Yam mosaic virus coat protein (YMV-CP) gene sequences of different isolates of yam mosaic virus (gi|7414928|emb|AJ244066.1, gi|14589250|emb|AJ305443.1, gi|14589246|emb|AJ305441.1, gi|14589244|emb|AJ305440.1, gi|14589242|emb|AJ305439.1, gi|14589238|emb|AJ305437.1, gi|14589234|emb|AJ305435.1, gi|7414926|emb|AJ244065.1, gi|7414924|emb|AJ244064.1, gi|7414922|emb|AJ244063.1, gi|7414920|emb|AJ244062.1, gi|7414918|emb|AJ244061.1, gi|7414916|emb|AJ244060.1 and gi|7414914|emb|AJ244059.1) were downloaded from NCBI. The sequences were aligned using Multalin (http://multalin.toulouse.inra.fr/multalin/) to identify conserved region. Primers were designed to amplify 161 bp fragment in the conserved region. Total RNA was extracted from YMV infected leaves using RNeasy® Plant Mini Kit (QIAGEN GmbH, Germany) and cDNA was synthesized from 1 µg of total RNA using iScript cDNA Synthesis Kit (BIO-RAD Laboratories, Inc.) according to the manufacturer’s instructions. One µl of the cDNA was then used for PCR analysis to amplify YMV-CP gene using the primers YMV-CP5P and YMV-CP3P (Table 1). After amplification, 5 µL of PCR product was resolved on 1% agarose gel stained with gel red.

The PCR product was purified using QIAquick PCR purification Kit (QIAGEN) and subsequently cloned into Gateway entry vector pCR™8/GW/TOPO® (Invitrogen, New Zealand), which contains attL1 and attL2 recombination sites. The ligated product was transformed to TOP10 E. coli competent cells and selected on LB medium containing spectinomycin (100 mg/L). Ten colonies were selected, plasmid DNA extracted and verified by Sanger sequencing using M13primers. The correct transformant containing the 161 bp YMV-CP was subcloned in the sense and antisense orientation to the pANDA35HK RNAi destination vector by LR clonase reaction using Gateway LR Clonase II enzyme mix (Thermo Fisher Scientific). The product was transformed to DH5α E. coli chemical competent cells and selected on LB plates containing 50 mg/L kanamycin. Plasmid was extracted from transformants using FastGene plasmid Mini Kit (NIPPON GENETICS Co., Ltd. Japan) and confirmed by digestion with Sac1 and Kpn1. One positive clone was selected and mobilized to Agrobacterium tumefaciens strain EHA105 by the Freeze-Thaw method and selected on LB plate containing 50 mg/L kanamycin and 25 mg/L Rifampicin. Plasmids were extracted from positive colonies using FastGene plasmid Mini Kit (NIPPON GENETICS Co., Ltd. Japan) and checked by PCR and by digestion with SacI and KpnI.

Leaves of Nicotiana tabacum (cv. Havana) were used for transformation using the Agrobacterium-mediated leaf disc method [27]. Agrobacterium tumefaciens strain EHA105 containing pANDA35HK-YMV-CP was grown overnight on a reciprocal shaker (120 cycles/min) at 28˚C in 20 ml LB medium containing 50 mg/L kanamycin and 25 mg/L Rifampicin. The overnight culture was centrifuged for 10 min at 10,000 rpm and re-suspended in 30 ml MS medium (inoculation medium) and left at room temperature for 30 min before inoculation. Leaves from in vitro plants were excised, cut into portions of 5 - 8 mm square and dipped in the bacterial suspension for 10 min with gentle shaking at room temperature. After inoculation, the explants were blotted dry and co-cultivated for 3 days in the dark on hormone free MS medium containing 100 µM acetosyringone and solidified with 8 g/L Agar, pH 5.8. At the end of the co-cultivation period, the explants were washed three times with sterilized distilled water and cultured on MS medium supplemented with 30 g/L sucrose, 8 g/L agar, 20 mg/L meropenem trihydrate (Meropenem; Dainippon Sumitomo Pharma, Osaka, Japan) [28] to eliminate bacterial carry over, and with hygromycin (40 mg/L) to select for transformants. The explants were sub-cultured at 2-week intervals to fresh medium of same composition. After 4 weeks of culture, adventitious shoots were excised and transferred to hormone-free MS medium containing 20 mg/L meropenem trihydrate and 40 mg/L hygromycin for development and rooting. Rooted plantlets were maintained in vitro in the growth room with subculture every 8 weeks.

Transgenic T₀ tobacco lines together with the control plants were analyzed for siRNA accumulation after challenge with YMV. Small RNAs (<200 bp) were extracted using ISOGEN II reagent (Nippon Gene). Thirty micrograms of small RNAs were electrophoresed on a 17% polyacrylamide gel containing 7 M urea and 10X Tris Borate EDTA (TBE). siRNAs were transferred to Immobilon-NY+ membrane (Millipore Corporation, Billerica, MA, USA) in a semi-dry cell (Semi-dry blotting apparatus NA-1512, Nippon Eido, Tokyo, Japan) for 1 h at10 V/400 mA and subjected to northern hybridization with a probe obtained by the in vitro transcription of the YMV-CP gene in the antisense and sense orientation using T7 RNA polymerase according to DIG Northern Starter Kit Version 10 (Roche) protocol. Prehybridization (30 min) and hybridization (overnight) were performed at 50˚C. Post-hybridization stringency washes and chemiluminescent detection of siRNA signals using CDP-star were performed following protocols outlined in the DIG Northern Starter Kit manual (Roche Diagnostic GmbH, Boehringer Mannheim, Germany).

In order to express virus-derived dsRNA in tobacco, a construct containing 161 bp DNA fragment of yam mosaic virus coat protein in the sense and antisense orientation was produced. The 161 bp fragment was derived from a conserved region between nucleotide 31 and 701 of YMV-CP gene sequences across different strains of YMV. The plasmid, pANDA35HK-YMV-CP contains hpt and nptII genes for selection of transgenic plants. The YMV-CP gene is regulated by 35 S promoter. To ensure stability of the inverted repeat in Escherichia coli, the two cDNA fragments were separated by a guslinker spacer sequence. The plasmid was mobilized to Agrobacterium tumefaciens strain EHA105 and used for tobacco transformation. A total of twenty transgenic plants were recovered. The T-DNA fragment of pANDA35HK carrying the YMV-CP gene in the sense and antisense orientation is shown in Figure 1.

Tobacco explants were successfully transformed with A. tumefaciens strain EHA105 harbouring pANDA35HK-YMV-CP, producing transgenic tobacco lines stably expressing YMV-CP gene. Screening of in vitro T₀ transgenic tobacco plants by PCR showed that 161 bp fragment of YMV-CP (Figure 2(a)), 557 bp fragment of the selectable marker gene (hpt) (Figure 2(b)) and 626 bp fragment of guslinker (Figure 2(c)) were amplified in all transgenic lines, but no signal was detected in the wild-type plant. To determine the copy number of the transgene, Southern blot analysis was performed. Result showed transgenic lines having different hybridization profile, with one, two or three transgene insert, suggesting that they regenerated from independent transformation event (Figure 3). Northern blot analysis was carried out to detect siRNA in inoculated wild-type and T₀ transgenic plants. Results show faint siRNA signals in the transgenic plants while no signal was detected in the mock inoculated wild-type plant (Figure 4).

After screening transgenic in vitro plants by PCR and Southern hybridization, positive lines were selected and transferred to the greenhouse for establishment and subsequently inoculated with YMV infection sap. Disease symptom severity on fully expanded leaves was recorded on a scale of 0-3 and disease index calculated using the method earlier described. When transgenic plants were inoculated with YMV, line L2 developed no symptoms on inoculated and emerging new leaves (Figure 5) comparative to the wild-type mock inoculated plants as all the leaves were grouped under grade 0 severity score (Figure 5), consequently, their disease indices were 0 and the plants were classified as being immune to YMV (Table 2) according to the disease rating. The remaining transgenic lines L3, L2, L5, L7, L8 and T10 developed mild symptoms, which were at a much reduced level of severity (grade 1). The disease indices calculated for these lines were very low (<25%), hence the lines were classified as being highly resistant to YMV (Table 2). Compared to the transgenic lines, all the emerging new leaves of the wild-type plants developed a range of symptoms, from mild (grade 1) to severe (grade 3) (Figure 5), with 97.8% of the new leaves having severe disease symptoms. The severity of the disease in the wild-type plants produced high disease indices which was 82.1% upon inoculation with YMV (Table 2). With the high disease index, the plants were classified as being highly susceptible (HS) to YMV. Plant height was recorded at 60 dpi. The mock inoculated wild type plants were significantly taller (P < 0.001) than the inoculated transgenic plants and the inoculated wild-type plants, with mean plant height ranging from 0.59 ± 0.05 m in YMV inoculated wild-type plants to 1.69 ± 0.03 cm in mock inoculated wild-type (Table 2).

Wild-type and transgenic T₀ plants inoculated with YMV were evaluated for virus accumulation on new un-inoculated leaves by RT-PCR. The result showed high accumulation of YMV on new un-inoculated leaves of wild-type plants. In contrast, reduced accumulation of YMV was detected on new un-inoculated leaves of T₀ transgenic plants (Figure 6).

Means with dissimilar lower case letters are statistically different (P < 0.001) on the basis of LSD test. MWT = mock inoculated wild-type plant; WTC = YMV inoculated wild-type plant; L1-L10 = transgenic lines; NA = not applicable; HS = highly susceptible; HR = highly resistant; I = immune. Symptom severity grades: 0-no symptom; 1-mild chlorotic pattern; 2-moderate mosaic; class 3-severe mosaic, distortion of the leaflets.

Following the germination of seeds from the T₀ transgenic tobacco plants to obtain T₁ plants, the T₁ plants were screened for the presence of the target transgene YMV-CP and the selectable marker gene HPT, before they were transferred to the greenhouse for disease assay (Figure 7). After four weeks of acclimatization in the greenhouse, the T₁ plants were inoculated with YMV infection sap. Lines L2, L3, L5, L6, L7, L8 and wild-type mock inoculated developed no symptoms on inoculated and emerging new leaves as the leaves were grouped under 0 severity score. Consequently, their disease indices were 0 and the plants were classified as being immune to YMV (Table 3) according to the disease rating. The remaining transgenic lines, L4 and L10, developed mild symptoms which were at a much reduced level of severity (grade 1) (Table 3). The disease indices calculated for these lines were very low (<25%), hence the lines were classified as being highly resistant to YMV (Table 3). Compared to the transgenic lines, all the emerging new leaves of the wild-type plants developed a range of symptoms, from mild (grade 1) to severe (grade 3). When the plants were inoculated with YMV, 100% of the new leaves had severe disease symptoms. The severity of the disease in the wild-type plants produced high disease indices which was 78.2% upon inoculation with YMV (Table 3). With this high disease index, the plants were classified as being highly susceptible (HS) to YMV. Plant height was recorded at 60 dpi. Mock inoculated plants were the tallest but they were not significantly different (P > 0.05) in height from all the transgenic plants except L4 and L10. The wild-type control plants inoculated with YMV were significantly shorter (P < 0.01) than all the other plants (0.45 ± 0.02) (Table 3).

Transgenic and wild-type T₁ tobacco plants inoculated with YMV were evaluated for virus accumulation on new un-inoculated leaves by qPCR. The relative expression of YMV-CP was significantly higher (P < 0.001) in wild-type plants, very little or no expression was observed in the transgenic lines (Figure 8).