Regulated Nuclear Trafficking of rpL10A Mediated by NIK1 Represents a Defense Strategy of Plant Cells against Virus

The NSP-interacting kinase (NIK) receptor-mediated defense pathway has been identified recently as a virulence target of the geminivirus nuclear shuttle protein (NSP). However, the NIK1–NSP interaction does not fit into the elicitor–receptor model of resistance, and hence the molecular mechanism that links this antiviral response to receptor activation remains obscure. Here, we identified a ribosomal protein, rpL10A, as a specific partner and substrate of NIK1 that functions as an immediate downstream effector of NIK1-mediated response. Phosphorylation of cytosolic rpL10A by NIK1 redirects the protein to the nucleus where it may act to modulate viral infection. While ectopic expression of normal NIK1 or a hyperactive NIK1 mutant promotes the accumulation of phosphorylated rpL10A within the nuclei, an inactive NIK1 mutant fails to redirect the protein to the nuclei of co-transfected cells. Likewise, a mutant rpL10A defective for NIK1 phosphorylation is not redirected to the nucleus. Furthermore, loss of rpL10A function enhances susceptibility to geminivirus infection, resembling the phenotype of nik1 null alleles. We also provide evidence that geminivirus infection directly interferes with NIK1-mediated nuclear relocalization of rpL10A as a counterdefensive measure. However, the NIK1-mediated defense signaling neither activates RNA silencing nor promotes a hypersensitive response but inhibits plant growth and development. Although the virulence function of the particular geminivirus NSP studied here overcomes this layer of defense in Arabidopsis, the NIK1-mediated signaling response may be involved in restricting the host range of other viruses.

Incorporated radioactivity in protein bands was quantified by phosphoimaging and protein loading by densitometry using the Multi Gauge V3.0 software (Fujifilm). Subcellular localization of proteins. For subcellular localization of proteins, Nicotiana tabacum leaves were agroinoculated with pK7F-L10, pYFP-L10, pK7F-L18, pYFP-L18, pK7F-NIK1, pK7FNIK2, pK7F-NIK3, pK7F-NIK1T474D or pK7F-NIK1G473V/T474A using Agrobacterium tumefaciens strain GV3101. Nicotiana tabacum plants were grown in a greenhouse with natural day length illumination. For experimental use, plants (about three week after germination) were transferred to a growth chamber at 21°C with a 16-hour light and 8-hour dark cycle. Each construct was mobilized in Agrobacterium tumefaciens strain GV3101 by freeze-thawing followed by selection on YEB plates containing the appropriate antibiotics. Agrobacterium-mediated transient expression in tobacco leaf epidermal cells was conducted as previously described [13,41]. About 72 hours post-agroinfiltration, 1-cm 2 leaf explants were excised and GFP and YFP fluorescence patterns were examined in epidermal cells with 40X or 60X oil immersion objective and a Zeiss inverted LSM510 META laser scanning microscope equipped with an argon laser and a helium laser as excitation source. For imaging GFP, the 458-488 nm excitation line and the 500 to 530 nm band pass filter were used. Excitation of YFP was at 514-560 nm and YFP emission was detected by using a 560-600 nm filter. Controls were performed to ensure clear separation of GFP and YFP signals.
The pinhole was usually set to give a 1 to 1.5 µm optical slice. Post-acquisition image processing was done using the LSM 5 Browser software (Carl-Zeiss) and Adobe Photoshop (Adobe Systems).

Protoplast isolation and co-immunoprecipitation of ectopically expressed proteins.
Protoplasts were prepared from leaves of 4 to 6 week old tobacco plants that had been agroinfiltrated with the constructions as indicated in the Figure 2, as described [13]. Frozen protoplasts were homogenized with two volumes of ice-cold buffer (150 mM Tris/HCl, 150 mM NaCl, 1.5 mM EDTA and 1.5% (v/v) Triton X-100, pH 7.5) supplemented with 0.1 mM PMSP. Cell homogenates from 2 x 10 6 cells from leaf protoplasts expressing NIG or NSP-YFP or both proteins were incubated with rabbit polyclonal antisera raised against GFP (Invitrogen) and protein A-sepharose. Immunoselected proteins were analyzed by SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were blocked in NaCl-Tris containing 0.05% (v/v) Tween 20 and 1% (w/v) nonfat dry milk, and then incubated with a rabbit anti-GFP or rabbit anti-rpL10 serum for 2 h at room temperature. Bound antibody was detected using an alkaline phosphatase-conjugated goat anti-rabbit IgG serum in conjunction with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad, UK) detection reagents.
Plant material, growth conditions, and genotyping. The Columbia (Col-0) ecotype of Arabidopsis thaliana was used as the wild type. The rpl10 and nik1 mutants were from the SALK Institute (SALK_010170). Seeds were surface sterilized and cold treated at 4°C for 2 days in the dark and then exposed to white light. Seedlings were grown at 22°C on plates  Photographs were taken with a UV filter. The images were processed using Adobe Photoshop. Tissue was harvested from infiltration zones and used for RNA extraction.
Total RNA from cells was extracted using an RNAeasy kit (Qiagen). The RNA was treated with RNase-free DNase (Promega) at 37 °C for 1 h and the DNAse was inactivated at 65 °C for 10 min. Reverse transcription reaction was done using the ImProm-IITm Reverse Transcription System (Promega) according to the manufacturer's instructions. A typical reaction consisted of 1 µl of the reverse transcription reaction, 0.5 mM each dNTP, 100 mM each sense and antisense gene-specific primers, 1 × PCR buffer, 1.5 mM MgCl2), and 1 unit of Taq DNA polymerase (Promega) in a total volume of 20 µl. The primers used are listed in