.
In A, progression of GFP silencing (indicated by dark red areas along the major veins) in a plant of N. benthamiana, line 16c, at 14 dpi with the TMV-GFP vector. Silenced areas were inoculated with AILV-V but no desilencing effects were observed at 30 dpi with AILV-V; rather the silenced areas expanded (in B) following the spread of TMV-GFP infection. In C Free GFP was expressed transiently in 16c N. benthamiana from the binary vector pBIN-mGFP4 carried by A. tumefaciens. Prior to agroinfiltration, leaves were mock-inoculated with buffer (Mock) or with AILV-V (AILV), PVY-SON41 (PVY), AILV-V and PVY- SON41 (AILV+PVY) and PVA-B11 (PVA). Upon ectopic expression of GFP, a thin border of dark red tissue was visible at 14 dpi in plants mock-inoculated indicating short-range movement of GFP silencing. This border was not produced in leaves of plants inoculated with AILV-V, suggesting a viral interference with cell-to-cell movement of the silencing signal. Green fluorescent areas visible in AILV+PVY, PVY and PVA infected plants indicate suppression of silencing driven by VSR coded by PVY-SON41 and PVA-B11.
Table 2.
Effect of suppressors of RNA silencing (VSR) derived from different RNA and DNA viruses and expressed as transgenes in lines of N. tabacum cv Xanthi on the development of symptoms and induction of the recovery phenotype upon infection of AILV-V.