Development of recombinant antibody mediated resistance against Tomato yellow leaf curl virus

In this study, we exploited the expression of specific scFv fragment in plant cells for suppression of disease symptoms caused by TYLCV infection. The C1, V1 and V2 genes encoding Rep, CP and MP, respectively, were cloned into the TOPO vector and subsequently into pGEX-5x3 and pMALc2x expression vectors. Recombinant proteins were expressed in E. coli as C-terminal fusion with GST or MBP and purified proteins obtained by affinity chromatography method. In addition, the amino terminal part of CP and Rep proteins were also cloned and expressed as fusion proteins with GST and MBP. Using phage display technology one scFv fragments against Rep (scFv-ScRep1) was isolated through panning of naïve Tomlinson I scFv phage library. In addition, an ARep phage display library constructed from total spleen RNA of a mouse immunized with MBP-Rep was analyzed by panning and another scFv fragment (scFv-ScRep2) was selected and characterized. The scFv fragments were expressed in pHENHI bacterial expression vector and purified by immobilized metal affinity chromatography. ELISA and Western blot analyses were used to analyze binding activities of bacterially expressed scFv. The observed high binding activity of bacterially expressed scFv-ScRep1 to both full length and C- terminal truncated of Rep protein indicated specific binding to the amino terminal end of Rep while scFv-ScRep2 interacted exclusively to intact Rep protein. Four more scFv fragments were developed from total RNA of murine hybridoma cells secreting specific MAbs against TYLCV virions. The pTRAkt plant expression vector was used to clone the generated scFv fragment genes individually and/or as an amino terminal fusion to GFP. Expression of scFv fragment constructs and their functionality within transiently transformed plant cells were analyzed. Blotting analyses showed detectable amounts of scFv-ScRep1, scFv-ScRep2, and NLS-scFv-ScRep1 presented in crude leaf extract of transformed plants. Further analyses proved binding ability of these scFv extracted from leaves against recombinant Rep. Fluorescence microscopy results confirmed expression and localization of scFv-ScRep1-GFP, scFv-ScRep2-GFP and scFv-NLS-ScRep1-GFP fusion protein within the cytoplasm and nucleus. Selected constructs were also used to generate stable transformations in entire N. benthamiana plants through leaf disc transformation. To determine the protection ability of transgenically expressed proteins, independent T0 progenies expressing different scFvs were challenged with pBIN19-2TYLCV-Ir construct. Early symptoms including leaf curling and size reduction of newly emerged leaves were observed on non-transgenic and sensitive transgenic plants 3-4 weeks after inoculation. Southern hybridization analyses confirmed reduction or complete suppression of viral DNA replication in the symptomless plants. Assessment of the resistance status within T0 inoculated plants was evaluated 5 weeks after inoculation. Infectivity assays of T0 progenies revealed that all inoculated NSR, HSC2, HSC3 and HSC4 lines as well as wild type plants are susceptible for the TYLCV challenge inoculation. However, SRG, SR and RW lines presented varying degrees of resistance from 8-28 percent. Inoculated plants were observed for disease symptoms development and assayed for presence of TYLCV DNA through molecular hybridization methods. Early symptoms in non-transgenic and sensitive plants appeared 2-3 wpi and developed further during the next weeks. Resistance response was evaluated at 4-5 wpi bases of symptom observation and DNA hybridization assays. A heterogeneous response was obtained within inoculated T1 progenies. These results indicated that all T1 progenies raised from RW14, RW22 and SR27 reveal typical TYLCV symptoms whereas SRG T1 plants expressing ScRep1-GFP recombinant protein showed a spectrum of symptoms ranging from a severely diseased to mild ones and the complete absence of any symptoms. The resistance phenotype was characterised by absence or remarkable reducing of disease symptoms and a concomitant substantial reduction or complete suppression of viral DNA replication. T1 plants developed from SRG28 and SRG18 lines revealed highest resistance. Further analyses indicated that individual SRG transgenic plants emit varying intensity of fluorescence under excitation by UV light. Usually, elevated amounts of ScRep1-GFP transcripts are directly correlated with higher fluorescence intensity emitted from transgenic lines. Together comparative Q-PCR and fluorescence intensity analyses with data obtained from virus resistant assays indicated that mostly plants with higher transcripts level consistently exhibited a higher degree of virus resistance