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. 2018 Mar 15:2018:8402839.
doi: 10.1155/2018/8402839. eCollection 2018.

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 , Ruoxue Meng  2
Affiliations

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

Luping Zheng et al. Can J Infect Dis Med Microbiol. .

Abstract

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 two-hybrid, 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).

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Figures

Figure 1
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.
Figure 2
Figure 2
Interactions between p2 and the three functional domains of NbFib2 as examined by yeast two-hybrid assay. An X-α-gal assay shows that p2 interacts with the three domains of NbFib2, respectively. pGAD-T + pGB-Lam is a negative control, and pGAD-T + pGBK-53 is a positive control.
Figure 3
Figure 3
p2 colocalizes with the three functional domains of NbFib2 in the leaves of N. benthamiana. (a) p2-CFP was coexpressed with NbFib2-1-YFP. (b) p2-CFP was coexpressed with NbFib2-2-YFP. (c) p2-CFP was coexpressed with NbFib2-3-YFP. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Possible nucleolus and cajal body described in the text are designated with red and blue arrows, respectively. Fluorescence was observed at 48 h postinfiltration. Scale bars, 10 μm.
Figure 4
Figure 4
p2 interacts with the three functional domains of NbFib2 in BiFC assay. (a) YC-p2 was coexpressed with YN-NbFib2-1. (b) YC-p2 was coexpressed with YN-NbFib2-2. and (c) YC-p2 was coexpressed with YN-NbFib2-3. The nucleus was stained with 4,6-diaminophenylindole (DAPI). Fluorescence was observed at 48 h postinfiltration. Scale bars, 10 μm.
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