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An E3 ubiquitin ligase from Nicotiana benthamiana targets the replicase of Bamboo mosaic virus and restricts its replication

Chen, I‐Hsuan, Chang, Jui‐En, Wu, Chen‐Yu, Huang, Ying‐Ping, Hsu, Yau‐Huei, Tsai, Ching‐Hsiu
Molecular plant pathology 2019 v.20 no.5 pp. 673-684
Bamboo mosaic virus, Nicotiana benthamiana, complementary DNA, fluorescent proteins, gene silencing, genes, loss-of-function mutation, polypeptides, precipitin tests, protoplasts, two hybrid system techniques, ubiquitin-protein ligase
One up‐regulated host gene identified previously was found involved in the infection process of Bamboo mosaic virus (BaMV), a single‐stranded positive‐sense RNA virus. The full length cDNA of this gene was cloned by 5′ and 3′‐rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved really interesting new gene (RING) domain and a transmembrane domain. The gene might function as an ubiquitin E3 ligase. We designated this protein in Nicotiana benthamiana as ubiquitin E3 ligase containing RING domain 1 (NbUbE3R1). Further characterization by using Tobacco rattle virus‐based virus‐induced gene silencing (loss‐of‐function) revealed that increased BaMV accumulation was in both knockdown plants and protoplasts. The gene might have a defensive role in the replication step of BaMV infection. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed in N. benthamiana plants. The wild‐type NbUbE3R1‐orange fluorescent protein (NbUbE3R1‐OFP), NbUbE3R1/△TM‐OFP (removal of the transmembrane domain) and NbUbE3R1/mRING‐OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting the accumulation of BaMV. The common feature of these constructs was the intact substrate‐interacting domain. Yeast two‐hybrid and co‐immunoprecipitation experiments used to determine the possible viral‐encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an up‐regulated gene, NbUbE3R1 that plays a role in BaMV replication.