5.3.5
MOLECULAR ANALYSIS OF POTATO VIRUS Y NIb GENE AND ENGINEERING OF VIRUS RESISTANCE IN TOBACCO AND POTATO

A LIPSKA-DWUŻNIK, AM CHACHULSKA and W ZAGÓRSKI

Institute of Biochemistry and Biophysics PAS, Pawińskiego 5a, 02-106 Warsaw, Poland

Background and objectives
Potato virus Y (PVY) is the type member of the family Potyviridae that is the largest and economically the most important family of plant viruses. PVY is a dangerous potato, pepper and tobacco pathogen in Europe, Mediterranean countries and North America, leading to significant crop losses. The conventional phytopathological classification of PVY isolates is based on primary hosts and on symptoms induced in indicator plants. Potato isolates are divided into three main strains: necrotic (PVYN), common (PVYO) and inducing potato stipple streak (PVYC), according to symptoms induced on tobacco and potato. A new isolate, called PVYN-Wilga, identified in 1984 in Poland, shows unusual features. Symptomatologically it is classified as a necrotic strain; however, immunological analysis did not follow the group assignment. This isolate is highly infectious and induces rather weak necrotic symptoms. Sequence analysis of part of the PVYN-Wilga genome showed near identity of this peculiar necrotic isolate to common isolates [1]. These results led us to re-evaluate conventional PVY classification by sequence analysis. Sequence comparison of PVYN-Wilga full-length RNA polymerase gene (NIb) with representatives of other strains was performed.

Non-structural viral genes are a promising source for inducing genetically engineered virus resistance [2]. Therefore construction of tobacco and potato plants carrying the PVYN-Wilga NIb gene was undertaken, and results of resistance tests will be presented.

Materials and methods
A cDNA fragment of PVY genome corresponding to the NIb sequence was cloned in the pBluescript vector. Appropriate subclones were obtained and sequenced. The whole sequence of the NIb gene was determined. This sequence was used for comparisons with the NIb sequences of PVY isolates differing in phenotypes. Comparisons were done with GAP, PILEUP, LINEUP and PRETTY programs from the GCG package.

The cDNA fragment encoding the whole NIb polymerase was used for plant transformation. The gene was transferred into the pROK binary plasmid under the 35S promoter and equipped with appropriate start and stop codons so that the whole gene could be translated in plants. That construct was introduced into tobacco (cultivar Xanthi) and potato (cultivar Irga) by Agrobacterium- mediated transformation. Transformed lines were regenerated on selective media and submitted to molecular analysis. Presence of the NIb gene in the genome of plants was determined by the PCR technique. Tests for resistance in selected transformants are in progress.

Results and conclusions
NIb gene sequence comparisons indicate that isolate PVYN-Wilga is close to isolates belonging to the common strain and distinct from necrotic ones, despite its phenotype-based classification. These findings are in support of our previous studies and indicate that the conventional phytopathological classification of PVY cannot be used as a universal criterion for grouping isolates and should be supported by sequence analysis.

We identified, in the NIb amino acid sequence, motifs characteristic of viral polymerases. Some of these motifs, possibly responsible for polymerase activity, are highly conserved in the virus kingdom. It suggests that polymerase sequence analysis can be helpful in the creation of a general classification of viruses. We obtained transgenic plants harbouring the whole NIb polymerase gene. We checked the presence of the gene in the genome of plants by the PCR technique. Selected tobacco and potato plants are currently being tested for PVY resistance. We would like to check if the whole polymerase gene present in the genome of plants can induce PVY resistance. This will provide information on the potential application of translatable and untranslatable (truncated) versions of the NIb gene for construction of resistant plants.

References
1. Chachulska AM, Chrzanowska M, Robaglia C, Zagórski W, 1997. Archives of Virology 142, 765-779.
2. Prins M, Goldbach R, 1996. Archives of Virology 141, 2259-2276.