Improving initial infectivity of the Turnip mosaic virus (TuMV) infectious clone by an mini binary vector via agro-infiltration

Abstract

Background: The in vivo infectious clone of Turnip mosaic virus (TuMV), p35S-TuMV, was used on plant pathology research for many years. To activate p35S-TuMV, the plasmid was mechanically introduced to the local lesion host Chenopodium quinoa. However, low infectivity occurred when the TuMV from C. quinoa was transferred to the systemic host Nicotiana benthamiana.

Results: To increase the efficiency of initial infectivity on N. benthamiana, the expression of the TuMV infectious clone by a binary vector that directly activates viral RNA through agro-infiltration is considered to be a good alternative. The size of the binary vector by agro-infiltration is usually large and its backbone has numerous restriction sites that increase difficulty for construction. In this study, we attempted to construct a mini binary vector (pBD003) with less restriction sites. The full-length cDNA of TuMV genome, with or without green fluorescence protein, was inserted in pBD003 to generate pBD-TuMV constructs, which were then individually introduced to N. benthamiana plants by agro-infiltration. Symptom development and ELISA positivity with TuMV antiserum indicated that the pBD-TuMV constructs are infectious. Moreover, the initial infectivity of a mild strain TuMV-GK, which contains an R₁₈₂K mutation on HC-Pro, constructed in the pBD003 vector was significantly increased by agro-infiltration.

Conclusion: Thus, we concluded that the newly constructed mini binary vector provides a more feasible tool for TuMV researches in areas, such as creating a mild strain for cross-protection, or a viral vector for foreign gene expression. In addition, the multiple cloning sites will be further cloned in pBD003 for convenience in constructing other viral infectious clones.

Keywords: Infectious clone; Initial infectivity; Mini binary vector; Turnip mosaic virus.

Figure 1

Construction of the pBD003 binary vector. Construction procedure of the mini binary vector - pBD003 contains an agrobacterium replication origin (pSa) and an E. coli. replication origin (Ori) for the plasmid replication in agrobacterium and E. coli, respectively. The Cauliflower mosaic virus (CaMV) 35S promoter (35S), the ribozyme (RZ) sequence, and the nopaline synthase terminator (nos) were constructed and inserted between the left border (LB) and right border (RB) on pBD003. The restriction sites frequently used in cloning on the vector backbone were mutated by PCR-mediated mutagenesis. The Kpn I restriction site was created between the CaMV 35S promoter and ribozyme sequence for further cloning or construction.

Figure 2

Schematic representation of constructing pBD-TuMV infectious clones. (A) Two TuMV in vivo infectious clones, p35S-TuMV-YC5 and p35S-TuMV-GFP (Lin et al., ; Niu et al., 2006), constructed on the pCaMVCN vector (Pharmacia) were designed for mechanical inoculation on C. quinoa plants. The two clones were used in the current study to construct a series of infectious constructs using a binary vector for agro-infiltration. (B) The full-length cDNA constructs of TuMV were constructed in the pBD003 binary vector (pBD-TuMV-YC5 and pBD-TuMV-GFP) for agrobacterium infiltration on N. benthamiana plants. The construction steps for cloning the TuMV full-length cDNA into the pBD003 binary vector are shown. The full-length TuMV cDNAs released from the p35S-TuMV series were constructed downstream of the CaMV 35S promoter (35S) by insertions in between suitable restriction enzyme sites of pBD003. The final constructs were designated pBD-TuMV-YC5 and pBD-TuMV-GFP, contained a complete cDNA copy of wild type TuMV and TuMV carrying GFP, respectively. The coding regions of the cDNA are indicated by an open box. The 5′ and 3′ non-coding regions of the cDNA are indicated by heavy lines. (C) R₁₈₂K mutation on HC-Pro of the TuMV was conducted by PCR-mediated mutagenesis. The mutated fragment was digested with MfeI/Kpn I and replaced the corresponding region of pBD-TuMV-GFP to generate pBD-TuMV-GK. An asterisk indicated the R₁₈₂K mutation position.

Figure 3

Infectivity assay of pBD-TuMV-YC5, pBD-TuMV-GFP and pBD-TuMV-GK. (A) The 3 constructs were directly mechanically introduced to C. quinoa plants, and local lesions appeared on the inoculated leaves at 7 dpi (upper panel). The virus culture of TuMV YC5 and Mock-inoculation were used as positive and negative controls, respectively. Bar, 0.5 cm. The GFP fluorescence expressed by pBD-TuMV-GFP and pBD-TuMV-GK is shown in the lower panel. Bar, 25 μM. (B) N. benthamiana plants were infiltrated with agrobacterium individually carrying pBD-TuMV-YC5, pBD-TuMV-GFP, and pBD-TuMV-GK. The virus culture of TuMV YC5 and Mock-inoculation were used as positive and negative controls, respectively. The photographs were taken at 14 dpi (upper panel). GFP expression was detected with a fluorescent microscope (lower panel).

Figure 4

Validation of the infectivity of pBD-TuMV-CY5, pBD-TuMV-GFP and pBD-TuMV-GK on N. benthamiana plants. The agro-infiltrated N. benthaminana plants were analyzed with western blotting (A) or indirect ELISA (B) at 7 dpi, using the antiserum to TuMV CP. The samples of agro-infiltrated leaves are individually indicated. The sample from leaves infected by TuMV virus culture (14 dpi) that was used as positive control is indicated as TuMV. The mock was inoculation was used as a negative control. The blank indicates the wells coated with buffer for basal-level control. The TuMV CP antiserum was used at 10,000x dilution for both assays. The samples significantly different with mock (p-value < 0.05) were indicated as “*”. Bars represent standard deviations (n = 3).

Figure 5

Comparison of TuMV initial infection efficiency between the agro-infiltration (pBD-TuMV-YC5) and mechanical inoculation (virus culture of TuMV YC5). Virus accumulated in vivo on inoculated or systemic leaves of N. benthamiana plants in which infection was initiated by either agro-infiltration or mechanical inoculation with TuMV infectious clones. The numbers indicated the days after inoculation (dpi). The virus titer was detected by ELISA with TuMV CP antiserum. The 12 dpi of TuMV-infected N. benthamiana plants (TuMV) were used as a positive control, whereas the mock-inoculated plants (Mock) were the negative control. The samples of 2, 4, 6, and 12 dpi significantly different with the sample of 0 dpi in the same inoculation method and leaves position (p-value < 0.05) are indicated as “*”. Significant difference between the agro-infiltration and the mechanical inoculation of the inoculated leaves under the same dpi (p-value < 0.05) is indicated as “a”. Significant difference between the agro-infiltration and the mechanical inoculation of the systemic leaves under the same dpi (p-value < 0.05) is indicated as “b”. Bars represent standard deviations (n = 3).