Function and Characterization of the Argonaute2 Gene in the RNA Interference Pathway in the Diamondback Moth

Author(s): Muhammad Salman Hameed, Xiaodong Jing, Wei Chen, Jinzhi Chen, Liette Vasseur, Gefu Wang-Pruski, Guang Yang

Argonaute2 (AGO2) is a core catalytic component of the RNA-induces silencing complex (RISC) that binds to small guide RNAs containing small interfering RNA (siRNA) and microRNA (miRNA). The guide RNA leads RISC to the complementary mRNA for gene suppression. We cloned the full length cDNA (2193 bp) of the Ago2 gene (PxAgo2) from diamondback moth (DBM, Plutella xylostella). The predicted PxAgo2 protein had an 83 kDa molecular weight with a theoretical isoelectric point of 9.39. The phylogenetic tree showed a high similarity of PxAgo2 with Bombyx mori Ago2 (BmAgo2). Western blot and RT-qPCR analyses showed a clear increase in the PxAgo2 mRNA and protein expression levels in the egg, 4th instar larva, pre-pupa, pupa and adult. The double-stranded RNA-mediated RNAi of PxAgo2 in DBM larvae was found 3 h after dsRNA injection, and the knockdown level was increased over time up to 36 h. PxAgo2 silencing recovered the expression of PxBurs-α (Bursicon-α in DBM) to the normal expression level, which was suppressed by dsPxBurs-α in a DBM cell line. The overexpression of PxAgo2 fundamentally enhanced the PxBurs-α silencing efficiency in DBM cells. Our findings reveal that PxAgo2 is involved in the dsRNA-regulated gene silencing mechanism and performs a crucial function in the RNAi process of DBM.