A-O7 A single amino acid substitution in the movement protein enables mechanical transmissibility of a geminivirus You-Xiu Zheng1, Chia-Hwa Lee1, Yu-Tsung Lin1, Wen-Shi Tsai2, Chin-Hsiang Chan1,3, Hsin-Mei Ku3 and Fuh-Jyh Jan1,4* 1 Department of Plant Pathology, National Chung Hsing University, Taichung 40227, Taiwan 2 Department of Plant Medicine, National Chia Yi University, Chiayi 60004, Taiwan 3 Department of Agronomy, National Chung Hsing University, Taichung 40227, Taiwan 4 Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan Abstract Begomoviruses of the Geminiviroidae are usually transmitted by whiteflies but rarely by mechanical inoculation. We used Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, to address this issue. While most ToLCNDV isolates are not mechanically transmissible to their natural hosts, a ToLCNDV oriental melon isolate (ToLCNDV-OM) identified in Taiwan is mechanically transmissible. To identify the viral factor that determines the mechanical transmissibility, we first performed genome swapping experiments with the DNA-A and DNA-B genomes between ToLCNDV-OM and non-mechanical transmissible ToLCNDV-CB (cucumber isolate). Mechanical transmissibility in melon and Nicotiana benthamiana was achieved only when a virus carried DNA-B from ToLCNDV-OM. Further pseudorecombination experiments mapped the mechanical transmissibility determinant to the movement protein (MP) gene. Mutational analyses identified the 19th amino acid (glutamate, E19) of ToLCNDV-OM MP as essential for mechanical transmissibility. This is the first identification of a specific geminiviral movement protein as a determinant of mechanical transmissibility. Keywords Begomovirus, Tomato leaf curl New Delhi virus, ToLCNDV oriental melon isolate, mechanical transmissibility, movement protein Figures and Tables Fig. 1. The recombinant construct between 5’ or 3’ end of MP gene. The different amino acid residues between ToLCNDV-OM and ToLCNDV-CB on MP protein determine the infectious ability. A total of eight amino acid residues were shown in MP protein and was separated by the restriction enzyme PstI. In the 5’-region included the difference at 3rd, 6th, 8th, and 19th and contrary the 3’-region contained the difference at 193rd, 225th, 233rd, and 262nd amino acid, respectively. Table 1. Inoculation results of ToLCNDV isolates and their mutants Mechanical inoculation Virus Infectious DNA Viral genome Tobacco Oriental melon Cucumber OM pOM2A + pOM2B + + + OM1 pOM2A +pOMB + + + OM4 pOM2A +pOMB-5’CBMP - - - OM5 pOM2A + pOMB-3’CBMP + + + OM6 pOM2A + pOMB-MPT3I + + + OM7 pOM2A + pOMB-MPE6D + + + OM8 pOM2A + pOMB-MPM8V + + + OM9 pOM2A + pOMB-MPE19G - - - CB pCB2A +pCB2B - - - CB1 pCB2A +pCBB - - - CB4 pCB2A + pCBB-5’OMMP + + + CB5 pCB2A + pCBB-3’OMMP - - - CB6 pCB2A + pCBB-MPI3T - - - CB7 pCB2A + pCBB-MPD6E - - - CB8 pCB2A + pCBB-MPV8M - - - CB9 pCB2A + pCBB-MPG19E + + + Acknowledgements We are grateful to Dr. Wen-Hsiung Ko, Professor Emeritus, University of Hawaii at Manoa and Dr. Chung-Jan Chang, Professor Emeritus, University of Georgia, Griffin Campus for their critical review of this manuscript. We also thank Dr. Shi-Dong Yeh for valuable discussion. This research was partially supported by grants from the National Science Council (NSC99-2628-B-005-012-MY3 and 101-2313-B-005-039-MY3), Executive Yuan, Taiwan. References cited 1. Chang, H. H., Ku, H. M., Tsai, W. S., Chien, R. C., and Jan, F. J. 2010. Identification and characterization of a mechanical transmissible begomovirus causing leaf curl on oriental melon. Eur. J. Plant Pathol. 127:219-228. 2. Levy, A., and Czosnek, H. 2003. The DNA-B of the non-phloem-limited Bean dwarf mosaic virus (BDMV) is able to move the phloem-limited Abutilon mosaic virus (AbMV) out of the phloem, but DNA-B of AbMV is unable to confine BDMV to the phloem. Plant Mol. Biol. 53:789-803. 3. Wege, C., and Pohl, D. 2007. Abutilon mosaic virus DNA B component supports mechanical virus transmission, but does not counteract begomoviral phloem limitation in transgenic plants. Virology 365:173-186.