Schematic diagram of ihpRNA construction with pRNAi-GG (Golden Gate) vector.

<p>(A) The cassette of pRNAi-GG. The duplicated 35S CaMV promoter, two copies of <i>ccdB</i> gene, the Pdk intron, four BsaI recognition sites with designed adaptors (cleavage site sequences of BsaI) are cloned between the HindIII and SacI of T-DNA vector pBI121. Adaptors with the same color have the same sequences but opposite orientation. A chloramphenicol-resistant gene (Cm^r) was contained in the Pdk intron. (B) One-step construction of ihpRNA. The target fragment of the gene of interest is PCR amplified using gene-specific primers carrying BsaI sites and adaptors complementary to the appropriate sequences on the vector. The purified PCR product is mixed, in one tube, with pRNAi-GG vector, BsaI enzyme and T4 ligase for a one-step restriction-ligation reaction.</p

Silencing of two marker genes GUS and GFP and endogenous NbPDS by transient expression of the ihpRNA constructs.

<p>(A) Silencing effect of GUS ihpRNA on transient expression of GUS gene. Agrobacterium cultures containing pBIN19-GUS were mixed with Agrobacterium containing pRNAi-GFP (left) or pRNAi-GUS (right), and infiltrated into different parts of the same leaves of <i>N. benthamiana</i> plants. (B) Silencing effect of green fluorescent protein (GFP) ihpRNA on transient expression of GFP gene. Agrobacterium cultures containing pBIN19-GFP were mixed with Agrobacterium containing pRNAi-GUS (left) or pRNAi-GFP (right), and infiltrated into different parts of the same leaves of <i>Nicotiana benthamiana</i> plants. (C) Real time PCR was performed to analyze the silencing effect of GUS, GFP and NbPDS genes. Error bars represent standard deviations (SD) of three independent experiments.</p

Efficiency of cloning of pRNAi-GG at different restriction-ligation times.

<p>The number of recombinant colonies for each transformation was counted. Restriction-ligation was performed with continuous incubation at 37°C or for 20–50 cycles (2 min 37°C+5 min16°C). Cloning for pRNAi-GFP and pRNAi-GFP/PDS were performed in duplicate. The negative control was performed without BsaI enzyme. NT, not tested.</p><p>For each transformation, 12 clones (when less than 12, pick all) were identified by PCR using vector specific primes P21 and P22, and insert reverse primer P12 (for pRNAi-GFP) or P16 (for pRNAi-GFP/PDS). All the clones showed the expected bands, as part of the results was shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038186#pone-0038186-g002" target="_blank">Fig. 2A</a>.</p

RT-PCR detection of ANRSV and its derivatives.

The upper non-inoculated leaves of N. benthamiana plants were assayed at 16 dpi. RT-PCR was conducted with primer set 8900F/9300R (S2 Table) that target viral CP region. (TIF)</p

HCPro2 interacts with CI and CP <i>in planta</i>.

(A) Y2H tests the interactions of HCPro2 with P3N-PIPO, CI and CP. The coding sequences of HCPro2, P3N-PIPO, CI and CP were cloned into pGBKT7-DEST or pGADT7-DEST for the expression of these proteins fused with GAL4 BD or AD domain. Yeast competent cells (Y2H Gold) were co-transformed to express the indicated pairs of proteins. The transformed cells were subjected to 10-fold serial dilutions and plated on the SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade mediums. The plates were cultured at 28°C for four to six days before photographing. Co-transformation of a pair of plasmids for simultaneous expression of AD-T7-T and BD-T7-53 was included as the positive control. (B) BiFC tests the interactions of HCPro2 with CI, CP and P3N-PIPO. The coding sequences of HCPro2, CI, CP and P3N-PIPO were individually engineered into pEarleyGate201-YN and pEarleyGate202-YC for the expression of these proteins fused with the YN or YC part of YFP. N. benthamiana leaves were co-inoculated for the expression of the indicated pairs of proteins. YFP signals (shown in green) were observed by fluorescence microscope at 72 hpi. Bars, 50 μm. (C, D) Co-IP tests the interactions of HCPro2 with CI and CP. The inoculated leaves of N. benthamiana plants for co-expression of GFP-HCPro2 / Myc-CI (C) or GFP-HCPro2 / Myc-CP (D) were sampled at 72 hpi for Co-IP assays using GFP-Trap Agarose. Total protein extracts prior to (Input) and after immunoprecipitation (IP) were analyzed by immunoblotting using anti-Myc and anti-GFP antibodies.</p

MYTH tests the interactions of HCPro2 with CI, CP and P3N-PIPO.

Yeast competent cells (NMY51) were co-transformed to express the indicated pairs of proteins. The transformed cells were subjected to 10-fold serial dilutions and plated on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade mediums. The plates were cultured at 28°C for four to six days before photographing. Co-transformation of a pair of constructs for simultaneous expression of soybean mosaic virus (SMV) P3-Cub-LexA and Nub-EIF4A [101] was included as the positive control. (TIF)</p

Silencing of <i>NbRbCS</i> or <i>NbFNR</i> in <i>N</i>. <i>benthamiana</i>.

(A) Phenotypic observation of NbRbCS- or NbFNR-silenced in N. benthamiana. N. benthamiana seedlings at 3- to 5-leaf stage were inoculated with pTRV1 along with pTRV2-NbRbCS (TRV-NbRbCS) or pTRV2-NbFNR (TRV-NbFNR), and photographed at 12 dpi. Co-inoculation of pTRV1 and pTRV2-GUS was included as the parallel control. Bars, 2.5 cm. (B, C) Real-time RT-qPCR analysis of NbRbCS or NbFNR mRNA transcript accumulation. The samples were collected at 12 dpi for the assay. Error bars denote the standard errors from three biological replicates. The average value for TRV-GUS was designated 1.0 to normalize the data. ***, P (TIF)</p

List of host proteins that are uniquely identified from co-purified products with 2×Strep-HCPro2 by LC-MS/MS.

List of host proteins that are uniquely identified from co-purified products with 2×Strep-HCPro2 by LC-MS/MS.</p

Real-time RT-qPCR analysis of viral genomic RNA accumulation.

The upper non-inoculated leaves of N. benthamiana plants were sampled at 12 dpi for the assay. RT-qPCR with primer set RS9200F/RS9350R (S2 Table) targeting viral CP region was performed. Error bars denote the SD from three biological replicates. The average value for pRS-G-2×Strep-HCPro2 was designated 1.0 to normalize the data. *, 0.01PP (TIF)</p

A working model depicting that NbRbCS is co-opted as the scaffold protein in mediating the assembly of viral intercellular movement complex.

NCLS, nucleus; CHL, chloroplast; PM, plasma-membrane; CW, cell wall; PD, plasmodesmata.</p