Identification of Functional Domain(s) of Fibrillarin Interacted with p2 of Rice stripe virus

Hindawi Canadian Journal of Infectious Diseases and Medical Microbiology Volume 2018, Article ID 8402839, 6 pages https://doi.org/10.1155/2018/8402839 Research Article Identification of Functional Domain(s) of Fibrillarin Interacted with p2 of Rice stripe virus Luping Zheng ,1,2 Jie He,2 Zuomei Ding,2 Chenlong Zhang,2 and Ruoxue Meng2 1 Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China 2 College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China Correspondence should be addressed to Luping Zheng; Received 4 October 2017; Accepted 6 December 2017; Published 15 March 2018 Academic Editor: Jialiang Yang Copyright © 2018 Luping Zheng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. p2 of Rice stripe virus may promote virus systemic infection by interacting with the full length of fibrillarin from Nicotiana benthamiana (NbFib2) in the nucleolus and cajal body (CB). NbFib2 contains three functional domains. We used yeast twohybrid, colocalization, and bimolecular fluorescence complementation (BiFC) assays to study the interactions between p2 and the three domains of NbFib2, namely, the N-terminal fragment containing a glycine and arginine-rich (GAR) domain, the central RNA-binding domain, and the C-terminal fragment containing an α-helical domain. The results show that the N-terminal domain is indispensable for NbFib2 to localize in the nucleolus and cajal body. p2 binds all three regions of NbFib2, and they target to the nucleus but fail to the nucleolus and cajal bodies (CBs). 1. Introduction Rice stripe virus (RSV), an economically significant pathogen of rice, is the member of the genus Tenuivirus. It is transmitted by the small brown planthopper (Laodelphax striatellus) in a persistent, circulative-propagative manner, affected by global warming [1, 2]. Nicotiana benthamiana (N. benthamiana) can be infected by RSV through mechanical sap inoculation [3]. The genome of RSV comprises four single-stranded RNAs, denoted as RNA1, RNA2, RNA3, and RNA4 in the decreasing order of their molecular weights [4]. Exception is RNA1 that is negative sense and encodes only one protein responsible for viral replication; all the other three RNA segments employ an unusual ambisense coding strategy and encode two proteins: one in the viral-sense RNA (vRNA) and the other in the viral complementary-sense RNA (vcRNA) [5, 6]. RNA2 encodes two nonstructural proteins p2 and pc2; p2 is a viral RNA-silencing suppressor and is involved in systemic viral movement by interacting with fibrillarin [7, 8], and pc2 shares many characteristics common to the glycoproteins [9, 10]. RNA3 encodes a nonstructural protein p3, another suppressor of gene silencing [11], and a structural protein pc3, which is a nucleocapsid protein connected with resistance to RSV [12, 13]. The nonstructural disease-specific protein (SP) and the movement protein pc4 are encoded by RNA4 [14, 15]. Many different viruses bind to the nucleolus to manipulate host-cell functions and recruit nucleoprotein to aid in virus infection. Fibrillarin, an important nucleolus protein, was reported to interact with viral proteins and regulate virus replication, movement, and so on. Fibrillarin from Nicotiana benthamiana (NbFib2) mediates assembly of Umbravirus ribonucleoprotein particles (RNPs), which are capable of long-distance movement and causing systemic viral infection [16]. Protein 1 (NS1) in Influenza A H3N2 subtype virus binds to the fibrillarin via the C-terminal nuclear localization signal 2 (NLS2) [17]. There is also a close relationship between fibrillarin and suppressors of gene silencing. For example, fibrillarin interacted with viral genome-linked protein (VPg) in Potato virus A (PVA) and the 2b silencing suppressor protein in Cucumber mosaic virus (CMV), respectively [18, 19]. In our previous works, we found that p2 of RSV targeted to NbFib2 to promote virus systemic movement [8]. NbFib2 is an evolutionarily conserved protein, it is usually consisted of three 2 Canadian Journal of Infectious Diseases and Medical Microbiology RBS GAR region “α-rich region” NbFib2 pGAD-p2 + pGBK-NbFib2-1 pGAD-p2 + pGBK-NbFib2-2 pGAD-p2 + pGBK-NbFib2-3 pGAD-p2 + pGBK pGAD + pGBK-NbFib2-1 pGAD + pGBK-NbFib2-2 pGAD + pGBK-NbFib2-3 pGAD-T + pGBK-Lam pGAD-T + pGBK-53 NbFib2-1 1 aa 130 aa NbFib2-2 131 aa 221 aa NbFib2-3 222 aa 314 aa Figure 1: Three functional domains of NbFib2. NbFib2-1: N-terminal fragment from 1 aa to 130 aa, containing a GAR region and a glycine- and arginine-rich domain. RBS means RNA binding sites. NbFib2-2: the central RNA-binding domain from 131 aa to 221 aa. NbFib2-3: the C-terminal fragment from 222 aa to 314 aa, containing an α-helical domain. domains, a glycine and arginine-rich domain (GAR), an RNA-binding domain, and an α-helical domain [20], but how p2 interacted with those motifs of NbFib2 is still unknown. In this study, the interactions between p2 and the three domains of NbFib2 are identified using yeast two-hybrid, colocalization, and BiFC methods. The results reveal that p2 binds to the three domains of NbFib2 in the nucleus but fails to target to the nucleolus and cajal bodies (CBs), and the GAR domain is necessary for NbFib2 to localize in the nucleolus and CBs. 2. Results and Discussion 2.1. p2 Interacts with Three Domains of NbFib2 in Yeast TwoHybrid Assay. NbFib2 is composed of three functional domains: N-terminal fragment, containing a glycine and arginine-rich (GAR) domain (NbFib2-1), the central RNAbinding domain (NbFib2-2), and C-terminal fragment, containing an α-helical domain (NbFib2-3) (Figure 1). As shown in Figure 2, yeast cells cotransformed with pGADT7 (pGAD)-p2 and pGBKT7 (pGBK)-NbFib2 grew and turned blue on SD medium containing X-α-gal but lacking adenine (Ade), histidine (His), leucine (Leu), and tryptophan (Trp) (SD/Trp−Leu−His−Ade−/X-α-gal+), and the cotransformants of pGAD-T/pGBK-53 and pGAD-T/pGBK-Lam were individually used as positive control and negative control. However, the cotransformants of pGBK/pGAD, pGBKNbFib2s/pGAD, pGBK/pGAD-NbFib2s, pGBK-p2/pGAD, or pGBK/pGAD-p2 failed to grow on SD/Leu−Trp−His−, although they grew well on SD/Trp−Leu− (Supplementary Figure S1). These results indicated that p2 of RSV interacts with the three domains of NbFib2 in yeast. 2.2. p2 Fails to Target to the Nucleus and Cajal Body in Colocation Assays. Colocalization result shows that only NbFib2-1 (GAR domain) can form bright spots in the nucleolus and cajal body (CB) (Figure 3(a)). The other two domains of NbFib2 (NbFib2-2 and NbFib2-3) also localize in nucleus, but they cannot agglomerate into small Figure 2: Interactions between p2 and the three functional domains of NbFib2 as examined by yeast (...truncated)