title: 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.
authors.affiliation: Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, a Key Laboratory of Beijing Municipality, China Agricultural University, Beijing, 100193, China.
authors.forename: Mingjun
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, a Key Laboratory of Beijing Municipality, China Agricultural University, Beijing, 100193, China.
authors.forename: Jiao
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, a Key Laboratory of Beijing Municipality, China Agricultural University, Beijing, 100193, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Feng
authors.affiliation: Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, a Key Laboratory of Beijing Municipality, China Agricultural University, Beijing, 100193, China.
authors.forename: Xianyou
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, a Key Laboratory of Beijing Municipality, China Agricultural University, Beijing, 100193, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Luo
authors.affiliation: Department of Plant Pathology, China Agricultural University, Beijing, 100193, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, a Key Laboratory of Beijing Municipality, China Agricultural University, Beijing, 100193, China.
authors.forename: Yuqin
authors.initials: Y
authors.lastname: Cheng
mesh_terms: D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism; D017790:Closterovirus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D010935:Plant Diseases / Q000821:virology; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000821:virology; D034741:RNA, Small Interfering / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism / Q000821:virology*
chemical_list: D060565:Argonaute Proteins; D034741:RNA, Small Interfering
keywords: Grapevine leafroll-associated virus 2; RNA silencing suppressor; argonaute protein; p24; self-interaction