Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus

Abstract

Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent long distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. Both N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.

Effect of Rice stripe virus NS3 on Transient Gene Expression and Transgene Co-Silencing

“…The open reading frame (ORF) consists of 636 nucleotides and 211 amino acids. The NS3 genes compared with YG-JN1 were selected from GenBank (NCBI) only with a high degree of similarity such as Y11095 (Liang et al, 2005), FJ602686 (Jonson et al, 2009), FN650803 (unpublished) and EF198724 (Xiong et al, 2009). Sanisbury and Lomonossoff, 2008).…”

“…Thus, transient expression systems which include with suppressors can be a powerful tool for the production of large amounts of functional proteins in a short time. The NS3 gene has been identified as a gene silencing suppressor not only in plants and cultured insect cell (Hemmes et 2007), but also in mammalian cells (Schnettler et al, 2008), through the binding and sequestering of siRNAs (Xiong et al, 2009). Here, we isolated the NS3 gene from a Korean isolate and reported its effect on both transient gene expression and transgene co-silencing in comparison with CMV 2b (Choi et al, 2008).…”

Analysis of rice stripe virus whole-gene expression in rice and in the small brown planthopper by real-time quantitative PCR

“…Zhang et al (2010) searched EST library for RSV gene sequences of viruliferous SBPH, and discovered that NS3 was the most abundant transcript. As a suppressor of gene silencing, NS3 can signifi cantly reduce the accumulation of siRNA in plant cells, and can bind 21-nt single-strand siRNA, siRNA duplexes, and long single-strand RNA (Xiong et al, 2009). Th e dominant expression of NS3 gene suggests that aft er invading the host cells, RSV must suppress the immune responses of plant and insect host.…”

“…RNA2 encodes NS2 protein, a silencing suppressor with other unknown functions (22.8 kDa) and NSvc2, a putative membrane glycoprotein (94 kDa) (Takahashi et al, 1993;Du et al, 2011). RNA3 encodes NS3, a suppressor of gene silencing (23.9 kDa) and a nucleocapsid protein (NCP or CP, 35 kDa) (Kakutani et al, 1991;Zhu et al, 1991;Xiong et al, 2009). RNA4 encodes a nonstructural disease-specifi c protein (SP, 20.5 kDa) and a movement protein NSvc4 (32.5 kDa) (Toriyama, 1986;Kakutani et al, 1990;Zhu et al, 1992;Xiong et al, 2008).…”

Suppression of NS3 and MP Is Important for the Stable Inheritance of RNAi-Mediated Rice Stripe Virus (RSV) Resistance Obtained by Targeting the Fully Complementary RSV-CP Gene

“…vRNA 2 encodes NS2 of unknown function, and vcRNA 2 encodes pC2 glycoprotein. RNA 3 encodes NS3, a viral suppressor of RNA-silencing (Xiong et al, 2009), and pC3, a viral NCP. RNA 4 encodes SP, a major non-structural protein that accumulates in infected plants, and pC4, a viral movement protein (MP; Xiong et al, 2008b).…”