Identification of the virulence factors and suppressors of posttranscriptional gene silencing encoded by Ageratum yellow vein virus, a monopartite begomovirus

Virus Research 149 (2010) 19–27

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Identification of the virulence factors and suppressors of posttranscriptional gene silencing encoded by Ageratum yellow vein virus, a monopartite begomovirus P. Sharma a,b,∗ , M. Ikegami a , T. Kon a a b

Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amemiyamachi, Aoba-ku, Sendai, Miyagi 981-8555, Japan Division of Corp Improvement, Directorate of Wheat Research, Karnal 132 001, India

a r t i c l e

i n f o

Article history: Received 12 May 2009 Received in revised form 17 November 2009 Accepted 30 December 2009 Available online 15 January 2010 Keywords: Ageratum yellow vein virus PTGS Sub-cellular localization Virulence GFP

a b s t r a c t Ageratum yellow vein disease (AYVD) is caused by the association of a Tomato leaf curl Java betasatellite [Indonesia:Indonesia 1:2003] (ToLCJB-[ID:ID1:03]) with a begomovirus component. Our previous results demonstrated that ToLCJB-[ID:ID:03] is essential for induction of leaf curl symptoms in plants and transgene expression of its ˇC1 gene in Nicotiana benthamiana plants induces virus-like symptoms. Here we show that Ageratum yellow vein virus-Indonesia [Indonesia: Tomato] (AYVV-ID[ID:Tom]) alone could systemically infect the plants and induced upward leaf curl symptoms. ToLCJB-[ID:ID1:03] was required, in addition to AYVV-ID[ID:Tom], for induction of severe downward leaf curl disease in N. benthamiana plants. However, DNA␤01fsˇC1, which encompasses a frameshift mutation, did not induce severe symptoms in N. benthamiana when co-inoculated with AYVV-ID[ID:Tom]. The infectivity analysis of AYVV-ID[ID:Tom] and its associated betasatellite encoded genes using Potato virus X (PVX) vector were carried out in N. benthamiana, indicate that the V2 and ˇC1 genes are symptom determinants. We have identified the DNA encoded V2 and its betasatellite, ToLCJB-[ID:ID1:03], encoded ␤C1 proteins as efficient silencing suppressors of posttranscriptional gene silencing (PTGS) by using an Agrobacterium co-infiltration or heterologous PVX vector assays. However, the results also showed weak suppression of gene silencing activities for C2 and C4 induced by GFP and mRNA associated with GFP was detected. Furthermore, confocal imaging analysis of ToLCJB-[ID:ID1:03] ␤C1 in the epidermal cells of N. benthamiana shows that this protein is accumulated towards the periphery of the cell and around the nucleus, however, V2 accumulated in the cell cytoplasm, C4 associated with plasma membrane and C2 exclusively targeted into nucleus. In this study, we identified as many as four distinct suppressors of RNA silencing encoded by AYVV-ID[ID:Tom] and its cognate betasatellite in the family Geminiviridae, counteracting innate antiviral response. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Geminiviruses consisting of circular single-stranded (ss) DNA encapsidated in twin (geminate) particles infect various important crop plants like cotton, cassava, maize and tomato with a considerable impact on agriculture and the economy of tropical and subtropical countries. The family Geminiviridae comprises four genera, Mastrevirus, Curtovirus, Topocuvirus and Begomovirus (Stanley et al., 2005). The majority of these diseases are caused by geminiviruses that are whitefly-transmitted and are placed in the genus Begomovirus. Within the last ten years there has been a fundamental shift in our understanding of the nature of begomoviruses. It has become evident that, in contrast to the situation

∗ Corresponding author at: Division of Corp Improvement, Directorate of Wheat Research, Agarsain Road, Karnal 132 001, Haryana, India. E-mail address: [email protected] (P. Sharma). 0168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.12.008

in the New World where all native viruses have genomes consisting of two components (bipartite; components designated DNA A and DNA B), in the Old World the majority of begomoviruses are monopartite and most of these associate with single-stranded DNA betasatellites mainly throughout Asia and parts of Africa (Jose and Usha, 2003; Kon et al., 2006; Saunders et al., 2004; Sharma and Rishi, 2007). Infectious clones of majority of the monopartite begomoviruses were unable to induce typical symptoms, therefore, requiring betasatellite molecules associated with them for inducing the characteristic disease symptoms (Briddon et al., 2001; Kon et al., 2006; Jose and Usha, 2003; Saunders et al., 2004). Recently, it has been shown that these betasatellite molecules encode a single gene, ␤C1 (13 kDa protein), which is pathogenicity determinant and RNA silencing suppressor (for review see in Sharma and Ikegami, 2008). RNA silencing is a sequence-specific RNA degradation process that leads to elimination of the targeted RNA mediated by cytoplasmic nucleases and plays a natural antiviral role in plants (Voinnet,

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2005; Waterhouse et al., 2001). In response to these types of host antiviral defenses, it is not unexpected that the majority of wellstudied plant viruses have devised counteracting mechanisms that interfere with them at different levels (Voinnet et al., 1999). Different viral suppressors act at distinct steps in posttranscriptional gene silencing (PTGS) and can help to elucidate the PTGS pathway (Dunoyer et al., 2004; Hammond et al., 2000). Viral suppressors are often determinants of pathogenicity, and their suppression activity is associated with the pathogenic determination (Brigneti et al., 1998; Chen et al., 2004; Kon et al., 2007; Diaz-Pendon and Ding, 2008). Since the discovery of RNA silencing, many plant viral proteins have been identified as suppressors of RNA silencing (reviewed in Sharma and Ikegami, 2008; Voinnet et al., 1999). The great diversity in sequence, structure and mechanism of action found for these proteins reinforces the importance of the studies directed to the identification of new RNA silencing suppressors and their mode of action for better understanding the basic mechanisms of RNA silencing and virus–host interactions (Voinnet, 2005). Ageratum yellow vein disease (AYVD) complex consists of a monopartite begomovirus component, Ageratum yellow vein virus-Indonesia [Indonesia:Tomato] (AYVV-ID[ID:Tom) DNA and a Tomato leaf curl Java betasatellite [Indonesia:Indonesia 1:2003] (ToLCJB-[ID:ID1:03]) component. ToLCJB-[ID:ID1:03] depends on DNA for its replication and encapsidation. ToLCJB-[ID:ID1:03], along with AYVV-ID[ID:Tom] DNA produces typical symptoms in Solanum lycopersicum (Kon et al., 2006). Nicotiana benthamiana inoculated with DNA produced upward leaf curling, upon coinoculation with its cognate satellite molecule, leads to severe downward leaf curling, vein swelling, stunted growth and systemic spread of the virus. Recent reports suggest that begomoviruses; African cassava mosaic virus-[Kenya:849:1982] (ACMV-[KE:844:82]) (ChowdaReddy et al., 2008; Vanitharani et al., 2004), Bhendi yellow vein mosaic virus-India [India:Madurai] (BYVMV-IN[IN:Mad] (Gopal et al., 2007) and Tomato leaf curl Java virus-A[Indonesia] (ToLCJVA[ID]) (Kon et al., 2007) encode multiple suppressors. To ascertain if encoding multiple suppressors is a general strategy in members of the family Geminiviridae, we have screened the AYVD genome. Here, we report that the V2 and ␤C1 protein encoded by AYVVID[ID:Tom] and its cognate ToLCJB-[ID:ID1:03] produces virus-like symptoms in N. benthamiana, when they were expressed from a Potato virus X (PVX) vector and suppresses very efficiently RNA silencing. Further, we demonstrate that C2 and C4 are involved mildly in suppressions of gene silencing. The results of the transient expression of GFP fused with ␤C1 protein suggest that this protein was localized to the cell periphery and around the nucleus, while C2 was targeted exclusively into the nucleus, C4 localized in membrane and V2 in the cell cytoplasm. AYVV-ID[ID:Tom] is the first begomovirus identified that encodes four distinct suppressors of RNA silencing in both of its genomic DNA and associated betasatellite. 2. Materials and methods

A partial dimmer of the ˇC1 gene mutant was constructed together with a partial repeat of AYVV-ID[ID:Tom] on the same binary vector pBI121 (Clontech) to produce clone pBToB1.4fs␤C1 (AYVV-ID[ID:Tom] with DNA␤01fs␤C1). The fidelity of mutant and infectious clones were confirmed by sequencing. The obtained constructs were separately mobilized into Agrobacterium tumefaciens strain LBA4404 by triparental mating (Ditta et al., 1980). A. tumefaciens cultures were grown at 28 ◦ C for 48 h (OD550 = 1), after which a fine needle was used to inject 0.2 ml of cultures into stems or petioles of plants at the five to six leaf stage. Seedlings of N. benthamiana were agro-inoculated with pBToB1.4, pBToB1.4␤01 and pBToB1.4fs␤C1. Inoculated plants were kept in an insect free glasshouse at a constant temperature of 25 ◦ C with supplementary lighting corresponding to a 16 h day length. Viral DNA detection was carried out as described previously (Ogawa et al., 2008). 2.2. Expression of V2, C2, C4 and ˇC1 from a PVX and pBIN based vectors The ˇC1 gene was PCR amplified using the ␤C1S primer (5 -ATCGATGACTATATCATATAGCAAC-3 ; the ˇC1 gene initiation codon is in bold, and the ClaI site is underlined) and the ˇC1C primer (5 -GTCGACTTATACGGTTACATGCTTG-3 ; the SalI site is underlined) and cloned into PVX based vector pPVX201, which was kindly provided by Dr. Baulcombe, United Kingdom, uses the unique ClaI–SalI sites, to produce PVX: ˇC1. The mutant ˇC1 gene contained a stop codon (TGA) immediately downstream of the initiation codon. The mutant ˇC1 gene was PCR amplified using the m␤C1S primer (5 -ATCGATGtgaATATCATATAGCAAC-3; the stop codon is in lowercase) and the ␤C1 primer used for PCR amplification of the ˇC1 gene, and cloned into pPVX201 to produce PVX:m␤C1. To produce PVX:V2, the V2 gene was amplified using the V2F primer (5 -ATCGATATGTGGGATCCTCTTTTGAAC-3 ) and the V2R primer (5 -GTCGACTCAGGGCTTCTGTACATTCTG-3 ) and cloned into pPVX201. Similarly, PVX:C2, was obtained from the C2 gene amplified using the C2F primer (5 -ATCGATATGCGGAATTCGTCACCCTCA-3 ) and the C2R primer (5 -GTCGACCTAAATACTCTTAAGAAACGC-3 ) and PVX:C4 from the C4 gene using the C4F primer (5 -ATCGATATGGGAGCCCTCATCTCCACG-3 ) and the C4R primer (5 -ATCGATTTACATTAAGAGCCTCTGACT-3 ; the ClaI site is underlined) and cloned into pPVX201. Additionally, the GFP gene (738 nucleotide) was amplified from total DNA of N. benthamiana line 16c using the GFP1F primer (5 ATCGATCATGAGTAAAGGAGAAGAAC-3 ) and the GFP1R primer (5 GTCGACATTTATTTGTATAGTTCATC-3 ) and cloned into pPVX201 to produce PVX:GFP. Primers mentioned above were used to amplify C2, C4, V2 and ˇC1 having Xba1/Sac1 restriction sites and the amplified products were cloned into same restriction sites on the binary vector pBIN2 to obtain pBIN:C2, pBIN:C4, pBIN:V2 and pBIN:␤C1 recombinant plasmids for leaf infiltration. GFP expressing binary plasmid pBIN:GFP and pBIN:HcPro were obtained from Dr Vicky Vance (University of North Carolina, Raleigh). All the inserts were sequenced to make sure that no sequence error was introduced by PCR.

2.1. Construction of infectious clones and plant inoculation 2.3. PTGS assay Infectious clones of AYVV-ID[ID:Tom] (pBToB1.4␤01), AYVVID[ID:Tom] with ToLCJB-[ID:ID1:03] (pBToB1.4␤01), and ToLCJB[ID:ID1:03] alone (pBTo␤01) were used (Kon et al., 2006). The mutant ˇC1 gene of DNA␤01 was constructed in frameshift (fs) ATG start codon (nt 545–547) mutation with the primer covering and flanking the mutation sites (underlined); PS␤C1F primer (5 -AAGCTTTATTTGTTTGATGG-3 ) and PS␤C1R primer (5 -AAGCTTGACTATATCATAT AGC-3 ). Polymerase chain reaction (PCR) amplified products were cloned into pGEM-T Easy (Promega, Madison, WI, USA) to produce full length clone pTo␤01fs␤C1.

Seedlings of transgenic N. benthamiana plant line 16c expressing GFP were mechanically co-inoculated as described previously (Baulcombe et al., 1995) and A. tumefaciens strain C58C1 were used for infiltration experiments. Agrobacterium cultures were prepared as described previously (Llave et al., 2000) using appropriate antibiotics. Before infiltration, Agrobacterium cells were mixed prior to infiltration by combining equal volumes of individual cultures. GFP fluorescence was detected using a 100-W long-wave UV lamp (Black Ray model B 100A; UV Products, Upland, CA, USA). Plants

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were photographed with a digital camera (CAMEDIA C-3030; Olympus, Tokyo, Japan) using a yellow filter (WRATTEN No. 8; Kodak, Tokyo, Japan) and images were processed using Adobe Photoshop software (Adobe System, CA). Total plant RNA was extracted from plant tissues using a TRIreagent (Sigma–Aldrich). For northern blot analysis of GFP mRNA, total RNA was separated on a 1.2% agarose/MOPS/formaldehyde gel in 1× MOPS buffer. The RNA was transferred overnight to a HybondN+ membrane (GE Healthcare Bio-Sciences, Piscataway, USA), and hybridized and detected using a DIG northern starter and detection kit (Roche Diagnostics, Switzerland). For RT-PCR, RNA (1 ␮g) was extracted from frozen and ground leaves with TRI-reagent according to the manufacturer’s instructions. The RNA was reverse-transcribed by using SuperScript RT-PCR 1st strand synthesis kit (Invitrogen, USA) and PCR amplified using primers specific for GFP (mentioned earlier) and ACTIN (sense 5 -CAATGAGCTTCGTGTTGCACCC-3 , complementary 5 -CCGGTGCCCTGAGGTCCTTTTCC-3 ) which generated 738 bp and 540 bp products from the corresponding transcripts. 2.4. Sub-cellular localization For sub-cellular localization studies, ToLCJB-[ID:ID1:03] ˇC1 gene PCR amplified from pBToX1.4ˇ01 while C2, C4 and V2 were amplified from pBToB1.4, were introduced into the N-terminus of the pGFP(S65T) binary plasmid, having SalI/NocI sites, was obtained from Dr. Yasuo Niwa (Shizuoka University, Japan). The orientation of inserted DNA was confirmed by sequencing. Particle–gun bombardment of the plasmid in plant cells was carried out with PDS-1000/He Particle Delivery System (Bio-Rad, USA) following standard protocol provided by the supplier. Tungsten particles (1 ␮m), a microcarrier loading quantity of 0.5 mg per target and

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a DNA loading ratio of 50 ␮g DNA/25 mg of tungsten were used in the particle bombardment and epidermal cells of N. benthamiana were bombarded at a pressure 1100 psi per shot. After bombardment, the transformed cells were placed in sealed Petri dish (90 mm in diameter) containing a small amount of MS liquid medium (1× MS salts, 3% sucrose) and incubated at 25 ◦ C for 12 h in an incubator. Transformed cells were visualized with a fluorescent microscope (Olympus x181/FV500 with a green helium/neon laser and the PLAP40X/NA 1.4 WDD 0.15 nm objectives) for capturing transmitted light images. Images were captured using LuminiVision software. 3. Results 3.1. Mutational analysis of the ˇC1 gene We have previously demonstrated that Tomato leaf curl Java betsatellite [Indonesia:Indonesia 2:2003] (ToLCJB-[ID:ID 2:03]), when co-inoculated with ToLCJV-A[ID], elicits severe leaf curling symptoms in N. benthamiana plants (Kon et al., 2007). In order to determine whether virulence caused by ToLCJB-[ID:ID1:03] is correlated with its ability, infectious clones of AYVVID[ID:Tom] (pBToB1.4), ToLCJB-[ID:ID1:03] with AYVV-ID[ID:Tom] (pBToB1.4␤01), and the ˇC1 gene mutant of ToLCJB-[ID:ID1:03] with AYVV-ID[ID:Tom] (pBToB1.4fs␤C1), were agro-inoculated into N. benthamiana. When AYVV-ID[ID:Tom] alone was agroinoculated into N. benthamiana, plants displayed upward leaf curl symptoms (Fig. 1A, top right panel). The N. benthamiana plants agro-inoculated with AYVV-ID[ID:Tom] and ToLCJB-[ID:ID1:03] developed severe downward leaf curl symptoms, swelling of veins and epinesty (Fig. 1A, bottom right panel). In contrast, N. benthamiana plants agro-inoculated with AYVV-ID[ID:Tom]

Fig. 1. Symptoms severity and detection of viral DNA levels associated with Ageratum yellow vein disease induced by agro-inoculation. (A) Nicotiana benthamiana plants that were mock-agro-inoculated (Mock), or agro-inoculated with AYVV-ID[ID:Tom] alone, AYVV-ID[ID:Tom] with DNA␤01m␤C1 (AYVV + DNA␤01m␤C1), or AYVV-ID[ID:Tom] with DNA␤01 (AYVV + DNA␤01). Plants were photographed 15 days postinoculation (dpi). (B) Detection of viral DNA in agro-inoculated N. benthamiana. The total plant DNA extracted from virally infected plants was separated on a 1% agarose gel and hybridized with cloned AYVV-ID[ID:Tom] genomic DNA (top panel) and DNA␤01 probes (bottom panel).

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Fig. 2. N. benthamiana plants that were mock inoculated, or inoculated by Potato virus X (PVX) vector or PVX:V2 and PVX:␤C1. At 10 days postinoculation (dpi), PVX induced no apparent symptoms except mosaic pattern, where as PVX:V2 included downward curling, mosaic, necrosis of leaf and stem, leading to death of plant at 15dpi. PVX:␤C1 showing severe curling, vein swelling, epinesty and crinkling were observed and plants were photographed 10 dpi. V2 and ␤C1 with non-sense mutation at the bottom panel (PVX:mV2 and PVX:m␤C1) were used as controls.

and DNA␤01fs␤C1 were systemically infected but developed less severe symptoms, as compare to wild type (Fig. 1A, bottom left panel). Total DNA was extracted from virus infected plants 2 weeks postinoculation and analyzed by Southern blotting (Fig. 1B). The results demonstrate that the ␤C1 protein required for enhancing the disease symptom and is a determinant of virulence. 3.2. Expression of AYVV-ID[ID:Tom] and its associated betasatellite encoded proteins from the heterologous PVX vector To establish firmly the involvement of individual AYVVID[ID:Tom] and ToLCJB-[ID:ID1:03] genes on symptom induction, they needed to be expressed separately from other AYVVID[ID:Tom] genes. Exogenous expression of individual AYVVID[ID:Tom] genes was accomplished by inserting them of PVX. This biologically active PVX vector can be used to obtain high level of foreign expression throughout the plant. To determine the effects of PVX-mediated expression of V2, N. benthamiana plants were inoculated with PVX:V2. Systemic symptoms with necrosis developed in upper leaves at four days postinoculation (dpi) and after 10 dpi infections typically resulted in severe necrosis of leaves and petioles

(Fig. 2) and eventually lead to death of the whole plant. Plant inoculated with PVX:␤C1 produced severe leaf curling and vein swelling type symptoms at 10 dpi, indicating that ␤C1 protein expression, when removed from the context of the helper AYVV-ID[ID:Tom], can markedly affect leaf development (Fig. 2). Plants infected with either PVX:␤C1 or PVX:V2 had virus-like symptoms resembling those associated with AYVV-ID [ID:Tom]/ToLCJB-[ID:ID1:03] infecting N. benthamiana. 3.3. Reversion of GFP silencing by AYVV-ID[ID:Tom] and its associated betasatellite molecule Given the facts that, agro-inoculation of AYVV-ID[ID:Tom] alone or with its cognate AYVV-ToLCJB-[ID:ID1:03] resulted in characteristic symptoms in N. benthamiana (Fig. 1), we determine whether virulence caused by AYVD is correlated with its ability to suppress gene silencing, agro-infiltration assay was performed with either the cloned viral DNA alone or the cloned viral DNA together with its cognate betasatellite into 16c seedlings carrying GFP transgene. GFP fluorescence in newly emerging leaves of GFP-silenced plants co-inoculated with AYVV alone or in combination with DNA␤01

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Fig. 3. PTGS by begomovirus infection. (A) Reversal of established silencing by AYVVD infection. GFP-transgenic N. benthamiana line 16c was infiltered with an A. tumefaciens strain harboring the cognate GFP. A plant silenced for GFP was agro-inoculated with AYVV-ID[ID:Tom] alone or AYVV-ID[ID:Tom] with ToLCJB-[ID:ID1:03] (DNA␤01) and photographed at 21 dpi (a and b). (B) Identification of V2, C2, C4 and ␤C1 as suppressors of RNA silencing by Agrobacteruim co-infiltration assay. The green fluorescence images of the co-infiltered leaves with the abaxial-side up were taken 6 dpi under a long-wave UV lamp. (C) Northern blot analysis of GFP mRNA extracted from the zones infiltered with an A. tumefaciens strain carrying 35S:GFP together with an A. tumefecanins strain carrying the empty binary vector (35S:–), 35S: HcPro (2), 35S:V2 (3), 35S:C2 (4), 35S:C4 (5), and 35S:␤C1 (6). The ethidium-stained gel shown below the blot provides an RNA loading control. (D) RT-PCR assay for the GFP mRNA. RT-PCR products for the GFP and Actin mRNA are shown in the top and bottom panels, respectively.

could be observed (Fig. 3A) at 25 dpi, indicating that the established 35S:GFP silencing had been suppressed. These experiments show that AYVV-ID[ID:Tom] alone and or with DNA␤01 are suppressors of established RNA silencing in infected plants. Further, we determine whether viral infection can interfere with the initiation of GFP silencing, GFP expressing 16c seedlings were agro-inoculated with either cloned viral DNA alone or cloned viral DNA in combination with the associated DNA␤01. By 20 dpi, the infected 16c seedlings were induced for GFP silencing via agro-infiltration. Plants co-inoculated with AYVV-ID[ID:Tom] plus DNA␤01 still exhibited a strong green fluorescence in the emerging leaves and to lesser extent by AYVV-ID[ID:Tom] alone. Some suppression of GFP silencing occurred in the lower infiltrated leaves, and incomplete suppression appeared in the intermediate leaves. However, green fluorescence was observed with the seedlings inoculated with AYVV-ID[ID:Tom] alone and DNA␤01 not only appears to interfere with established silencing but also prevents the establishment of silencing by 35S:GFP in infected plants (data not shown). 3.4. Identification of RNA silencing suppressors encoded by AYVV-ID[ID:Tom] and its cognate betasatellite molecule To identify potential RNA silencing suppressor in the AYVVID[ID:Tom] and its associated ToLCJB-[ID:ID1:03] open reading frames (ORFs), C2, V2, C4 and ˇC1 were tested. The initial screening was carried out by using an Agrobacterium co-infiltration assay as described by Voinnet et al. (1999). Thus, to assess

the suppressor properties of the different AYVV-ID[ID:Tom] and ToLCJB-[ID:ID1:03] genes, N. benthamiana line 16c leaves were coinfiltered with 35S:GFP expressing A. tumefaciens and a second strain containing the desired AYVV-ID[ID:Tom] coding sequences under the control of 35S promoter in a pBIN2 vector. Suppression of GFP silencing was monitored and the leaves were photographed seven days after infiltration (dpi). Co-infiltration of 35S: GFP with empty pBIN61 vector or a plasmid (35S:HcPro) expressing the HcPro suppressor of silencing from TEV was used as negative and positive control, respectively. Examination of infiltered leaves at 7 dpi showed that in tissues infiltered with 35S:GFP plus the empty vector, green fluorescence decreased as a consequence of RNA silencing activation. By contrast, in tissues co-infiltered with 35S:GFP plus 35S:HcPro, intense green fluorescence was observed (Fig. 3B). When leaves were infiltered with a mixture of suspension carrying GFP as a construct harboring C2, V2, C4 and ␤C1, fluorescence in case of V2 and ␤C1 were comparable with that of positive control of HcPro. Co-infiltered leaves of C2 and C4 showed GFP fluorescence at the beginning but later it became milder as compare to that of V2 and ␤C1 (Fig. 3B). Consistent with these observation, northern blot analysis and RT-PCR revealed that at 7 dpi, the steady state levels of GFP mRNA were very low in leaves agro-infiltered with 35S:GFP plus empty vector. In contrast, very high level of GFP mRNA accumulation was evident in leaves infiltered with 35S:GFP plus V2 and 35S:GFP plus HcPro followed by expression of 35S:GFP plus ␤C1 (Fig. 3C). However, silencing suppression by C2 and C4 was weaker as measured by both the intensity of green fluorescence and

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Fig. 4. Suppression by AYVV-ID[ID:Tom] and ToLCJB-[ID:ID1:03] expressed from the heterologous vector PVX of RNA silencing triggered by GFP mRNA. (A) Photographs taken under UV light of N benthamiana 16c plants expressing GFP and inoculated with GFP plus either in combination with PVX or with recombinant constructs at 15 dpi. Northern blot analysis of PVX:GFP (1), PVX:GFP + PVX:C4 (2), PVX:GFP + PVX:␤C1 (3), PVX:GFP + PVX:C2 (4), PVX:GFP + PVX:m␤C1 (5), PVX:GFP + PVX:C2 (6). (B) Analysis of GFP mRNA using northern blot hybridization. Ethidium bromide staining of rRNA was used as loading control.

the accumulation levels of GFP mRNA in the infiltered leaves. These results were confirmed by RT-PCR analysis of GFP transcripts. In a positive control, co-expression of HcPro promoted accumulation of even high amounts of the GFP transcripts (Fig. 4D). Equal efficiency of all RT-PCR reactions was confirmed using a constitutively expressed ACTIN gene (Fig. 3D). Next, PVX derived vector was used to express the different AYVD proteins in planta, reasoning that suppression ability of proteins with a mild intracellular suppression activity could be overlooked if the transient expression under a 35S promoter does not allow expression at sufficient levels to see their effect in the infiltered patch. However, the coding sequences tested in the coinfiltration assay described above, were expressed from the PVX vector and tested individually in N. benthamiana line 16c inoculated with PVX:GFP. Fig. 4A shows that mock inoculated plants maintained green fluorescence, as that of PVX vector inoculated plants under UV light when photographed at 15 dpi. In contrast, PVX: GFP inoculated plants showed decreased green fluorescence (Fig. 4A) and consequently appeared red. GFP-transgenic plants inoculated with PVX:m␤C1 plus PVX:GFP showed systemic spread of silencing (Fig. 4A). Plants inoculated with PVX:␤C1 plus PVX:GFP showed severe downward curling and maintained green fluorescence under UV light (Fig. 4A). However, PVX: V2 plus PVX:GFP inoculated plants showed more severe symptoms than those of PVX:␤C1. Instead of the normal mild mosaic symptoms by PVX, PVX:V2 induced necrosis on the systemically infected leaves and stem, leading to death of the plants at 20 dpi. However, at 15 dpi, before the systemic necrosis had developed, the newly emerging leaves of PVX:V2 infected plants were green fluorescent under long-wave UV light. GFP-transgenic plants inoculated with either PVX:C2 plus PVX:GFP or PVX:C4 plus PVX:GFP had decreased UV light when photographed at 15 dpi. Persistence of the GFP fluo-

rescence in co-inoculation with V2 and ␤C1 expressed from PVX correlated with high steady state levels of GFP mRNA, in contrast to the marked reduction observed in leaves co-inoculated with recombinant C2, C4 and m␤C1 (Fig. 4B). These results demonstrated that V2 and ␤C1 are strong suppressors of gene silencing; however, C2 is mild suppressor and C4 show very weak activity to suppress PTGS in N. benthamiana expressing GFP. 3.5. Sub-cellular localization Sub-cellular localization of AYVD encoded V2, C2 and ␤C1 were examined using autoflourescence tag GFP after post-bombardment in epidermal cells of N. benthamiana. Each ORFs were fused at the N-terminus of sGFP (Niwa et al., 1999), thus obtaining V2:GFP, C2:GFP and ␤C1:GFP. The GFP fluorescence was observed with a confocal microscope. Fig. 5 shows that GFP alone was distributed uniformly in the nucleus and the cytoplasm while C2:GFP fluorescence was localized to the cell nucleus (Fig. 5), as that of positive control (GFP:CP), which is consistent with previous findings (Kon et al., 2007; Trinks et al., 2005; van Wezel et al., 2001). Sub-cellular localization of V2:GFP was distributed in cell cytoplasm and showed bright inclusion body along the cell periphery (Fig. 5). This association of AYVV-ID[ID:Tom] V2 with cytoplasm and punctuate spots in cell periphery is reminiscent of the localization of V2 of Tomato yellow leaf curl virus-Israel [Dominican Republic] (TYLCV-IL[Do] and East African cassava mosaic Cameroon virus-Cameroon[Cameroon:1998] (EACMCV-CM[CM:98]) (Rojas et al., 2001; Chowda-Reddy et al., 2008), however unlike the TYLCVIL[Do] (Rojas et al., 2001), AYVV-ID[ID:Tom] V2 did not move to neighboring cells. To test whether ToLCJB-[ID:ID1:03] ␤C1 protein targets to the cell nucleus or cytoplasm, a construct was synthesized by inserting ␤C1 sequence to the N-terminus of sGFP

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Fig. 5. Sub-cellular localization of GFP fusion proteins in epidermis cells of tobacco. GFP alone is distributed throughout cell (left panel at top), C2:GFP exclusively localized into the nucleus (middle panel, top), V2:GFP showed distribution around the cell cytoplasm (left panel at bottom), C4 localized within plasma membrane, as shown by white arrows (right panel at top) and ␤C1 localized around the nucleus, punctuate spots distributed cell periphery and cytoplasmic strand in tobacco (middle panel at bottom). Images shown are projections of several confocal sections under green filter, while V2 observed under yellow filter. GFP:CP captured under cyan filter was used as positive control after 12 h post-bombardment, exclusively localized in the cell nucleus. The fluorescence image of GFP is superimposed on the DIC, DsRed and GFP of the same cell.

(Niwa et al., 1999), thus obtaining ␤C1:GFP. Transient expression of ␤C1:GFP fusion protein co-localized at the cell periphery and around the nucleus (Fig. 5). However, the inflorescence in a large number of cells was much stronger in the cytoplasmic strands than nuclei. Our findings that C4 of AYVV-ID[ID:Tom] localize with the plasma membrane (Fig. 5) is consistent with the findings of Fondong et al. (2007), however, we could not detect the protein in peri-nucleus as shown for EACMCV-CM[CM:98] AC4. 4. Discussion One of the most common strategies used by plant viruses to counteract the antiviral RNA silencing defense response of plants consist of encoding suppressor proteins. To efficiently counteract an RNA silencing defense response, some of the members in the Geminiviridae family have been shown to encode multiple RNA silencing suppressors (reviewed in Sharma and Ikegami, 2008; Vanitharani et al., 2005). Similarly, our data suggest that another member of this family, the begomovirus AYVV, also adopts the strategy of encoding several viral proteins to counteract the plant RNA silencing defense response. In the light of previous studies that showed the ability of Tomato yellow leaf curl China betasatellite [China:Yunnam 10:2000] (TYLCCNB-[CN:Yn10:00] ␤C1 protein binds to DNA, suppress PTGS and target the nucleus (Cui et al., 2005). However, it has not shown whether V2, or C4 proteins encoded by Tomato yellow leaf curl China virus-Boshan1 [China:Yunnam10: Tobacco:2000] (TYLCCNVBao1 [CN:Yn10:Tob:00]) and Ageratum yellow vein China virus acts as suppressors of PTGS. The aim of present work was to understand the functions of V2 and ␤C1 of AYVD. The AYVV-ID[ID:Tom] co-infection with cognate ToLCJB-[ID:ID1:03] could reverse established GFP and could block the onset of GFP silencing in new leaves of 16c plants. In this experiment, the intensity of the silencing

suppressor activity of ␤C1 appears to be similar to that of other suppressor protein for example potyviruses CMV 2b, HcPro and p19 tombusvirus (Brigneti et al., 1998; Kasschau and Carrington, 1998; Roth et al., 2004). However, a ␤C1 mutant of ToLCJB[ID:ID1:03] failed to induce disease symptoms when co-inoculated with AYVV-ID[ID:Tom] and consequently did not play role in silencing suppression. Present data suggest that ␤C1 protein functions as a suppressor of RNA silencing and that it is a pathogenesis protein that plays a vital role in symptom induction by suppression of the silencing defense in plants. Many plant RNA viruses have associated with satellite RNAs that are completely dependent on the virus for replication, movement and encapsidation. Some satellites RNAs are known to exacerbate viral symptoms or induce symptoms distinct from those induced by the helper virus alone, but most attenuate symptoms, whether or not they contain the expressed ORF (Collmer and Howell, 1992). Betasatellites associated with begomoviruses contains a functional ˇC1 gene and have been demonstrated to play a vital role in inducing symptoms (Briddon et al., 2003; Jose and Usha, 2003; Kon et al., 2006; Sharma and Rishi, 2007; Ogawa et al., 2008). In present study, we found that ␤C1 protein functions as a virulence determinant and induce virus-like leaf curl symptoms in N. banthamianum plants in the absence of virus infection, while mutated ␤C1 decreases symptoms drastically. Identifying host proteins that interact with a viral suppressor of RNA silencing is a very promising approach to take advantage of viral suppressors to elucidate the silencing pathway. Indeed, Yang et al. (2008) have shown that the TYLCCNB-[CN:Yn10:00] pathogenesis ␤C1 interacts with asymmetric leaves (AS1) to cause alterations in leaf development resulting in the manifestation of disease symptoms and suppresses a subset of jasmonic acid responses. However, previous reports suggested that ˇC1 genes with first methionine codon mutation remain fully pathogenic (Cui et al., 2004; Saunders et al., 2004). Our localization experimental studies showed that GFP:␤C1 protein was confined to the cell periphery and around the nucleus

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in tobacco. Recently, we have reported that the ␤C1 encoded by ToLCJB-[ID:ID 2:03] associated with tomato leaf curl disease in Java (ToLCD) is localized towards the periphery of the cell. Together, these patterns of localization are similar to that of TYLCV-IL[Do] and Maize streak virus (MSV) encoded by V1 protein, known to mediate cell-to-cell movement (Kotlizky et al., 2000; Rojas et al., 2001). However, Cui et al. (2005) demonstrated that ␤C1 encoded by DNA␤ associated with TYLCCNB-[CN:Yn10:00] is exclusively targeted into the cell nucleus, as ␤C1 has a nuclear localization signal (NLS-45 PALAKKK51 ) and mutation of this NLS resulted in the loss of nuclear localization. AYVD ␤C1 lacks this sequence, providing a possible explanation for their sub-cellular targeting. Therefore, colocalization of ␤C1 around the nucleus and cytoplasmic strands (the present study) might play a role in cell-to-cell movement. However, further studies are directed to understand the precise role of ␤C1 in movement. Kumar et al. (2006) have shown by using yeast one hybrid system that ␤C1 of BYVMV-IN[IN:Mad] encompasses nuclear export signal (NES) and interact with tomato karyopherin␣ protein. Our results show that infections with AYVV-ID[ID:Tom] alone could reverse established GFP or to block the onset of GFP silencing in N. benthamiana 16c plants. Therefore, we tested all AYVVID[ID:Tom] proteins for suppression of RNA silencing; we observed this activity with the V2, C4 and C2 proteins. However, viral encoded silencing suppressors often do not share homology in their sequence or in other functions during its life cycle (reviewed in Sharma and Ikegami, 2008) and we did not detect the suppressor activity in rest of the proteins encoded by AYVV-ID[ID:Tom] (data not shown). The observation that the expression of V2 from the heterologous virus (PVX) enhances the symptoms severity (Fig. 2) and plant eventually died three to four days after the systemic symptoms were observed. This enhancement of virulence, including death of N. benthamiana has also been reported after the expression of several other plant silencing suppressors from a PVX vector (Brigneti et al., 1998). A direct association between virulence enhancement and increased PVX accumulation (data not shown) due to suppression of RNA silencing cannot be established for V2 because PXV:V2 infected plants died rapidly. A possible clue could be that V2 suppresses silencing at an early stage of RNA silencing, as demonstrated recently in case of Tomato yellow leaf curl virus-Israel[Israel:Rehovot:1986] (TYLCV-IL[IL:Reo:86] (Zrachya et al., 2007). In contrast, V2 encoded by BYVV-IN[IN:Mad] did not show any suppressor activity (Gopal et al., 2007). Using point mutations, Chowda-Reddy et al. (2008) have shown that AV2 pathogenicity and its ability to suppress RNA silencing is dependent upon a conserved putative protein kinase C (PKC) phosphorylation motif. In addition, it has been shown that V2 of ToLCJV-A[ID] encode virulence determinant, which elicits hypersensitive response (HR) (Sharma and Ikegami, 2010). It is worth noting that AYVV-ID[ID:Tom] V2 protein when expressed from PVX vector showed necrosis (Fig. 2). Any HR response encoded by AYVVID[ID:Tom] V2 needs to be further confirmed. The C2 protein of begomoviruses is multifunctional. It is the transcription factor required for the expression of late viral genes (Sunter and Bisaro, 1991), can be a pathogenicity factor (van Wezel et al., 2001), a suppressor of PTGS (van Wezel et al., 2002; Gopal et al., 2007; Kon et al., 2007) and also interacts with and inactivates SNF-1 related kinase (Hao et al., 2003) and adenosine kinase (Wang et al., 2005). Infiltration and inoculation studies showed that AYVV-ID[ID:Tom] C2 might be a mild suppressor. If it were strong suppressor of PTGS, it should have maintained the enhanced GFP expression when co-infiltered with GFP. Recent study showed that BYVV-IN[IN:Mad] C2 is a weak suppressor and predominantly a trans-activator but both activities may be essential for viral replication and trans-activation (Gopal et al., 2007). Preliminary evidence of N-terminus sequence analysis of AYVV-

ID[ID:Tom] C2 protein shows that this region contains a stretch of Arginine residues (24 KRRTTRRRR32 ) and the zinc finger domain, may imply transcriptional dependent activation mechanisms of silencing suppression, as has been demonstrated in other geminiviruses (Dong et al., 2003; Trinks et al., 2005; van Wezel et al., 2001). How a weak suppressor is initially recognized remains to be elucidated. The steps in the RNA silencing pathway targeted by V2, and ␤C1 proteins remain to be elucidated. Experiments with other several suppressor proteins, such as Hc-Pro, P19, AC4 of ACMV and turnip yellow mosaic virus p69, have shown that they affect plant development by interference with miRNA mediated silencing pathways that share components with the siRNA mediated antiviral pathway (Chapman et al., 2004; Chellappan et al., 2005; Chen et al., 2004; Dunoyer et al., 2004). In Arabidopsis, these pathways are affected by interfering with the dicing activity of DCLs (DCL1, DCL2, and DCL3) required for miRNA and siRNA biogenesis (Zamore et al., 2000). In the present study, the over expression of V2 and ␤C1 elicited viruslike symptoms suggesting that these proteins may also play role in developmental regulation by interfering with miRNA pathways. Thus, it may be possible that V2 and ␤C1 proteins may affect the activity of the Dicer-like proteins in plants that function in silencing suppression and down regulate transcription of a host protein that acts in the PTGS of a pathway in the cytoplasm or could activate transcription of a host PTGS inhibitor. The precise mechanism by which V2 and ␤C1 regulates miRNA levels will be the focus of our future research. Previously, we have isolated ToLCJV-ID[ID:05] and AYVVID[ID:Tom:05] and their associated betasatellites (Kon et al., 2006) from the same tomato infected plants in Indonesia. Evidence for geminivirus gene silencing activity suggests that at least four proteins have this capacity (for review see in Sharma and Ikegami, 2008). Surprisingly, the PTGS suppressor function of these proteins is not conserved among all geminiviruses. For example, C4 protein encoded by AYVV-ID[ID:Tom:05] code for suppressor activity (this study), while C4 of ToLCJV-ID:[ID:05], another tomato infecting monopartite, is not (Kon et al., 2007). Probably, none of these suppressors has been characterized as being as strong as those encoded by RNA viruses, such as P19 (Voinnet, 2008), which may in part support the choice of a multiprotein strategy adopted by begomoviruses. The existence of a range of molecularly unrelated suppressors of PTGS for geminiviruses illustrates the differential evolution of geminiviruses encodes suppressors of gene silencing. It remains to be discovered whether this is a mere reflection of the removed plasticity of geminivirus genomes or an indication of a powerful selection pressure to be able to counteract RNA silencing. Acknowledgments This work was funded by Grant-in-Aids from Japan Society for the Promotion of Science for a Postdoctoral Fellowship awarded to first author. We are thankful to David Baulcombe, Cambridge University, for providing the PVX vector and N. benthamiana line 16c plants and Yasuo Niwa, University of Shizuoka for providing us pGFP(S65T) plasmid, Vicky Vance, University of North Carolina for pBIN:HcPro construct P.S. has designed, conducted experiments and prepared MS, T. Kon has helped in making infectious clones in Section 2.1. M. Ikegami has provided the necessary facilities. Finally, we are thankful to Drs HL Sharma, N. Sharma (HPKV, Palampur) for critical reading and Project Director (DWR) for encouragement during preparation of this manuscript. References Baulcombe, D.C., Chapman, S.N., Santa-Cruz, S., 1995. Jellyfish green fluorescent protein as a reporter for virus infections. Plant J. 7, 1045–1053.

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