1.11.32
AMINO ACID MUTATIONS IN THE HELPER COMPONENT PROTEASE AND THE COAT PROTEIN OF PLUM POX POTYVIRUS (PPV-NAT) ALTER SYMPTOM EXPRESSION IN NICOTIANA BENTHAMIANA PLANTS

M VARRELMANN and E MAISS

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

Background and objectives
The coat protein (CP) of potyviruses is responsible for the 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 the CP and the 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 nonfunctional 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. 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 N. benthamiana plants (line 27/4) expressing the coat protein of an aphid-transmissible strain of PPV (PPV-AT).

Results and conclusions
None of the virus mutants with amino acid changes in the CP affecting particle assembly gave raise to symptoms in N. benthamiana 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. 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. In addition to its function in the aphid transmission process, the distinct amino-acid motifs in CP and HCpro are able to influence symptom severity.

References
1. Maiss E et al., 1992. Journal of General Virology 73, 709-713.
2. Maiss E. et al., 1989. Journal of General Virology 70, 513-524.