1.1.4
INVESTIGATIONS ON P1, P2 AND P3 NON-STRUCTURAL PROTEINS OF BARLEY MILD MOSAIC VIRUS (BaMMV) IN INFECTED BARLEY CELLS

INVESTIGATIONS ON P1, P2 AND P3 NON-STRUCTURAL PROTEINS OF BARLEY MILD MOSAIC VIRUS (BaMMV) IN INFECTED BARLEY CELLS

VW FOMITCHEVA1, Z SUBR2, F EHRIG1, E PROLL1 and T KUEHNE1

1Federal Centre for Breeding Research on Cultivated Plants, Institute for Resistance Research and Pathogen Diagnostics Aschersleben, PO Box 1505, 06435 Aschersleben, Germany; 2Department of Plant Virology, Institute of Virology, Slovak Academy of Sciences, Dúbavská 9, 84246 Bratislava, Slovakia

Background and objectives
BaMMV is a member of the Bymovirus group which is transmitted to barley seedlings under natural conditions by the soilborne fungus Polymyxa graminis. It has a bipartite genome with 7262 nt (RNA1) and 3524 nt (RNA2) in the case of isolate BaMMV-ASL. Although the virus has been known for more than 20 years in Europe, little is known about the mechanism of the specific uptake and preservation of the virus by the vector. The RNA2-encoded protein P2 is assumed to play a key role in this process. For several years it has been known that mutants of the wild-type virus displaying deletions in the P2 gene may occur spontaneously. The sizes of the lost fragments are very different and range from approximately 100 nt up to more than 1000 nt. Contradictory results exist in the literature regarding the fungus transmissibility of deleted BaMMV isolates [1, 2]. The aim of this work was to further elucidate the biological functions of the non-structural proteins P1, P2 and P3 (encoded by RNA1) using different serological methods.

Materials and methods
Starting from almost full-length clones of the two genomic RNAs, the corresponding genes were recloned in pThioHis and pTrxFus vectors (Invitrogen) and expressed in E. coli as fusion proteins with thioredoxin. Recombinant proteins were purified by PAGE and polyclonal antisera were raised in rabbits.

Results and conclusions
The antisera reacted specifically with the homologous antigens in bacterial extracts and in sap of virus-infected barley plants. This was demonstrated by Western blotting and direct tissue print immuno-assay. Immuno-electron microscopical and Western blot analyses of purified virus particles and crude plant sap (only IEM) revealed no data indicating integration of P1 and P2 molecules into the virus capsid. The proteins obviously do not serve as receptors for the interaction with the fungal vector. In our previous cytological investigations, very characteristic, regular foldings of proliferated endoplasmatic reticulum were observed in ultrathin sections of leaf and root cells of BaMMV infected plants. Those structures did not occur in plants harbouring an isolate with a large deletion in the 3’ region of the P2 gene, although the replication rates of the wild type and the mutant viruses were almost the same. Results of immunocytological analysis applying the specific antisera for P1, P2 and P3 will be presented.

References
1. Adams MJ, Swaby AG, Jones P, 1988. Annals of Applied Biology 112, 133-141.
2. Dessens JT, Meyer M, 1996. Virology 212, 383-391.