Cucumber Mosaic Virus-Encoded 2B Suppressor Inhibits Arabidopsis Argonaute1 Cleavage Activity

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Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense

Supplementary Materials

Figure Legends

Figure S1. Differential stability of FNY2b and Q2b. (A) Q2b transcripts and protein expression levels in 35S-Q2b transgenic lines. (B) Q2b-3HA transcript and protein expression levels in 35S-Q2b-3HA lines. (C) FNY2b-3HA expression levels in 35S-FNY2b-3HA lines. Upper panels (A and B) show transcript levels. Lower panels (A and B) show protein levels. Polyclonal antibody to Q2b (A) and monoclonal antibody to HA (B and C) were used for western blot assays. Note that the majority of detected Q2b and Q2b-3HA proteins (A and B) appeared as truncated forms. A cross-reacting band (asterisk *) was used as a loading control.

Figure S2. Intracellular localization of FNY2b-CFP and YFP-AGO1. The indicated proteins were transiently expressed in N. benthamiana cells. Note that the CFP-FNY2b protein was localized predominantly in the nucleus but was also found in the cytosol. The YFP-AGO1 was distributed throughout the nucleus and cytoplasm in 70-80% of the expressing cells (YFP-AGO1 I), but in 20-30% of the cells YFP-AGO1 was localized in discrete cytoplasmic foci (YFP-AGO1 II). Co-localization of FNY2b-CFP and YFP-AGO1 was observed whether in the nucleus or in cytoplasmic foci (2b-CFP/YFP-AGO1, a and b). YFP and CFP only were used as controls. DIC, differential interference contrast in light microscope mode.

Figure S3. Flag-AGO1 recruits trans siRNA (siR480/255) in vivo and ss siRNA in vitro. (A). Inflorescence tissues of Flag-AGO1/ago1-36 and WT Col-0 (control) plants were used for immunoprecipitation with Flag antibody. Immunoprecipitates were incubated with single-stranded 32P-labeled 21 nt siRNAs (siRNAPDS) bearing photoreactive dT residues at the two 3 positions or siRNALUC bearing iodo-U in the 5’ position. Mixtures were UV cross-linked as described in Supporting Information. Cross-linked products were resolved by 6% SDS-PAGE. (B) ta-siRNAs are associated with Flag-AGO1. Immunoprecipitates were obtained as in (A). Small RNA blot assays were performed with RNAs recovered from Flag-AGO1 immunoprecipitates and control immunoprecipitates using an antibody to 6His. Oligonucleotide probes complementary to siR480/255 were used.

Figure S4 Prolonged incubation of FNY2b and AGO1 enhances inhibition of AGO1 cleavage activity. Flag-AGO1 immunoprecipitates were incubated overnight at 4°C (instead of 1 h in other experiments in Fig. 6) without (buffer only, lane 3) or with the indicated protein (lanes 4-6) before addition of 32P-cap-labeled in vitro transcripts of At4g29770. Immunoprecipitates of Flag-AGO1/ago1-36 inflorescences using 6His antibody were used as a negative control (lane 2). SUMO-FNY concentration (0.1 and 1 mM) used here was less than the one used in Fig. 6.

Materials and Methods

DNA construction

The majority of constructs were made using the Gateway system (Invitrogen) (Zhang et al. 2005). Several destination vectors (DC in our nomenclature) were created for transient expression in Nicotiana benthamiania and stable Arabidopsis transformation. The binary vector pHyg001 and pBA002 (Zhang et al. 2005) for constitutive expression in plants was modified to obtain pHyg-DC, pBA-DC, pBA-6myc-DC and pBA-DC-CFP. The vector pER8 and pER10 (Zuo et al. 2000) for estradiol-inducible expression in plants under the control of the XVE system was modified to obtain pER8-6myc-DC and pER10-YFP-DC, respectively. Most of the cDNA or DNA fragments were cloned into pENTR/D vectors and then transferred to the appropriate vectors by recombination using the LR Clonase enzyme (Invitrogen).

Full-length and truncated forms of AGO1 cDNA were cloned into pENTR vector using following sets of primers: AGO1: For 5’–CACCATGGTGAGAAAGAGAAGAACGGATGC-3’ and Rev 5’- TCAGCAGTAGAACATGACACGCTTCACA-3’. AGO1NT: AGO1 For and Rev 5’- AGGCAGTATACGAGCCTCAACAGAAGCC-3’. AGO1CT: For 5’-CACCATGGCTTCTGTTGAGGCTCGTATACTGCC-3’ and AGO1 Rev. AGO1 (1-187 aa): AGO1 For and 5’-TTTACCAGGCCTCATTGGAAAC-3’. AGO1 (185-371 aa): 5’-CACCATGAGGCCTGGTAAAGGACAGAGTG-3’ and 5’- GCCCATCTGTGTAGGACGAAT-3’. AGO1 (366-567 aa): 5’-CACCATGCGTCCTACACAGATGGGCTTATCA-3’ and AGO1 NT Rev. AGO1 (559-675 aa): AGO1 CT For and 5’-ATCAATTTCTTTTCCTTGGGAGAG-3’. AGO1 (674-836 aa): 5’-CACCATGGATCTGCTTATTGTCATTCTGCCC-3’ and 5’- ATGCCCAGTTGATCTACGGAAGGC-3’. AGO1 (829-1048 aa): 5’-CACCATGGCCTTCCGTAGATCAACTGGG-3’ and AGO1 Rev.

FNY2b and Q2b cDNAs were cloned using the following primers: FNY2b: 5’-CACCATGGAATTGAACGTAGGTGCA-3’ and 5’- GAAAGCACCTTCCGCCCATTC-3’. Q2b: 5’- CACCATGGATGTGTTGACAGTAGTGGTG-3’ and 5’- AAACGACCCTTCGGCCCATTCG-3’. A series of pET28-SUMO derived vectors were constructed as follows. PCR fragments of FNY2b, Q2b, P19, P20, and P0 were amplified using the following pairs of primers. FNY2b: 5’-GGTGGATCCATGGAATTGAACGTAGGTGCAATG-3’ and 5’-GTGCTCGAGTTATCAGAAAGCACCTTCCGCCCATTC-3’. Q2b: 5’-GGTGGATCCATGGATGTGTTGACAGTAGTGG-3’ and 5’-GTGCTCGAGTTATCAAAACGACCCTTCGGCCCATTC-3’. P19: 5’-GGTGGATCCATGGAACGAGCTATACAAGG-3’ and 5’-GTGCTCGAGTCACTCGCTTTCTTCTTTGAAGGCTTC-3’. P20: 5’-GGTGGATCCATGCGAGCTTACTTTAGTGTTAATG-3’ and 5’-GTGCTCGAGCTACACGCAAGATGGAGAGACTAA-3’. P0 5’-GGTGGATCCATGCAATTTCTCGCTCACGATAAC-3’ and 5’-GGCAAGCTTTCATACAAACATTTCGGTGTAGATCG-3’. The PCR fragments were digested with BamHI/XhoI for 2b, p19 and P20, and BamHI/HindIII for P0. The resulting fragments were ligated into the pET28-SUMO backbone to yield pET28-SUMO-2b, -P19, -P20, and -P0. Similarly, BamHI/XhoI-digested fragments of 2b, metacaspase, At1g08370 and At5g13570 were introduced into pGEX4T-1 to produce pGEX4T-2b. -metacaspase, -At1g08370 and -At5g13570.

To create pER8-6myc-AGO1, PCR fragment of 6xmyc was obtained with a set of primers: 5’-GACGGCGCGCCGTATCGATTCAAAGCTATGG-3’ and 5’-CCATCGATTTCGAACCCTAGGTACCGAATTC-3’. The PCR product was digested with AscI/AvrII. PCR fragment of the N-terminal part of AGO1, which was amplified with primers of 5’-GCCCCTAGGGGATCCATGGTGAGAAAGAGAAGAACG-3’ and AGO1 (1-187 aa) Rev, and treated with AvrII/KpnI. The remaining part of AGO1 was obtained from KpnI/PacI-digestion of pER8-AGO1. The three fragments above were ligated into an AscI/PacI–derived pER8-DC-6myc backbone (Zhang et al. 2005) to produce pER8-6myc-AGO1.

Target genes containing miRNA165/166 or ta-siRNA480 complementary sequences were amplified from Arabidopsis cDNAs using PCR and the PCR fragments and then cloned into pENTR vectors (Invitrogen). The following sets of primers were used. At4g29770/siR480: 5’-CACCTAATACGACTCACTATAGGGTATTTACGATGGCCCGTCA-3’ and 5’-TCATACTTCTCGTAGACACTTTGAC-3’

PHAVOLUTA (PHV): 5’-CACCTAATACGACTCACTATAGGGCTACAGGAACTGCTGTCGAC-3’ and 5’-CGATCTTTGAGGATTTCAGCGAC-3’.

Phytoene desaturase (PDS) fragment containing an artificial siRNAPDS binding site was subcloned into pENTR vector from PX7-PDSRNAi (Guo et al. 2003) using a pair of primers 5’-CACCTAATACGACTCACTATAGGGCTGAATGAGGATGGAAGTGTC-3’ and 5’-GGAACTCCCACTAGCTTCTCC-3’.

In vitro pull-down assays and co-immunoprecipitation experiments

In vitro pull-down assays and in vivo co-immunoprecipitation were done as described (Zhang et al. 2005). Briefly, two micrograms of 6His or GST-tagged target proteins were pre-absorbed for 1 h at room temperature in 1 ml of binding buffer (50 µl amylose resin, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.2% glycerol, 0.6% Triton X-100, 0.5 mM b-mercaptoethanol). The mixture was cleared by centrifugation at 12,000 g for 2 min. The resulting supernatant was transferred to a fresh tube containing 2.0 µg of the MBP-tagged bait proteins. After incubation at room temperature for 2 h, 50 µl amylose resin beads were added and the incubation continued in the same conditions. Finally, after vigorous washes for 6 times, pulled-down proteins were resolved by SDS-PAGE and detected by western blot.

For co-immunoprecipitation experiments, N. benthamiana leaves were collected 2 days after agroinfiltration. For Arabidopsis plants, two-week-old 35S-FNY2-3HA/XVE-6myc-AGO1 transgenic seedlings were transferred into a medium supplemented with MG132 (50 µM) in the presence or absence of ß-estradiol (25 µM) for 20 h. Total proteins were extracted in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 0.2% Triton-100 supplemented with a EDTA-free protease inhibitor cocktail (Amersham Pharmacia). Protein extracts were immunoprecipitated with monoclonal antibody to myc or MBP at 4°C for 2 h. Protein A beads were then added and incubated for another hour. Beads were washed 4 times with the same buffer before an equal volume of 2x SDS-loading buffer was added. Western blots were analyzed with a monoclonal antibody to HA to detect co-immunoprecipitated FNY2b-3HA, and/ or with a monoclonal antibody to phyB to detect control proteins in the IP fraction of Arabidopsis extracts. For CMV (FNY strain) infection experiments, materials were collected 7 dpi. Similar procedures were performed except in western blot analysis, polyclonal antibodies to FNY2b and CP were used to detect co- immunoprecipitated FNY 2b and control protein CMV CP, respectively.

Subcellular localization experiments and confocal microscopy

Three-week old tobacco plants (Nicotiana benthamiana) were agroinfiltrated by using a syringe without needle as previously described (Zhang et al. 2005). Plants were maintained for 2 days at 24oC (16h light/8h dark). Confocal images for YFP and CFP were captured with an Axiovert 200 inverted, LMS 510 multiphoton confocal Microscope (Zeiss). An Argon laser was used, and the chlorophyll autoflorescence was filtered out by using a 540/520 nm filter. Images were processed using Spot Advance and Adobe Photoshop softwares.

UV- Photo-Crosslinking

UV photo-crosslinking of RNA oligos containing 3’-dTdT or 5’-iodo-uridine to

Flag-AGO1 immunoprecipitates was performed as described (Qi et al. 2005; Rivas et al. 2005). Briefly, 25 µl reactions were assembled as in mRNA cleavage assays except that no target mRNA was added, and that the siRNA (~2 nM) was 5’ end-labeled. After 30-min incubation at 25 0C, reaction mixtures were placed on ice and irradiated with UV with a wavelength of 254 nm (for 3’ dTdT-containing oligos) or 312 nm (for 5’-iodo-U containing oligos). Reactions were then mixed with 2x SDS loading buffer, boiled for 5 min and resolved on a 6% SDS-polyacrylamide gel.

References

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