Figure 1.
Schematic representation of the tospovirus tripartite RNA genome.
Figure 2.
Folding prediction of A-U rich hairpin structures from tospovirus S RNA IGR: TSWV (left panel) and TYRV (right panel).
Figure 3.
Production of vsiRNAs from tospoviral S, M and L genomic RNA segments.
(A) Total RNA from healthy (lane 1) and TSWV infected N.benthamiana (lane 2); genomic RNA from TSWV RNPs (lane 3). As a size marker (m), ssRNA Ladder (NEB) was used. (B) Genomic RNA from TYRV RNPs, undiluted (lane 1) and diluted 1x (lane 2). As a size marker (m), RiboRuler High Range RNA Ladder (Thermo Scientific) was used. Left panel presents agarose gel. Right panel presents Northern blot hybridized with radiolabeled siRNAs purified from TSWV or TYRV-infected N. benthamiana.
Figure 4.
Distribution of vsiRNAs on TSWV ambisense S RNA segment.
(A) Schematic representation of TSWV S RNA segment. Intergenic region (IGR), with predicted hairpin structure (AU box), is indicated in red. PCR fragments spanning S RNA (S1 to S6) respective basepair sizes are indicated; dotted lines roughly demark positions of primers used. (B) Ethidium bromide staining of agarose gel containing fragments S1 to S6 (upper panel), and corresponding Southern blot hybridized to radiolabeled siRNAs purified from TSWV-infected N. benthamiana (lower panel). (C) Relative signal strength of siRNAs on each genomic cDNA fragment. Standard error of mean (SEM) from two independent experiments is indicated.
Figure 5.
Distribution of vsiRNAs on TYRV ambisense S RNA segment.
(A) Schematic representation of TYRV S RNA segment. Intergenic region (IGR), with predicted hairpin structure (AU box), is indicated in red. PCR fragments spanning S RNA (Y1 to Y7) respective basepair sizes are indicated; dotted lines roughly demark positions of primers used. (B) Ethidium bromide staining of agarose gel containing PCR fragments Y1 to Y7 (upper panel), and corresponding Southern blot hybridized to radiolabeled siRNAs purified from TYRV-infected N. benthamiana (lower panel). Below, fine mapping of fragment Y1. (C) Relative signal strength of siRNAs on each genomic cDNA fragment.
Figure 6.
Dicer-mediated cleavage of hairpin transcripts (HP) from TSWV S RNA IGR-encoded hairpin sequence.
Radioactively labeled HP transcripts (lane 2) were incubated in the presence of dicer containing Drosophila melanogaster (Dm) embryo extracts and cleavage products (lane 1) subsequently resolved on 8% denaturing acrylamide gel. As positive control, 114-nt dsRNA (lane 4) was included to verify dicer activity from Dm extracts (lane 3). As size marker, radiolabeled 21nt siRNAs were included (lane 5).
Figure 7.
Agroinfiltration leaf patch assays of GFP gene constructs containing 3′ hairpin trailer.
(A) Schematic representation of GFP constructs containing the different 3′ trailer sequences analyzed. The noHP sequence consists of a partial N gene sequence in antisense polarity. (B) Transient GFP expression after agroinfiltration of GFP constructs in absence of RSS. As only very low levels of fluorescence were visual at first (left), leaf disks were further analysed on binocular stereomicroscope M3Z, Leica (right). (C) Similar as panel B, but in the additional presence of TSWV NSs. Fluorescence in panels B and C was quantified and depicted in the graphs underneath. Standard error of mean (SEM) from three leaf disks is indicated.
Figure 8.
Production and distribution of siRNAs from GFP constructs containing various 3′ trailer sequences.
Small RNAs purified from transient expression of GFP constructs were probed on Southern blots containing PCR fragments spanning the respective construct sequence. (A) Schematic view of constructs and PCR products spanning the sequence. The noHP sequence consists of a partial N gene sequence in antisense polarity. Southern blot analysis of constructs: (B) GFP-HPTSWV; (C) GFP-HPTYRV; (D) GFP-noHP; (E) GFP. Ethidium bromide-staining of PCR products are shown below. (F) Graphical representation of the siRNA signal strength corresponding to the 3′ trailer sequences and normalized to the signal strength of the 3′ half of GFP (FP) of each construct. Abbreviation: G: 5′ half of GFP; FP: 3′ half of GFP; HP: A-U rich hairpin structure (from IGR of TSWV and TYRV S RNA); noHP: part of TSWV N gene.