A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC

Abstract

In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine.

Figure 1.

SPMMV P1 inhibits AGO1, but not AGO2. (A) N. benthamiana leaves were agroinfiltrated with 35S:TAS1c, 35S:amiR173, 35S:HA-AGO1, 35S:HA-AGO1-DAH and 35S:Flag-P1_1-395 as indicated. TAS1c mRNA cleavage was analyzed by Northern blotting. Protein extracts were analyzed by SDS-PAGE followed by anti-HA or anti-Flag Western blotting to detect HA-AGO1/HA-AGO1-DAH or Flag-P1_1–395, respectively. Ponceau staining shows equal loading of proteins. Top panel, mean (n = 3) relative TAS1c transcript level and ±SD (lane 2 = 1.0 for TAS1c transcript). One representative blot from three biological replicates is shown. (B) N. benthamiana leaves were agroinfiltrated with 35S:TAS1c-A388T, 35S:amiR173-5΄A, 35S:amiR173, 35S:HA-AGO2, 35S:HA-AGO2-DAD and 35S:Flag-P1_1-395 as indicated. TAS1c-A388T mRNA cleavage was analyzed by Northern blotting. Protein extracts were analyzed by SDS-PAGE followed by anti-HA or anti-Flag Western blotting to detect HA-AGO2/HA-AGO2-DAD or Flag-P1_1–395, respectively. Ponceau staining shows equal loading of proteins. Top panel, mean (n = 3) relative TAS1c-A388T transcript level and ±SD (lane 2 = 1.0 for TAS1c-A388T transcript). One representative blot from three biological replicates is shown. (C) 5΄ RACE product of TAS1c (lane 2) and and TAS1c-A388T (lanes 5 and 6) cleaved by amiR173/AGO1 and amiR173-5΄A/AGO2 complexes, respectively. In lanes 1, 3 and 4, amplification of fragments different from the miRNA cleaved products is observed. Amplification of N. benthamiana actin mRNA is used as control. (D) N. benthamiana leaves were agroinfiltrated with 35S:TAS1c-A388T, 35S:amiR173-5΄A, 35S:HA-AGO2 and 35S:Flag-P1_1-395, as indicated. N. benthamiana AGO2 mRNA expression was normalized to that of the endogenous elongation factor 1 (EF1), and represented as relative AGO2 mRNA expression. A mock-infiltrated control sample was used as the calibrator (relative AGO2 mRNA expression = 1.0). Three independent biological replicates of each treatment were carried out. For each biological replicate, two parallel samples were analyzed. The * indicates statistically significant differences between groups according to a one-way ANOVA followed by Bonferroni post hoc test (*P < 0.05).

Figure 2.

AGO2 interacts with SPMMV P1. 35S:mGPF4, 35S:HA-AGO1, 35S:HA-AGO2, 35S:amiR173, 35S:amiR173-5΄A were co-agroinfiltrated with 35S:Flag-P1_1–395, as indicated. Input and IP protein fractions were analyzed by SDS-PAGE followed by anti-HA, anti-Flag or anti-GFP Western blotting to detect HA-AGO1/HA-AGO2, Flag-P1_1–395 and GFP, respectively. Ponceau staining shows equal loading of proteins.

Figure 3.

Mutational analysis of the zinc finger domain present in SPMMV P1. (A) 35S:P1-C85A, 35S:P1-C88A, 35S:P1-C91A, 35S:P1-C103A and 35S:P1-C106A respectively were coinfiltrated with 35S:mGFP4. Photos were taken under UV light at 3 days post agroinfiltration. (B) GFP mRNA was detected by Northern blotting. Extracts of infiltrated leaves were subjected to SDS-PAGE followed by immunoblotting with the anti-GFP antibody. Flag-tagged P1 proteins were detected by Western blotting using the M2-anti Flag antibody. Ponceau staining shows equal loading of proteins in both Western blots. Top panel, mean (n = 3) relative mGFP4 transcript level and ±SD (lane 3 = 1.0 for mGFP4 transcript). One representative blot from three biological replicates is shown.

Figure 4.

The putative zinc finger is required for P1 activity. (A) 35S:Flag-P1_1-395, 35S:P1-C85A/C88A, 35S:P1-C85A/C91A, 35S:P1-C88A/C91A, 35S:P1-C88A/C103A, 35S:P1-C88A/C106A, 35S:P1-C91A/C103A, 35S:P1-C91A/C106A and 35S:P1-C103A/C106A were co-agroinfiltrated with 35S:mGFP4. Photos were taken under UV light at 3 days post agroinfiltration. (B) GFP mRNA was detected by Northern blotting. Extracts of infiltrated leaves were analyzed by SDS-PAGE followed by immunoblot with anti-GFP antibody. Flag-tagged P1 proteins were detected by Western blotting using anti-Flag antibody in both Western blots. Ponceau staining shows equal loading of proteins in both Western blots. Top panel, mean (n = 3) relative mGFP4 transcript level and ±SD (lane 3 = 1.0 for mGFP4 transcript). One representative blot from three biological replicates is shown.

Figure 5.

The zinc finger in SPMMV P1 is not required for AGO1 binding. (A) P1 binds to AGO1-DAH. 35S:amiR173, 35S:HA-AGO1, 35S:HA-AGO1-DAH, 35S:Flag-P1_1-395 were agroinfiltrated in N. benthamiana leaves as indicated. Flag IP was carried out, then input and IP fractions were analyzed by Western blot to detect P1_1–395, AGO1 and AGO1-DAH proteins. Ponceau staining shows equal loading of proteins. (B) 35S:Flag-P1_1-395, 35S:P1-C88A/9C1A, 35S:P1-C88A/C103A, 35S:P1-C88A/C106A, 35S:P1-C91A/C103A, 35S:P1-C91/C106A and 35S:P1-C103A/C106A were coinfiltrated with 35S:HA-AGO1-DAH. Input and IP protein fractions were analyzed by SDS-PAGE followed by anti-HA or anti-Flag immunoblot to detect HA-AGO1-DAH or Flag-P1_1–395 proteins, respectively. Ponceau staining shows equal loading of proteins. One representative blot from three biological replicates is shown.

Figure 6.

Target RNA binding is inhibited by SPMMV P1. 35S:TAS1c1, 35S:mGFP4, 35S:amiR173, 35S:HA-AGO1, 35S:HA-AGO1-DAH, 35S:Flag-P1_1–395 and 35S:P1-C88A/C103A respectively were infiltrated into N. benthamiana leaves as indicated. To detect protein–protein interactions native extracts were analyzed, while for RNA immunoprecipitations infiltrated leaves were crosslinked with formaldehyde prior to extract preparation. Input and IP protein fractions were analyzed by SDS-PAGE followed by anti-HA or anti-Flag Western blot to detect HA-AGO1/HA-AGO1-DAH or Flag-P1_1–395 proteins, respectively. Ponceau staining shows equal loading of proteins. Input and IP RNA fractions where analyzed by semi-quantitative RT-PCR to detect TAS1c or actin mRNAs. One representative blot from three biological replicates is shown.

Figure 7.

Model for P1 silencing suppression mechanism. (A) The AGO1-sRNA binary complex binds target RNA leading to RNA cleavage or translational inhibition. (B) P1 interferes with target RNA association in a competitive way. (C) P1 interferes with target RNA association in a non-competitive way.

Figure 8.

Model for SPMMV pathogenicity based on P1 inhibition of target RNA binding by pre-assembled AGO1 complexes. P1 binding to AGO1 complexes loaded with SPMMV-derived vsiRNAs could inhibit SPMMV RNA targeting by preventing its association with pre-assembled AGO1 complxes. In addition, P1 binding to AGO1/miR403 complexes could prevent their association with AGO2 mRNA binding, leading to derepression of AGO2 mRNA. AGO2 would overaccumulate and load SPMMV vsiRNAs to target complementary SPMMV RNAs. For simplicity, P1 interaction with AGO2 is omitted in this model.