Potato leafroll virus : molecular analysis and genetically engineered resistance

Abstract

The nucleotide sequence of the genomic RNA of potato leafroll virus (PLRV) was elucidated and its genetic organization deduced (Chapter 2). Six open reading frames (ORFs) were shown to be present on the genome. Both the PLRV coat protein gene and the RNA- dependent RNA polymerase gene were identified by interviral sequence comparison. The PLRV genomic organization was shown to be highly similar to that of beet western yellows virus (BWYV) and except for the ORF1 products all PLRV and BWYV coded proteins displayed an extensive amino acid sequence homology.In order to obtain resistance following the principle of pathogen-derived resistance, the PLRV coat protein gene was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter and used to transform potato (Chapter 3). Upon analysis of the transgenic plants obtained it was shown that, although transgenic transcripts were abundantly present in the plant tissues, the presence of transgenic coat protein could not be detected. The transgenic potato plants were shown to be susceptible to PLRV infection but contained significant lower virus titers as compared to infected wild-type potato plants. To enhance the translational expression of the coat protein gene the sequences flanking the start codon were modified to a theoretically optimized context (Chapter 4). Potato plants were transformed with the altered coat protein gene and analyzed for the presence of transgenic coat protein. Despite of the induced mutations transgenic protein could not be detected. The results from inoculation experiments with PLRV were identical to those obtained with the transgenic plants containing the unaltered coat protein gene, the transgenic plants containing less viral antigen than infected wild-type plants.To investigate the role of the PLRV ORF1 product (P1) in the viral infection process and to define its intracellular location in infected plant cells, the protein was expressed in Escherichia coli and in the baculovirus expression system and used to raise an antiserurn (Chapter 5). Expression of P1 proved to be difficult, possibly due to a toxic effect imparted by the protein. Using an antiserurn raised against a recombinant P1 fusion protein, it was determined that P1 did not accumulate in infected plant tissues to detectable levels.To further investigate the function of the ORF1, its sequence was transformed into potato (Chapter 6). Surprisingly, the transgenic plants expressing detectable levels of ORF1, transcripts displayed an altered phenotype closely resembling that of virusdiseased plants. Plants expressing a modified and therefore untranslatable, version of the ORF1, sequence were phenotypically indistinguishable from wild-type control plants, indicating that the expression of the P1 protein induced virus disease-like symptoms. The transgenic potato plants containing the ORF1, sequence were analyzed for possibly acquired resistance (Chapter 7). Upon infection one plant line showed to be highly resistant while all other plant lines were susceptible to PLRV-infection similar to wild-type plants. The resistance obtained expressed itself as near immunity, only under high inoculation pressure a low percentage of the plants became infected