Viral source
CBSV strain Nampula, from the region of Nampula, Mozambique (accession number HM346953) was obtained as Nicotiana benthamiana infected material from the Food and Environment Research Agency (FERA York-UK). CBSV strain Tanza, collected from a region in Tanzania was obtained from the infectious clone CBSV_Tanza IC (Duff-Farrier et al., 2019). UCBSV strain Kikombe, collected from a region in Tanzania, accession No. KX753356, was obtained from the infectious clone UCBSV “Kikombe” IC (Duff-Farrier et al., 2019).
For synergism studies, Tobacco mosaic virus strain U1, (accession No. V01408.1) and Potato virus Y (PVYO) strain Ordinary, (accession No. EF026074) were used, both being propagated in N. tabacum.
Construction of plasmids
Each encoded gene in CBSV Nampula strain and HC-Pro from PVY Ordinary strain were amplified by PCR, adding start and stop codons ATG and TAA, using primers described in supplemental Table S3. PCRs were performed using Phusion High-Fidelity polymerase (ThermoScientific™). Viral amplicons were cloned into the pJET 1.2 (Themo Scientific™), then sequenced and cloned into pCambia2300_EC, under the 35S promoter and the tNOS terminator regulatory elements.
For the construction of the modified P1 expression vector and P1 from CBSV Tanza expression vector, the entire P1 from CBSV_Tanza_P1_LAAA IC and P1 from CBSV_Tanza IC were excised and cloned into pCambia2300_EC independently.
Homologous recombination in yeast
The P1 region from the CBSV_Tanza IC was mutated through PCR-based site directed mutagenesis and homologous recombination in yeast. The yeast homologous recombination was performed as described in Gietz and Woods, (2002).
The infectious clone was digested using PshAI, the homologous recombination in yeast was performed using overlapping fragments created with the following primers; P1_LRRA_1st_FW with P1_LRRA_1st_RV and P1_LRRA_2nd_FW with P1_LRRA_2nd_RV (supplemental Table S4) and according to the strategy presented in Supplemental Figure S1.
The mutated infectious clone, CBSV_Tanza_P1_LAAA IC was recovered from Escherichia coli and confirmed by PCR (Supplemental Figure S2), utilizing primers for six regions of the infectious clone (Supplemental Table S4). The CBSV_Tanza_P1_LAAA infectious clone was then used as a template for the cloning of the CBSV P1_LAAA into pCambia2300_EC, creating the expression vector P1_Tanza_LAAA_pCambia2300_EC, which was recovered from E. coli and confirmed by restriction digestion (Supplemental Figure S3). The induced mutation for the LAAA was confirmed by sequencing the P1 region (Supplemental Figure S4)
Agrobacterium-mediated transient silencing-suppression assay
Viral constructs in pCambia2300_EC were transformed into Agrobacterium tumefaciens LBA4404 through electroporation. The preparation of A. tumefaciens for infiltration was performed as described in Johansen and Carrington (2001). A culture of A. tumefaciens transformed with pCambia2300_EC containing the sequence of the green fluorescent protein (GFP) was included for each viral construct during silencing suppression assays. Cultures for each viral construct and GFP construct were mixed in a proportion 1:1. Infiltration was performed with 0.2 mL of the suspension at the abaxial surface of the leaves with a syringe with no needle. Assays were performed using wild type N. benthamiana and the transgenic 16c line in N. benthamiana, which constitutively express the GFP under the 35S promotor of the Cauliflower mosaic virus (CaMV). For each assay of silencing suppression activity, six plants were infiltrated. P1 from UCBSV and HC-Pro from PVY were used as positive controls for their known silencing suppressor activity (Gallois and Marinho, 1995; Mbanzibwa et al., 2009). A single agroinfiltration with the GFP construct into the 16c line N. benthamiana was used as positive control for the gene silencing of the GFP in planta.
GFP imaging
The expression levels of GFP were monitored five days after agroinfiltration into leaves of N. benthamiana 16c line, as described in Johansen and Carrington (2001). GFP was visualised using a trans-illuminator UVP VisiBlue VB-26V, fluorescence emission was photographed with a Canon EOS T2i camera, operated with an exposure time of 15 seconds, ISO value of 800 and F value of 8.0. For epi-illumination, pictures were taken with an excitation filter of 425/60 nm and a barrier filter of 480 nm utilized for visualization of GFP in a Leica MZFLIII fluorescence stereomicroscope.
Virus inoculation
N. tabacum plants were inoculated with TMV and PVY from frozen stocks virus infected leaf material. The leaf material (2 g) was ground at room temperature in 5 mL sterile deionised water with a pestle and mortar. Young opened leaves of N. benthamiana and N. tabacum were dusted with 600 mesh carborundum powder (Fisher Scientific™) at the adaxial surface of the leaf, and freshly ground inoculum was gently rubbed onto these leaves.
For viral interaction assays with CBSV and/or TMV and CBSV and/or PVY, three biological replicas per infection were established in groups of single and mixed infections, inoculations were performed on the same leaf at the same time. Single and mixed infections were monitored for 26 days looking at symptom development and measuring viral titre.
Viral interaction assays
N. tabacum transgenic lines expressing P3, CI, 6k1, 6k2, VPg, NIa, NIb, Ham1 and CP from CBSV were infected with TMV or PVY, using three biological replicas from each independent transgenic line. Infected plants were monitored and sampled for subsequent ELISA analysis at: 0 and 2 DPI from the inoculated leaf; then at 4, 6, 8, 10, 14, 18, 22 and 26 DPI from systemic infection in upper leaves. TMV and PVY titres from infections in transgenic N. tabacum were compared to infections in wild type N. tabacum.
Transformation of N. tabacum plants.
N. tabacum plants were transformed using A. tumefaciens LBA4404 to express each individual encoded gene from CBSV. Using the methodology described in Gallois and Marinho (1995) disc leaves were transformed then selected in Murashige & Skoog (MS) media supplemented with kanamycin 100 µg/mL and 6-benzylaminopurin (6-BAP) 1 mg/L for generation of new shoots. Shooting callus pieces were placed in MS media supplemented with kanamycin 100 µg/mL and α-naphthalene acetic acid (NAA) 0.1 mg/L for the generation of roots.
Confirmation of N. tabacum transgenic lines
Transgenic lines of N. tabacum were maintained in MS media supplemented with kanamycin 100 µg/mL. RT-PCR was carried out using primers in Table S4, in order to verify the presence of transgene mRNA transcripts in all transformed plants. Relative expression of each viral transcript was measured through Q-RT-PCR. Reactions were carried out using RevertAid First-Strand cDNA Synthesis Kit (ThermoScientific™) for the synthesis of cDNA and Maxima® SYBR Green/ROX (Thermo Fisher Scientific) for the quantitative PCR reactions. Primers and conditions for this analysis are listed in supplemental Table S4. Confirmed transgenic lines and relative expression for each transgene are listed in supplemental Table S1. For each transgene used in this study, three independent transgenic lines were used and propagated as technical and biological replica during experimentation. Transgenic expression of the CBSV P1 gene resulted in very low rates of plant growth, which prevented the use of lines expressing P1 during virus-transgene interaction assays (data not shown).
Semi-quantitative viral titre accumulation analysis.
For the detection of TMV and PVY, an enzyme-linked immunosorbent assay (ELISA) was performed using double sandwich antibody (DAS-ELISA) kits for TMV and PVY mono-cocktail (BIOREBA AG), for the detection of CBSV a triple sandwich antibody (TAS-ELISA) kit from DSMZ was used. Leaves were ground in sample extraction buffer with a ratio of 1:20 (wight/volume). ELISAs were performed in a medium-binding 96-wells microtiter plates (Greiner). Colorimetric reactions were measured after two hours of colour development, using a microtiter plate reader (Spectra Max 190 from Molecular Devices) with a filter for a wavelength of 405 nm.
Data analysis and experiment designing.
The absorbances obtained, reading microtiter plates for ELISA, were normalised to the positive control from each ELISA kit. Analyses for quantification and definition of threshold were performed as described by the manufacturer of the relevant ELISA kits. Values of absorbance were transformed to percentages, taking a mix of the positive control reaction from each kit as the 100 percent of the respective measured virus, this adjustment was performed per microtiter plate, to normalize the data to the same consistent measurement each time, since absorbances vary from plate to plate.
At least two transgenic lines were obtained per CBSV gene in this study, from each transgenic line generated in N. tabacum, outlined in supplemental Table S1, three clones were then used per line as biological replicas, and two wells with the same sample in the microtiter plate were used as technical replicas, from which average was used to calculate the mean ± standard error, to estimate significant differences. When infections were performed with different versions of CBSV infectious clones or when transgenic lines and wild type N. tabacum were inoculated with PVY or TMV a Kruskal-Wallis test was performed to analyse differences in viral titres between treatments , then treatments were analyses in pairs with Dunn post hoc test per day. For the estimation of significant differences between single and mixed infections in N. tabacum and N. benthamiana plants, a Wilcoxon test was performed. For both analyses the statistical significance was taken at P <0.05.