Fig 1.
Differential response of grafted and non-grafted tomat\o genotypes to the infection of TSWV-CiPz.
(A) Recovery from disease symptoms shown by Sl-Ma (left) but not from Sl-UC (right) at 28 dpi with TSWV-CiPz. (B) disease symptoms shown by Sl-Me (left) and Sl-Pu (right) grafted on Sl-Ma at 21 dpi with TSWV-CiPz. Sl-Me is a tomato variety carrying the Sw-5 resistance gene to standard strains of TSWV. Sl-Pu is a tomato variety susceptible also to ordinary strains of TSWV. (C) Root development in plants of Sl-Me grafted on Sl-Ma compared with roots of non-grafted plants of Sl-Ma and Sl-Me.
Fig 2.
Accumulation of TSWV-CiPz RNA varies among Solanum genotypes and grafted and self-grafted plants.
Load of viral RNA was estimated in Solanum spp genotypes (green bars), grafted (scion/rootstock) plants (violet bars) and self-grafted plants (blue bars) at 21dpi, by quantitative dot blot hybridization. Bars represent means of two independent experiments of spot intensity values of RNA M and were calculated on the basis of a standard curve generated by serial dilutions of a plasmid preparation containing the fragment of the RNA M targeted by the probe. Each bar represents average of three biological replicates for each of the two experiments and error bars on lines represent the standard error among replicates. Acronyms and symbols as in Table 1.
Table 1.
Disease symptoms observed in the Solanum spp genotypes and in grafted plants upon infection with TSWV-CiPz.
Fig 3.
Localization of TSWV-CiPz RNA in grafted tomato plants.
Localization of TSWV-CiPz RNA at 19 dpi by tissue print hybridization in cross sections of principal root, below, point and above the graft junction, inoculated leaf and top leaf of Sl-UC (UC82), Sl-Ma (Mand) and graft combinations. Mock = negative control plants mock-inoculated with buffer. TSWV C+ = plasmid preparation (50 ng) containing the fragment of the RNA M targeted by the probe.
Fig 4.
Detection of sRNA and mapping on the TSWV-CiPz genome.
(A) silver staining of the total RNA (Total) and the sRNA-enriched preparations (sRNA) extracted from Sl-Ma and Sl-UC plants mock-inoculated (mock) or at 21 dpi with TSWV-CiPz (infected). M is a 100 bp DNA ladder (Promega, Madison, WI, USA). P is a custom 21 bp RNA oligonucleotide used as marker. (B) Northern blots of RNA extracted from purified preparations of TSWV-CiPz and TYRV hybridized with siRNA probes derived from the sRNA-enriched preparations obtained from Sl-Ma and Sl-UC plants mock-inoculated (mock) or at 21 dpi with TSWV-CiPz (infected) (see panel A). (C) ethidium bromide staining of RNA extracted from purified preparations of TSWV-CiPz and TYRV prior to transfer onto nylon membranes, demonstrating equal loading. Hybridization signals of sRNAs labeled with (γ-32P) ATP with virus genomic RNAs S, M and L pointed by arrows are visible only in RNA preparations from TSWV.
Fig 5.
Differential modulation in the expression of RNAi-hallmark enzymes in grafted and non grafted tomato plants.
Variations in the expression level of AGO1, AGO2, AGO4 (A), DCL1, DCL2, DCL4 (B), PAZ, RDR1 and RDR6 (C) genes (orthologs of Arabidopsis thaliana) in leaves and roots of grafted and non-grafted tomato plants at 21 dpi with TSWV-CiPz (infected) or mock-inoculated (mock). For each gene, RQ values are expressed as mean ± standard error of 3 plants (biological replicates) with a technical replicate for each plant. Different letters represent statistically significant differences of means according to factorial analysis of variance (ANOVA) (P ≤ 0.05) (Tukey test). Acronyms for tomato genotypes as in Table 1.
Fig 6.
Photo-bleached phenotype is differentially expressed in Sl-Ma and Sl-UC plants.
Photo-bleached phenotype induced in Sl-Ma (A) and Sl-UC (B) at 10 dpa with A. tumefaciens carrying pTRV1+ pTRV2-PDS. In each panel, helathy (left), agroinfiltrated with A. tumefaciens carrying pTRV1+ pTRV2-PDS (central) and agroinfiltrated with A. tumefaciens carrying empty plasmids (right). Panel (C) shows photo-bleached phenotype induced in stem and flower sepals of Sl-Ma at 30 dpa.
Fig 7.
VIGS of RDR1 and RDR6 in Sl-Ma and Sl-UC plants.
Relative quantity (RQ) of RDR1 and RDR6 transcripts (columns) in samples of Sl-Ma and Sl-UC plants collected at 14 dpa with A. tumefaciens carrying pTRV1+ pTRV2-RDR1 (Sl-Ma TRV-R1 and Sl-UC TRV-R6) and pTRV1+ pTRV2-RDR6 (Sl-Ma TRV-R1 and Sl-Ma TRV-R6). RQ 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 RDR1 and RDR6 transcripts was deduced by the formula expression 2-ΔΔCt. Columns represent mean RQ values from three biological replicates 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 shows also estimates of the accumulation of TSWV-CiPz RNA (red line) in agroinfiltrated plants. After collection of leaf samples, plants were inoculated with TSWV-CiPZ on the first and second true leaves above remnants of cotyledons and load of viral RNA estimated at 19 dpi with dot blot hybridization. Vertical bars on line represent standard deviations among replicates.
Table 2.
List of primer pairs.