Plant material and growth condition
Plant-related experiments, such as agro-infiltration and viral inoculation were performed in a greenhouse (16 h light/8 h darkness, 20°C to 25°C). The seeds of C. quinoa and N. benthamiana were sown in peat soil. After germination, seedlings were transferred to soil and fertilized with Peters 20-20-20 once a week.
Construction of a mini binary vector
The strategy for constructing a mini binary vector with the 35S promoter and nos terminator is summarized in Figure 1. The pSa origin was amplified by polymerase chain reaction (PCR) with the primer pair FP-pSa-SpeI (5′- TCTTATCACTAGT AAGCCCGAGAGGTTGCCGCC -3′) and RP-pSa-NheI (5′- GGAGGGTAGGCTAGC GTTATCCACGTGAAACCGC -3′), which contain Spe I and Nhe I (underlined), respectively, from pGreen (Hellens et al., 2000) and was cloned into the pGEM-T vector (Promga) to generate pGEM-pSa. Next, the kanamycin resistance gene (KanR) was amplified from pBI121 binary vector with the primer pair FP-8-6-SacII (5′- GCCTGTGATCATCCGCGG TTTCAAAATCGGCTCCG -3′) and RP-AvrII (5′- GGTTTTTTTGTTTGCAAGCCTAGG CAGATTACGCGC -3′), which contains Sac II and Avr II (underlined), respectively. The PCR fragment of KanR gene was digested with Avr II/Sac II and ligated to Nhe I/Sac II-digested pGEM-pSa vector to generate pGEM-pSa-Kan. To remove the unnecessary DNA backbone, the primer pair FP-Kan-KpnI-AvrII (5′- GCTCCCGGTACC GCCAGGCGGCCTAGG TTTCAAAATCGG -3′) containing Kpn I and Avr II (underlined), and RP-pGEMT-ORI-KpnI (5′- GCCTCACTGATTAAGCATTGGTACC TGTCAGACC -3′) containing Kpn I, (underlined) was used to amplify the 2,916 bp fragment of pGEM-pSa-Kan that included the Ori-pSa-KanR sequence by PCR. The PCR product was digested with Kpn I and self-ligated to generate pSAK. Thereafter, the pSAK plasmid was digested with Ase I/Eoc RI to remove the restriction enzyme sites and the digested product was treated with Mung Bean nuclease to produce blunt ends and then self-ligated to generate pSAK-dMSC. The left border (LB) and CaMV 35S promoter sequence were amplified from pCaMVCN (Pharmacia) with the primers FP10-4-NcoI-KpnI (5′- CCGGCCGCCATGGGGTACC CCTCTCCAAATGAAAT -3′) and RP9-7-NheI (5′- GCGATGATCACAGGCTAGC AACGCTCTGTCATCG -3′), which contain Kpn I/Nco I, and Nhe I (underlined), respectively. The PCR product was then digested with Kpn I/Nhe I and ligated with Kpn I/Avr II-digested pSAK-dMSC vector to generate pSAK-35S-Pro. Finally, the DNA region including the ribozyme, nos, and right border (RB) was amplified from pTRV2 plasmid (Liu et al., 2002) with the primers FP1-KpnI (5′- ACTTGGTACC GTCTGTACTTATATCAGTACACTGACGAG -3′) and RP-NOS-RB-AvrII (5′- TTGGCACCTAGG TACAAATGGACGAACGGATAAAC -3′), which contain Kpn I, and Avr II (underlined), respectively. The PCR product was digested with Kpn I/Avr II and ligated with the Kpn I/Nhe I-digested pSAK-35S-Pro vector to generate the mini binary vector pBD003 of 4,141 bp.
Construction of in vivo full-length cDNA clones of TuMV for agro-infiltration
The strategy for constructing the pBD-TuMV serial in vivo infectious clones is summarized in Figure 2. Two in vivo TuMV infectious clones, p35S-TuMV-YC5 and p35S-TuMV-GFP, constructed on the pCaMVCN vector were provided by Dr. Shyi-Dong Yeh (Figure 2A) (Lin et al., 2009; Niu et al., 2006). The 3′-end fragment of the TuMV was amplified by PCR with the primers FP-TuCP9353-KpnI (5′-AACCGGTACC GACCATACATGCCACGATATGGTCTTC-3′) and RP-POLYA-AvrII (5′-AGGTCGACGCGGCCGCCTAGG TTTTTTTTTTTTTTT-3′), which contain Kpn I, and Avr II (underlined), respectively. The PCR fragment was digested with Kpn I/Avr II and then ligated to the Kpn I/Nhe I-digested pBD003 vector to generate pBD-TuCP. Next, the Eco RV/Kpn I-digested fragment from p35S-TuMV-YC5 was ligated into the Eco RV/Kpn I-digested pBD-TuCP vector to generate pBD-TuMV5′3′-YC5. The primers FP-TuMV7804-KpnI (5′- GGAAAGGTACC CGTGGATGATTTCAACAAC -3′) containing Kpn I (underlined), and MTuCP8854 (5′- GCCTCTCTCGTTCCTTTTCT -3′) were used to amplify the region between the NIb and CP genes from p35S-TuMV-YC5 and p35S-TuMV-GFP, respectively. The Kpn I/Mlu I-digested fragment from p35S-TuMV-YC5 was ligated with the same restriction enzyme-digested pBD-TuMV-5′3′-YC5, to generate pBD-TuMV5′3′X-YC5. In addition, the Kpn I/Mlu I-digested fragment from p35S-TuMV-GFP was ligated with Kpn I/Mlu I-digested pBD-TuMV-5′3′-YC5 to generate pBD-TuMV-5′3′X-GFP. Finally, the fragment that was digested by Kpn I/Xho I from p35S-TuMV-YC5 was ligated with the same enzyme-digested pBD-TuMV-5′3′X-YC5 and pBD-TuMV-5′3′X-GFP to generate pBD-TuMV-YC5 and pBD-TuMV-GFP, respectively (Figure 2B).
For the mild strain of TuMV construction, the R182K mutation was introduced on the HC-Pro gene by PCR mutagenesis on p35S-TuMV-YC5, following the method demonstrated by Lin et al. (2009). The HC-ProR182K sequence was digested with Mfe I/Kpn I and ligated with the same enzyme-digested pBD-TuMV-GFP to generate pBD-TuMV-GK (Figure 2C).
Infectivity assay of in vivo full-length cDNA clones
Local lesion host C. quinoa and systemic host N. benthamiana were used for the in vivo infectious clone infectivity assay. An aliquot of 10 μl (1 μg/μl) of individual constructs were mechanically introduced to leaves of C. quinoa with carborundum-dust, and the development of local lesions was recorded at 7 days post-inoculation (dpi). The mock (control) was mechanically inoculated with 10 μl ddH2O. For the agrobacterium mediated transformation activity assay, the Agrobacterium tumefaciens strain C58C1, which carries pBD-TuMV serial in vivo infectious clones, was incubated in an LB medium supplemented with kanamycin (100 μg/μl) at 28°C for 16 h. Thereafter, agro-infiltration was applied following the standard protocol (Llave et al., 2000). The symptoms and GFP fluorescence were observed after 6 days of infiltration.
Purification of recombinant TuMV CP and production of antisera
The TuMV coat protein (CP) gene was amplified from the p35S-TuMV-YC5 infectious clone with primers PTu-CP-NdeI (5′- GTGTTTATCATATG GCAGGTGAGACG -3′) and MTu-CP-XhoI (5′- CAACTTCACTCGAG CTATAACCCCTTAACGC -3′), which contain Nde I, and Xho I (underlined), respectively. The PCR fragment was digested with Nde I/Xho I and ligated with the same restriction enzyme-digested pET-28b to generate pET-TuCP. The pET-TuCP was transferred into E. coli BL21 (DE3), which was treated with 0.5 M IPTG in LB medium for recombinant his-TuCP production. The recombinant his-TuCP was purified with Ni-NTA column by fast protein liquid chromatography (FPLC) (AKTApurifier, GE Healthcare) and used for antisera production that were produced from New Zealand white rabbits, as described by Lin et al. (2002). The titer of TuMV CP antisera was analyzed by Enzyme-linked immunosorbent assay (ELISA) and western blotting.
Enzyme-linked immunosorbent assay (ELISA)
For verification of virus infection, each sample represented 3 inoculated leaves and 3 systemic leaves collected from each of the 3 repeated plants, with 3 leaf-discs (0.6 cm in diameter) punched from each leaf. These collected samples were assayed by indirect ELISA using the polyclonal antiserum to the TuMV CP. Goat anti-rabbit immunoglobulin G conjugated with alkaline phosphatase (Amersham Biosciences) was used as the secondary antibody, and p-nitrophenyl phosphate (Sigma) was used as the substrate for color development. Results were recorded by measuring absorbance at 405 nm (which were stained for 5 min after the addition of the substrate) using an ELISA reader (Perkin Elmer).
The analysis of variance (ANOVA) was performed at first to test the levels of absorbance under different conditions. It was followed by the pairwise comparison between two conditions of interest using Tukey’s honest significant difference (HSD) test.
Western blot analysis
The systemic leaves from inoculated plants were homogenized in 20 volumes (wt/vol) of denaturing buffer (50 mM Tris–HCl, pH 6.8, 4% SDS, 2% 2-mercaptoethanol, 10% glycerol, and 0.001% bromophenol blue). After incubation at 100°C for 5 min, extracts were clarified by centrifugation at 8,000 g for 3 min. Total proteins were separated by SDS gel electrophoresis, and western blots were analyzed using the antiserum to TuMV CP. Gels were stained with Coomassie brilliant blue R250, and levels of the large subunit of RUBISCO (molecular mass, 55 kDa) were used as loading controls.