The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants

Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases.


Introduction
Geminiviruses constitute a large family of plant viruses which infect a wide range of crops and cause enormous losses worldwide. They possess monopartite or bipartite genomes, comprising circular, single-stranded DNAs and are encapsidated in paired icosahedral particles [1,2]. The family Geminiviridae is classified into seven genera, Begomovirus, Mastrevirus, Curtovirus, Becurtovirus, Eragrovirus, Topocuvirus and Turncurtovirus [3].
Cassava is a major food and commercial crop in Africa and India and cassava mosaic disease is the major constraint to cassava cultivation [4,5]. The disease is caused in India by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) [6,7]. Transgenic expression of pathogen-derived genes is widely used to obtain geminivirus resistance in crop plants [8]. Strategies of engineering viral resistance by expressing full-length or truncated viral proteins such as replication associated protein, coat protein and movement protein are effective in controlling viral infection in model as well as crop plants [9][10][11][12][13][14]. Gene silencing strategies using sense RNA, anti-sense RNA and double-stranded RNA have been successfully used to generate geminivirus resistance [15][16][17][18][19].
There are fewer examples with respect to the use of non-viral protein genes to develop virus resistance. Padidam et al. [20] reported that M13 bacteriophage gene 5 protein, which binds ssDNA, was effective in restricting Tomato leaf curl virus infections. Sera [21] reported that expression of an artificial zinc finger protein targeting the replication origin of Beet severe curly top virus efficiently blocked viral DNA replication in transgenic Arabidopsis plants. Sunitha et al. [22] expressed the A. tumefaciens Ti plasmid virE2 gene in tobacco and showed a reduction of viral DNA accumulation in leaf discs agroinoculated with Mungbean yellow mosaic virus (MYMV) partial dimers. Here, we report the efficacy of the virE2 gene in restricting SLCMV infection in transgenic Nicotiana benthamiana plants. This report shows that the virE2 gene is a good non-viral gene that can be used to develop broad spectrum geminivirus tolerance.

Plasmid Constructs
Construction of pCAM-virE2: The Agrobacterium tumefaciens virE2 gene with an intron, under the transcriptional control of CaMV 35S promoter, was cloned as a BamHI/PstI fragment in the corresponding sites of pCAMBIA1380 to yield pCAM-virE2. pCAM-virE2 was mobilized into the A. tumefaciens strain GV3101 which has the disarmed vector pPM6000 [22].
The pPZP-SLCMV-At1.6A and pPZP-SLCMV-At1.8B with the DNA A and DNA B partial dimers, respectively, were introduced independently into the A. tumefaciens strain Ach5 by triparental mating [24] or electroporation (Bio-Rad, Hercules, CA, USA) and the transconjugants or transformants were confirmed by Southern blot analysis.

N. benthamiana Transformation
Leaf discs from axenically-grown N. benthamiana plants were transformed with A. tumefaciens (pCAM-virE2) as described by Horsch et al. [25]. Transgenic shoots were selected on Murashige and Skoog (MS) [26] shoot induction medium containing 4 µM 6-benzylaminopurine, 0.5 µM α-naphthaleneacetic acid, 50 mg/L hygromycin and 250 mg/L cefotaxime. The shoots were subjected to root induction for two days in half-strength liquid MS medium containing 20 µM indolebutyric acid. The shoots were then kept on half-strength solid MS basal medium containing 25 mg/L hygromycin, 250 mg/L cefotaxime and 150 mg/L timentin for root development.

Southern Blot Analysis
Total plant DNA was extracted [27] and estimated by fluorometry. DNA was digested with suitable restriction enzymes and electrophoresed in a 0.8% (w/v) agarose gel in 1 X TNE (40 mM Tris-acetate, pH 7.5, 20 mM sodium acetate, 2 mM EDTA) or 1 X TBE (Tris-borate-EDTA) buffers. DNA was transferred [28] to the Zeta-probe nylon membrane (Bio-Rad, Hercules, CA, USA). DNA was labeled with [α-32 P]dCTP using the Megaprime DNA labeling system (GE Healthcare UK Ltd., Little Chalfont, UK) to prepare probes. Hybridizations were carried out at 65°C and high stringency post hybridization washes were performed [29].

VirE2 Complementation Assay
Control N. benthamiana leaf discs were infected with wild type A. tumefaciens strain A348 (with the wild type Ti plasmid pTiA6) and the mutant A. tumefaciens strain A348mx358 which harbors pTi358 with Tn3 HoHo1 insertion in the virE2 gene [30]. For VirE2 complementation assay, leaf discs from 3-week-old control or virE2 transgenic N. benthamiana plants were preincubated for two days on MS shoot induction medium and infected with A. tumefaciens strains grown to A600 = 1 in AB minimal medium [31]. The leaf discs were placed on MS shoot induction medium for co-cultivation. After two days, the leaf discs were transferred to a hormone-free MS medium with 250 mg/L cefotaxime.

Northern Blot Analysis
Young leaf tissue (0.5 g) of N. benthamiana plants was used for RNA extraction. Total RNA was extracted using the Trizol method as per instructions provided by the manufacturer (Sigma-Aldrich, St. Louis, MO, USA). RNA was estimated in a spectrophotometer. Total RNA (10 µg) was electrophoresed in a 1.2% (w/v) agarose gel containing 1% formaldehyde (v/v) in 1 × MOPS (3-(N-morpholino)propane sulphonic acid) buffer. Northern blot analysis was performed as described by Pawlowski et al. [32].

Agroinfection
Agroinfection of 3-week-old N. benthamiana plants was performed with the A. tumefaciens strain Ach5 harboring the partial dimers of SLCMV-[Attur2] DNA A and DNA B (two strain method). The A. tumefaciens (pPZP-SLCMV-At1.6A) and A. tumefaciens (pPZP-SLCMV-At1.8B) cultures were grown in AB minimal medium to A600 = 1 and cells were centrifuged at 28 °C at 1100× g. The pellets were resuspended in AB minimal medium (pH 5.6) containing 100 µM acetosyringone. The cultures were mixed (1:1) and agroinfection was performed by inoculating 10 µL of the bacterial mixture above the node of the first fully expanded leaf from the top. The stem was immediately pricked with a 30 G needle [33,34].

Densitometry Analysis
Integrated density values (IDV) of the autoradiogram were determined by using the AlphaEase™ software (Version 5.5, Alpha Innotech Corpoartion, San Leandro, CA, USA).

Characterization of N. benthamiana Plants Transformed with the A. tumefaciens virE2 Gene
The binary plasmid pCAM-virE2 (Figure 1), which harbors the virE2 gene of the octopine type Ti plasmid under the transcriptional control of the CaMV 35S promoter [22], was used to transform N. benthamiana leaf discs. pCAM-virE2 contains the hygromycin phosphotransferase gene (hpt) as plant selectable marker.   (Figure 2b). Plants E1, E2 and E7 displayed hybridization to junction fragments of the same size (2.0 kb). Thus, the plants E1, E2 and E7 were inferred as single-copy transgenic plants which regenerated from the same transformed callus. The remaining transgenic plants E3, E5 and E6 showed individual single junction fragments confirming that all are single-copy transgenic plants.

Complementation of the A. tumefaciens virE2 Mutation by VirE2 Expressed in Transgenic N. benthamiana Plants
The function of the VirE2 protein in the transgenic N. benthamiana plants was evaluated by its ability to complement the virE2 mutation of the A. tumefaciens strain A348mx358 [35]. Leaf discs of untransformed, control N. benthamiana plants infected with the wild type A. tumefaciens A348 harboring pTiA6 formed tumours when kept on hormone-free MS medium ( Figure 3). Leaf discs of untransformed N. benthamiana plants, infected with the virE2 mutant A. tumefaciens (pTi358), did not form tumors. Leaf discs of all six virE2 transgenic N. benthamiana plants, E1, E2, E3, E5, E6 and E7 infected with the virE2 mutant A. tumefaciens strain A348mx358, efficiently formed tumors on hormone-free MS medium ( Figure 3). Thus, the transgenically expressed VirE2 complemented the virE2 mutation of the A. tumefaciens strain A348mx358. The results showed that the plant-expressed VirE2 protein is functional. Of the four independent transgenic plants E3, E5, E6 and E7, the plant E5 did not set seeds. Therefore, the plants E3, E6 and E7 were forwarded to T1 generation and the T1 plants (virE2 PCR-positive) were used for northern blot analysis and SLCMV infection analysis. Northern blotting with the virE2 probe showed that all the three transgenic plants E3, E6 and E7 accumulated the 2.1-kb virE2 transcript (Figure 4).

SLCMV-Infected N. benthamiana Plants Displayed a Reduction in Viral Disease Symptoms and Viral DNA Level
The effect of VirE2 expression in transgenic N. benthamiana plants E3, E6 and E7 on SLCMV disease symptoms and viral DNA accumulation was studied. T1 plants were raised and the transgenic T1 plants were identified by PCR with the virE2 primers (data not shown). The transgenic plants were phenotypically similar to the control N. benthamiana plants. Three-week-old virE2 transgenic and control plants were agroinoculated with the A. tumefaciens strain Ach5 harboring the partial dimers of SLCMV-[Attur2] DNA A and DNA B. Two weeks post-infection, control plants infected with partial dimers of SLCMV showed severe stunting and downward leaf curling. These symptoms were noticeably reduced in the E3, E6 and E7 transgenic plants (Figure 5a,b). A discrete reduction in downward leaf curling was observed in all the transgenic plants.

Discussion
Several transgenic approaches based on viral and non-viral genes have been used to achieve geminivirus resistance [8]. We studied the efficacy of a non-viral protein, A. tumefaciens VirE2, to develop tolerance against SLCMV. VirE2 is a ssDNA binding protein [36] which binds to ssDNA in a cooperative manner and ensures that the complete ssDNA is coated with the protein. This protects the Agrobacterium T-strand from nuclease attack during the transfer process. VirE2 contains two bipartite nuclear localization signals and both are required for targeting VirE2 to the nucleus [37][38][39][40]. These properties of virE2 prompted us to select the gene for engineering tolerance against SLCMV which has a ssDNA genome.
Agrobacterium VirE2 functions in the plant cell during T-DNA transfer. Citovsky et al. [35] showed that transgenic tobacco plants which expressed virE2 complemented a virE2 mutation in A. tumefaciens and restored tumourigenesis. We found that all virE2 transgenic plants E1, E2, E3, E5, E6 and E7, generated by us, complemented the virE2 mutation in the A. tumefaciens strain A348mx358 and caused tumourigenesis of leaf discs in a hormone-minus medium ( Figure 3). Thus, the VirE2 protein expressed in the transgenic plants is functional. The E3, E6 and E7 plants accumulated the virE2 transcript. The three plants exhibited a reduction in disease symptoms when challenged by agroinoculation with the partial dimers of SLCMV-[Attur2] DNA A and DNA B. The level of SLCMV DNA was reduced to 95%, 52% and 75% in the transgenic plants E3, E6 and E7, respectively, in comparison to the SLCMV DNA levels in the control plants. In a previous report [22] we showed that leaf disc-agroinoculation of VirE2-expressing N. tabacum plants displayed a reduction in MYMV DNA levels. The MYMV agroinoculation experiments in the previous report [22] were limited to only leaf discs of virE2 transgenic tobacco plants. Therefore, information was not generated on whether MYMV disease symptoms were reduced by virE2 in the whole plants. In this report, the susceptibility of N. benthamiana plants to SLCMV permitted us to agroinoculate the whole virE2 transgenic plants. The results clearly show that VirE2 reduced the SLCMV symptom level and brought down the SLCMV DNA accumulation in virE2 transgenic N. benthamiana plants. In the current report, we have used N. benthamiana plants, rather than leaf discs, for agroinfection with SLCMV. These findings show that plant-expressed VirE2 reduces the levels of both MYMV and SLCMV and suggest that virE2 may be useful for controlling geminiviruses in general and perhaps other DNA viruses as well.
Local and systemic spread of geminiviral DNA is essential to establish infection in different parts of a plant. In bipartite begomoviruses, nuclear shuttle protein (NSP) and movement protein (MP) play important roles in viral movement. NSP helps in the transport of geminivirus DNA from the nucleus to the cytoplasm, whereas MP facilitates the viral movement between the cells [41][42][43][44]. Two models, relay race model [44,45] and couple skating model [43,46], have been proposed for geminivirus movement. As per the couple skating model, the viral ssDNA bound to NSP shuttles between the nucleus and the cytoplasm. The complex then interacts with MP to cross the cell boundary. The Tomato leaf curl virus (ToLCV) genome modified to express the M13 phage ssDNA binding protein g5p [20] developed only mild symptoms and did not spread efficiently in N. benthamiana plants. Binding of g5p with the ssDNA of ToLCV may have competed with the NSP binding to ssDNA and thereby reduced the spread of the viral DNA. As in the case of g5p, the ssDNA binding protein VirE2 also might cooperatively bind to SLCMV ssDNA in the nucleus, thus preventing NSP binding and shuttling to the cytoplasm for cell to cell and systemic movement. It would very useful to study whether VirE2 can out-compete NSP in an in vitro ssDNA binding assay. Our reports show that A. tumefaciens VirE2, with the unique features of ssDNA binding and nuclear localization, is very effective in both MYMV [22] and SLCMV and holds promise to develop broad spectrum geminivirus tolerance.