Discussion: The C2 protein of geminiviruses plays a variety of roles. All positional homologues of this protein in begomoviruses have three

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1 Discussion: The C2 protein of geminiviruses plays a variety of roles. All positional homologues of this protein in begomoviruses have three functional domains: a basic domain with a bipartite nuclear localization signal (NLS) at the N terminus, a nonclassical zinc finger in the middle and an acidic activation domain at the C terminus (Chowda-Reddy et al., 2009; Jeske, 2009; Sunter & Bisaro, 1997). TYLCV C2 binds to ssdna in a sequence non-specific manner (Noris et al., 1996). MYMV AC2 causes toxicity when expressed transgenically and the effect can be overcome by mutating the NLS or zinc finger motif or truncating the activation domain (Rajeswaran et al., 2007). More than thirty genes were found to be co-activated when the transcriptome profile of the Arabidopsis protoplast transiently expressing AC2 protein of MYMV and AC2 protein of ACMV were compared. AC2 protein of ACMV is a suppressor of PTGS and the transgenic expression of AC2 in tobacco plants causes phenotypic changes in plant leaves and flowers. The transcriptome analysis of the transgenic tobacco expressing ACMV AC2 revealed that 1118 genes were differentially expressed in leaves, whereas in flowers 251 genes were differentially expressed. The expression of genes which are involved in the biosynthesis of ethylene and jasmonate and subsequently the genes which are involved in pathways regulated by these hormones were found to be upregulated. Genes involved in the biosynthesis of chlorophyll, photosynthesis, translational factors and ribosomal protein subunits were down regulated (Soitamo et al., 2012). Hypersensitive response (HR) is one of the ways by which the plant defense is able to overcome the pathogen by a type of programmed cell death. NSP of ToLCNDV can induce HR in Nicotiana tabacum and Lycopersicum esculentum plants. The C2 protein of ToLCNDV was found to prevent HR. The zinc finger and NLS motifs are important to overcome HR (Hussain et al., 2007). In the present study, the role of BYVMV C2 protein in symptom development and replication were determined. Two stop codons were introduced in the C2 ORF and an infectious 1.2 mer C2 mut A PTR was created. N.benthamiana plants infiltrated with A PTR and β PTR showed severe leaf curling and enations similar to the results obtained by Jose and Usha, (2003). But when co-infiltrated with C2 mut A PTR and β PTR, no symptoms were found even at 45 dpi. Even the mild leaf crumpling which appears in the plants infiltrated with wild type A PTR alone does not appear when the

2 C2 ORF is mutated. This shows that C2 may play a role in symptom development. The plants infiltrated with β PTR alone did not produce any symptoms. Southern hybridization using probe specific for DNA A revealed that the viral DNA replication was drastically affected when the C2 ORF was mutated and as a result the intensity of the band was very weak when compared to the sample from plants coinfiltrated with the wild type APTR and β PTR. No viral DNA was detected when the plants were infiltrated with C2 mut APTR alone, whereas viral DNA replication could be detected at higher levels when infiltrated with wild type APTR alone. The level of betasatellite accumulation was very low in plants co-infiltrated with the PTR containing the mutant version of C2, when compared to the plants co-infiltrated with PTR with the wild type C2. No betasatellite was detected in plants infiltrated with β PTR alone. Quantitative real-time PCR was used to find the viral load in infiltrated samples. Plants infiltrated with the wild type A PTR alone contained 2.83 x 10 7 copies whereas C2 mut A PTR alone infiltrated plants contained only x 10 2 copies, indicating the effect of the mutant version of C2. There was a 100 fold reduction in the copy number of DNA A in plants co-infiltrated with the C2 mut A PTR and β PTR compared to those where the wild type A PTR was used along with β PTR (1.65 x 10 6 copies vs 1.08 x 10 8 copies) showing that the betasatellite of the virus could compensate for the C2 mutation. The Ct value denotes the cycle threshold. The Ct values for the A PTR alone and C2 mut A PTR alone samples were 9.17 and respectively reflecting the drastic reduction in the viral load when C2 ORF was mutated, as Ct values are inversely proportional to the viral load. The effect of mutant C2 in the viral replication can be overcome to a greater extent when co-infiltrated with betasatellite which is reflected in the Ct values of the samples wild type A PTR + β PTR (9.17), C2 mut A PTR + β PTR (15.12). Therefore it may be inferred that betasatellite plays a role in overcoming the reduction in replication due to mutation of C2 ORF. In BCTV, mutations in the C2 ORF were introduced using termination codons or by frame shifting, resulting in the production of truncated versions of the C2 protein. The mutations did not have any effect on the infectivity of the virus and induced severe symptoms in N.benthamiana and B.vulgaris (Stanley et al., 1992). Mutations in the C2 of curtovirus SCTV induced milder symptoms and accumulated viral DNA at a reduced level compared to wild type virus (Baliji et al., 2007). Stop codon was introduced in BSCTV C2 ORF to create a virus lacking the C2 protein. Arabidopsis

3 plants were inoculated with mutant BSCTV and analyses revealed that the methylation of BSCTV genome was enhanced due to the lack of C2 protein (Zhang et al., 2011). Cotton leaf curl Burewala virus (CLCuBuV) is a begomovirus associated with Cotton leaf curl Multan betasatellite, and is capable of infecting resistant varieties of cotton. Sequence analysis of several isolates of CLCuBuV revealed that it lacked a functional C2 protein with one or two stop codons present in the C2 ORF leading to the production of a truncated version of C2 containing only 35 amino acids (Amrao et al., 2010). This resistance-breaking strain has become prevalent in north-western parts of India also (Rajagopalan et al., 2012). It is indeed very interesting to observe the fact that a functional C2 is absent in a resistance-breaking strain. Post transcriptional gene silencing (PTGS) is one of the processes by which the host tries to overcome pathogens and the pathogen tries to evade the pathogen defense mechanism by encoding suppressors of PTGS. Transient agro co-infiltration assay was used to study the suppression of PTGS by the truncated versions of C2 lacking either the activation domain or the NLS motif. In the plants infiltrated with Agrobacterium expressing GFP alone, the fluorescence was reduced drastically at 10 dpi. This is due to the triggering of a local RNA silencing mechanism. The positive control used for this study is HcPro which has been demonstrated to be a strong suppressor of PTGS (Llave et al., 2000). Therefore when N.benthamiana plants were co-infiltrated with GFP and HcPro there was no loss of fluorescence. The leaves of plants co-infiltrated with GFP and BYVMV C2 displayed a weak fluorescence. This is consistent with the results obtained by Gopal et al., (2007), where they have demonstrated that BYVMV C2 is a weak suppressor of PTGS. When the plants were co-infiltrated with Agrobacterium expressing GFP + C2 Tr4 or GFP + C2 del NLS there was considerable reduction in fluorescence when compared to the leaves of plants co-infiltrated along with the fulllength C2. Though the fluorescence in the Tr4 co-infiltrated region appears less when compared to the region co-infiltrated with C2 del NLS, it is very difficult to find the differences visually, as the wild type C2 itself is only a weak suppressor of PTGS. AC2 proteins of EACMCV and ICMV which are bipartite old world begomoviruses, have been demonstrated to be suppressors of PTGS (Vanitharani et al., 2004). The AC2 protein of MYMV acts as a strong suppressor of PTGS. However, when the NLS domain or the Zinc finger motif or the activation domain was truncated the PTGS suppression activity of the protein was abolished (Trinks et al., 2005). The

4 AC2 protein of TGMV which is a bipartite new world begomovirus, is a weak suppressor of silencing. C33A mutation in the zinc finger like CCHC motif of TGMV AC2 did not affect the PTGS suppression activity (Yang et al., 2007). C2 protein of TYLCCNV which is a monopartite begomovirus with a betasatellite, has been shown to be a strong suppressor of PTGS. When three cysteine residues present in the putative zinc finger motif were mutated the PTGS suppression activity was lost (van et al., 2002). Similarly when the stretch of four arginines present in the NLS motif was mutated the protein lost the ability to suppress PTGS (Dong et al., 2003). The C2 protein of TYLCSV acts as a weak suppressor of PTGS (Luna et al., 2012), whereas the C2 protein of TYLCV Is does not function as a suppressor of PTGS (Zrachya et al., 2007). Rand et al., (2011), have demonstrated that the karyopherin α1 promoter can be induced using auxin. When N.benthamiana plants were co-infiltrated with C2 and karyopherin α1 promoter driving the expression of GUS, blue color development was observed, suggesting that C2 was able to trans-activate the karyopherin α1 promoter. Further analysis is required to substantiate this trans-activation capability. C2 acts as a transcriptional activator and trans-activates the transcription of late viral genes (Haley et al., 1992; Sunter and Bisaro, 1992). In the case of the curtoviruses BSCTV and SCTV, the transcription activation domain was absent in C2 and hence it did not function as a transcriptional activator (Baliji et al., 2007; Hormuzdi and Bisaro, 1995). Trans-activation of the host genes was also demonstrated in the case of TGMV AC2, where the activity of the cytokinin-responsive promoters was increased (Baliji et al., 2010). E. coli based protein expression vectors permit the production of recombinant fusion protein in which specific affinity tags are added to the protein of interest. In the present study the 6X His tag was used to purify the viral proteins. The 6XHis tag is poorly immunogenic, small and does not affect the secretion, folding, compartmentalization, structure and function of the recombinant protein. The 6X His tag was fused in-frame to the N terminus of the viral proteins C2 and βc1. The expression of the viral proteins C2 and βc1 was very weak and hence no difference was observed between the uninduced and induced E.coli BL21 cells. Positive signal was detected from the supernatant of the induced E.coli BL21 cells for both the proteins C2 and βc1 by Western blotting using anti His antibody. This confirmed the

5 expression of the viral proteins, albeit at a lower concentration. The proteins were obtained in the soluble form in the supernatant. The expression of the viral proteins was comparatively better at 30 o C for 4h than 37 o C for 4h. Since the viral proteins were obtained in a soluble form, non-denaturing conditions were used to purify the viral proteins using Ni-NTA agarose beads. Both the viral proteins C2 and βc1 were purified by affinity chromatography using Ni-NTA agarose beads. The over-expression of CP of MSV (Liu et al., 1997), TYLCV (Palanichelvam et al., 1998), SLCV (Qin et al., 1998), ToLCBV- [Ban 5] (Kirthi and Savithri, 2003), ACMV (Unseld et al., 2004) and MYMIV (Malik et al., 2005) have been reported. The recombinant expression of Rep protein of TYLCV (Laufs et al., 1995b) TGMV (Settlage et al., 1996; Pant et al., 2001), BC1 and BV1 of SLCV (Pascal et al., 1994), BC1 of BDMV (Noueiry et al., 1994) have demonstrated. The C2 proteins of TYLCV (Noris et al., 1996) and AC2 protein of Potato yellow mosaic virus (Sung and Coutts, 1996) have been expressed as fusion with 6x His tag. The TGMV AC2 and BCTV C2 have been expressed as GST fusion. The TGMV AC2 has also been expressed as 6X His fusion in Sf9 insect cells (Wang et al., 2003). The ACMV AC4 and EACMCV AC2 were expressed as 6x His tag fusions and the purified proteins were used to check their ability to bind with mirna. The ACMV AC4 was able to bind single stranded forms of mirna, whereas EACMCV AC2 did not bind to mirna (Chellappan et al., 2005). The βc1 protein of betasatellite associated with TYLCCNV has been expressed as fusion with 6x His tag (Cui et al., 2005) and with GST tag (Yang et al., 2008). The BYVMV proteins C2 and βc1 will be used subsequently to study the interaction with mirna and to study the interaction with other host proteins using pull down assay. The proteins were expressed at a small scale in this present study and conditions need to be optimized for large scale expression of the viral proteins, which will be useful for studying the structural features of the protein. Conclusion: BYVMV C2 protein plays an important role in symptom development. When Nicotiana benthamiana plants were infected with BYVMV containing mutated version of C2 and betasatellite, no symptoms were observed even at 45 dpi. The C2 protein also helps in the replication of the virus, which is evident from the fact that there is a drastic reduction in the level of viral DNA when the plants are infected with BYVMV having mutant C2 as determined by quantitative real time PCR analysis. But when BYVMV