Characterization of silencing suppressor p24 of Grapevine leafroll‐associated virus 2

Abstract

Grapevine leafroll-associated virus 2 (GLRaV-2) p24 has been reported to be an RNA silencing suppressor (RSS). However, the mechanisms underlying p24's suppression of RNA silencing are unknown. Using Agrobacterium infiltration-mediated RNA silencing assays, we showed that GLRaV-2 p24 is a strong RSS triggered by positive-sense green fluorescent protein (GFP) RNA, and that silencing suppression by p24 effectively blocks the accumulation of small interfering RNAs. Deletion analyses showed that the region of amino acids 1-188, which contains all predicted α-helices and β-strands, is required for the RSS activity of p24. Hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, previously shown to be critical for p24 self-interaction, are also crucial for silencing suppression, and western blotting results suggested that a lack of self-interaction ability results in decreased p24 accumulation in plants. The mutants showed greatly weakened or a lack of RSS activity. Substitution with two basic residues at positions 2 or 86, putatively involved in RNA binding, totally abolished the RSS activity of p24, suggesting that p24 uses an RNA-binding strategy to suppress RNA silencing. Our results also showed that W54 in the WG/GW-like motif (W54/G55) is crucial for the RSS activity of p24, whereas p24 does not physically interact with AGO1 of Nicotiana benthamiana. Furthermore, p24 did not promote AGO1 degradation, but significantly up-regulated AGO1 mRNA expression, and this effect was correlated with the RSS activity of p24, indicating that p24 may interfere with microRNA-directed processes. The presented results contribute to our understanding of viral suppression of RNA silencing and the molecular mechanisms underlying GLRaV-2 infection.

p24G1 Encoded by Grapevine Leafroll-Associated Virus 1 Suppresses RNA Silencing and Elicits Hypersensitive Response-Like Necrosis in Nicotiana Species

“…Leaves of GFP-expressing transgenic N. benthamiana line 16c [ 37 ] were infiltrated with cultures harboring pGD–GFP and pGD–p24 G1 . Coinfiltration of pGD–GFP with an empty vector (pGD), or with pGD–p19 [ 27 ] expressing p19 of TBSV, a well-characterized suppressor, was used as negative and positive controls, respectively. Leaf patches expressing p24 G1 showed strong GFP fluorescence three days post-infiltration (dpi), similar to that with expression of p19 ( Figure 1 a).…”

“…Basic aa residues are critical for protein nuclear transport [ 40 ], as well as for viral RSS binding of siRNA and suppression of RNA silencing [ 10 , 12 , 27 ]. p24 G1 contains a predicted NLS in the aa 122–139 region ( Figure S4a ) that is rich in basic residues ( Figure S3a ), and our results showed that mutant ∆122–139 almost lost its nuclear localization, and failed to bind ds siRNA, block RNA silencing and induce a necrotic response in N. benthamiana ( Figure 4 , Figure 5 , Figure 6 and Figure 7 ).…”

“…The young fully expanded leaves of N. tabacum , and wt and line 16c N. benthamiana plants were used for agroinfiltration. Agrobacterium tumefaciens GV3101 was transformed with each plasmid and agroinfiltration was performed according to previously described methods [ 27 ]. For RSS activity and pathogenicity assays, agroinfiltrated plants were monitored every day.…”

“…Extraction of protein from plant tissues and Western blot analysis were performed as detailed previously [ 27 ]. Antibody/antisera and dilutions used in the Western blot analysis were as follows: anti-GFP antibody (1:5000, ComWin, Beijing, China), and anti-p24 G1 (1:2000, noncommercial), anti-CP of PVX (1:2000, noncommercial) and anti-NbPR10 (1:1000, noncommercial) antisera.…”

“…ORFs 1a and 1b encode two replication-associated proteins, and the other eight ORFs encode a small transmembrane protein of 7 kDa (p7), a heat-shock protein 70 homolog, a polypeptide of 55 kDa (p55), coat protein (CP), two divergent copies of the CP (CPd1 and CPd2) and two polypeptides of 21 kDa (p21) and 24 kDa (p24) with unknown function [ 24 ]. In the genus Closterovirus , citrus tristeza virus encodes three distinct RSSs: p20, p23 and CP [ 25 ]; beet yellow virus (BYV) and GLRaV-2 encode one RSS: p21 [ 26 ] and p24 [ 27 ], respectively. Within the genus Ampelovirus , GLRaV-3 encodes one RSS, p19.7 [ 28 ]; pineapple mealybug wilt associated virus-2, which is closely related to GLRaV-1 and GLRaV-3, encodes four RSSs—p20, CP, p22 and CPd [ 29 ].…”