Structural and Genetic Requirements for the Biogenesis of Tobacco Rattle Virus-derived Small Interfering RNAs

Livia Donaire1, Daniel Barajas1, Belén Martínez-García1, Llucia Martínez-Priego1, Israel Pagán2 and César Llave1*

Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain1

Departamento de Biotecnología and Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain2

SUPPLEMENTAL FIGURE LEGENDS

FIG.S1.Predicted fold-back secondary structuresfor siRNA-containing regions along the positive-strand TRV2-PDS genome as determined by the mfold program. The numbers under each diagram show the nucleotide position according to the pTRV2 sequence. siRNA sequences of sense (black lines) and antisense (red lines) polarities are shown. Sequences of cloned TRV-derived siRNAs are listed in Table S1 in the supplemental material.

FIG.S2.Predicted fold-back secondary structures within the 3’UTR of TRV2 using the mfold program. Structure #1 corresponds to the full-length 3’ UTR region. Structure #2 corresponds to the 268-terminal nts of the 3’ UTR. Structure #3 corresponds to the putative stem-loop A. The putative stem-loops A, B and C described in the main text are highlighted in grey.siRNA sequences of sense (black lines) and antisense (red lines) polarities are shown. Sequences of cloned TRV-derived siRNAs are listed in Table S1 in the supplemental material.

FIG. S3.Spatial distribution profile of TRV1-derived siRNAsin Arabidopsisdcl mutants. DNA blots of PCR-amplified DNAfragments shown in Fig. 3 were probed with 5’-radiolabeled siRNAs purified fromsingle, double and triple dcl mutants infected withTRV. A DNA fragment corresponding to the -glucoronidase gene (GUS) was used as a negative hybridization control. The ethidium bromide-stained agarose gel is shown as a loading control. DNA blots from three independent experiments are shown.

FIG. S4. Spatial distribution profile of TRV1-derived siRNAs in Arabidopsisrdr mutants.DNA blots of PCR-amplified DNA fragments shown in Fig. 3 were probed with 5’-radiolabeled siRNAs purified from single, double and triple rdr mutants infected with TRV. Hybridization signals were measured by densitometry using Quantity One-4.2.3 (BioRad) from three independent experiments. Histograms below the DNA gel blots represent the percentage of the intensities of the hybridization signals corresponding to each PCR fragment relative to the total fragments. Mean values and standard deviations are shown. Figure content is as described in the legend for Fig. 3.

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