JIA-2018-09

2035 PAN Yuan et al. Journal of Integrative Agriculture 2018, 17(9): 2031–2041 3.3. P6 IBs traffic along microfilaments and colocal- izes with P1 at the cell periphery In view of our finding that P6 IBs and chloroplast movement are independent, we explored whether the P6 IBs move freely, or if their movement depends on cytoskeleton structures. We used laser scanning confocal microscopy to take pictures every 12 s and observed P6 IB trafficking throughout the length of the actin microfilaments (Fig. 3-A). Actin monomers are dynamic that can rapidly assemble to form microfilaments, which can in turn be simultaneously be degraded by other plant proteins. However, in our experiments, the actin microfilaments remained stable despite the fact that IB fluorescence traversed from one end to the other of the microfilaments (Fig. 3-A). CaMV P6 is thought to be involved in the transport of virus particles, and CaMV P1 is also considered to be a movement protein (Baughman et al . 1988). P6 and P1 also interact in vitro as determined by yeast two-hybrid and pull-down experiments (Hapiak et al . 2008). In SVBV, laser scanning confocal microscope showed that at 3 dpi, SVBV P6 IBs co-localized with SVBV P1 at periphery of the cell (Fig. 3-B). Therefore, we speculate that P6 moves along the microfilament network to interact with P1 at the plasmodesmata (PD). 3.4. The P6 C-terminal 25 aa affect nuclear localiza- tion and inclusion body formation To explore key amino acids responsible for P6 nuclear localization, we used a prediction program available at http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form. cgi, this program identified two putative nuclear localization signal (NLS) regions present at 193–223 and 402–426 aa of the 514 aa P6 protein (Fig. 4-A). To validate this prediction, we constructed two P6 deletion mutants, M1 (deletion 193–223 aa) and M2 (deletion 402–426 aa). The M1 and M2 mutants were fused to GFP and agroinfiltrated into N . benthamiana leaves, respectively. The results indicate that the P6 M1 mutant protein could assemble into amorphous IBs with different sizes, but these differed in their distribution. The larger IBs accumulated in, or adjacent to, the nucleus, and the smaller IBs were distributed throughout the cytoplasm (Fig. 4-B). However, the M2 mutant protein lacking the 25 aa C-terminal region was present only at the cell periphery, and there was no evidence for nuclear import or formation of cytoplasmic IBs (Fig. 4-C). We substituted the SV40 NLS into P6 for the 402–426 aa region to construct the chimeric mutant M5. The M5-GFP fluorescence concentrated in the nucleus, showing that the SV40 NLS could restore the M2 nuclear localization function (Fig. 4-C). Analysis of the 402–426 aa P6 polypeptide revealed a polar region (16 of 25 aa) that consisted of one acidic and seven basic amino acids. Furthermore, a secondary structure prediction of the 25-aa polypeptide indicated that the region mainly contains α-helices, which is similar to the CaMV P6 key region required for IB formation (Lutz et al . 2015). To obtain further information about the function of the P6 25 residue peptide in IBs, we constructed two additional P6 mutants, M3 (1–402 aa) and M4 (1–426 aa). Visualization of infiltrated tissue showed that the GFP fluorescence of both the M3 and M4 mutants was distributed at the cell periphery without any IB formation, as is the case for the M2 mutant (Fig. 4-D). This result indicates that 402–426 aa is necessary but is not sufficient for P6 formation of IBs; hence, other P6 regions likely contribute to IB generation. Fig. 2 P6 inclusion body (IB) accumulation in the nucleus, and colocalization with the cytoskeleton and endoplasmic reticulum (ER). Transient expression was performed in Nicotiana benthamiana leaves via Agrobacterium infiltration. A, P6 import into the nucleus. a, P6-GFP; b, DAPI stained nucleus; c, overlay of a and b. B, P6 IB and cytoskeleton colocalization. P6 IBs were marked with the red arrowheads. a, GFP labeled microfilaments; b, coexpression of P6-GFP with GFP-ABD2- GFP; c, P6-GFP; d, mCherry labeled microtubules, mCherry- MAP65-1; e, overlay of c and d. C, P6 IBs co-localized with ER. a, P6-GFP; b, mCherry labeled ER, mCherry-HDEL; c, overlay of a and b. Magnification bar for all images is 50 μm. A B C a b c c d a b e a b c