1.11.62
AMINO ACID MUTATIONS IN THE HELPER COMPONENT PROTEASE AND THE COAT PROTEIN OF PLUM POX POTYVIRUS (PPV-NAT) ALTER SYMPTOM EXPRESSION AND VIRUS REPLICATION IN NICOTIANA BENTHAMIANA PLANTS

M VARR-ELMANN and E MAISS

Institute for Plant Diseases and Plant Protection, University of Hannover, Herrenhaeuser Str. 2, D-30419 Hannover, Germany

Background and objectives
The coat protein (CP) of potyviruses is responsible for encapsidation of the viral RNA and is involved in replication and aphid transmission of the virus. The helper component (HCpro) has a protease activity and is essential for aphid transmission. In addition, the CP as well as the HCpro play roles in the movement of the virus from cell-to-cell. To test the participation of CP and HCpro of PPV on replication and symptom expression, different mutations were generated in the CP and HCpro genes and introduced into a full-length clone of PPV (35PPV-NAT) [1].

Materials and methods
The core region of potyvirus CPs contains two amino acid motifs supposed to be associated with particle assembly. These motifs were rendered non-functional by replacing the original amino acids with ones of different chemical properties (3015RQ>DV, 3059DF>KI; amino acid numbering according to PPV-NAT [2]). These two mutations were introduced separately and together into the CP gene. The HCpro of PPV-NAT possesses two amino acid motifs involved in aphid transmission. Even in this case, the motifs were changed (360KI>EF; 619TK>AE) and introduced separately and together into the HCpro gene. The different mutated full-length constructs were tested for infectivity via particle bombardment on Nicotiana benthamiana plants and transgenic Nicotiana benthamiana plants (line 27/4) expressing the coat protein of PPV-AT.

Results and conclusions
None of the mutations in the CP gene affecting particle assembly displayed systemic infections in N. benthamiana plants. No virus could be detected either in ELISA or in ISEM in these plants. In transgenic N. benthamiana plants, expressing the viral CP, all of the defective CP-mutants were complemented by the transgenic CP, as indicated by the appearance of a systemic infection. In the case of transgenic complementation these different CP-mutants showed divergent and milder symptoms compared to the wild type PPV-NAT. These findings strengthen previous results on the role of the coat protein in cell-to-cell movement and particle assembly of potyviruses.

None of the different mutations in the HCpro influenced the ability of PPV to infect N. benthamiana plants systemically, but the double mutation and the mutation 619TK>AE produced very severe symptoms with local lesions followed by the appearance of vein necrosis, completely differing from the wild-type PPV symptoms. Beside its function in the aphid transmission process, the distinct amino acid motifs in the HCpro are able to influence symptom severity.

References
1. Maiss E. et al., 1992. Journal of General Virology 73, 709-13.
2. Maiss E. et al., 1989. Journal of General Virology 70, 513-24.