Interaction Between Rice Protein Pip2-2and Rice Stripe Virus-Encoded Proteins

Rice stripe virus (RSV) causes a serious rice viral disease in China and East Asia. RSV-infected plants are dwarfed, with withered sterile spikes, resulting in significant losses of production. RSV is primarily spread by the small brown rice planthopper (SBPH), and its genome has four single-stranded RNAs.The nucleolus is an important cell structure, responsible for rRNA transcription and assembly of ribosomes. Pathological studies reveal that the nucleolus also plays important roles in viral infection. Viruses can interact with the nucleolus or its components to facilitate the transportation of viral particles, transcription and translation of the viral genome.In our previous work, Yeast two-hybrid (Y2H) screening showed that a rice protein, named Pip2-2, interacted with RSV p2protein. In this study we used Y2H and bimolecular fluorescence microscopy to find Pip2-2not only interact with p2protein, but also interact with pc2protein. And we used microscopy to demonstrate that GFP-fused Pip2-2(Pip2-2-GFP) localized at the nucleus, but with two different configurations:there were dispersed granules in the nucleus and a single granule that co-localized with Fib2, a nucleolus marker protein. A potential nuclear localization signal (NLS) was identified in the N-terminus of Pip2-2and when this was mutated, localization to the nucleolus was lost.To examine the biological function of Pip2-2in RSV infection, we over-expressed Pip2-2in N. benthamiana plants systemically using the PVX vector, and then inoculated the plants with RSV. Results showed that at20dpi of RSV, RSV symptoms on plants infected with PVX empty vector were more serious than those on the control plants infected with PVX-Pip2-2. Real-time PCR showed that RSV RNAs from the PVX-Pip2-2-infected plant accumulated at a higher level than in control plants. This result suggests that Pip2-2may function as a resistance factor during RSV infection.To further confirm the function of Pip2-2, transgenic rice over-expressing Pip2-2was obtained and challenged with RSV. On T2generation plants over-expressing Pip2-2, RSV symptoms were alleviated and there was low mortality compared to that in the control plants, supporting the view that Pip2-2functions as a resistance factor. We also tried to down-regulate Pip2-2expression in plants but it was difficult to regenerate the plants from calli, and those plants that did grow were weak and fruitless, indicating that Pip2-2also plays key roles in rice development.Taken together, the results showed that the expression of Pip2-2was induced by RSV infection. By interacting with RSV p2and pc2, Pip2-2may affect their normal function in RSV infection, hence conferring resistance against RSV when it is overexpressed in plants.