Lettuce necrotic yellows virus (LNYV) is a major problem in most Australian lettuce growing areas, with up to 20 % losses to farmers in dry years. The extensive use of herbicides and insecticides to remove the virus' weed reservoirs and aphid hosts is economically and environmentally undesirable. Four grower-preferred commercial crisphead lettuce lines with genetic resistance to other pathogens were selected for genetic improvement to confer resistance to LNYV.
A transformation and regeneration system was developed utilising the marker green fluorescent protein (GFP), which worked with high efficiency for the four cultivars. GFP expression was stable in these plants with integration of the gene construct into the lettuce genome, and this was conferred to the next generation.
To confer resistance to LNYV, the nucleoprotein gene was isolated and cloned into a plant gene expression vector which was
specifically designed to minimise the use of 3rd party intellectual property. Both a translatable (N) and an untranslatable (UN) version of the nucleoprotein gene in the construct was generated and transformed into the plant varieties 'Seagreen' and 'Centenary'. Fifteen N and fourteen UN independent lines were generated for 'Seagreen' and nineteen N and eighteen UN lines were created for 'Centenary', with both single and multi-copy integrants identified. N protein expression levels varied between transgenic and in some lines the transgene was silenced.
Resistance screening of transgenic lettuce plants was attempted using the aphid vector Hyperomyzus lactacae, in which the virus is circulative and propagative. Aphids carrying the strain of LNYV that the nucleoprotein gene was isolated from could not transmit LNYV to lettuce. Naturally LNYV-infected H. lactucae were sourced from the Gatton agricultural district and used in
a number of resistance screening trials, both supervised feeding and cage trials. No resistant or immune lines could be definitively identified, but six hues were infected by the virus, and were classed as susceptible. The sequence of the LNYV nucleoprotein gene of the Gatton isolate was found to be substantially different from the gene used in the resistance construct, so plants may not be protected against this strain. The presence of LNYV-specific siRNAs in infected N. glutinosa (an indicator host) was confirmed, which demonstrates that LNYV RNA can act as inducer and target of RNA-silencing and that transgenic resistance provided by this mechanism should be achievable for rhabdoviruses.
A detailed safety assessment and risk management analysis was complied for the Genetic Manipulation Advisory Committee and two environmental releases were approved. Selected transformed lines were tested in small-scale field trials for agronomic
performance, progeny analysis and resistance to LNYV. The growth habit, shape, size, appearance and colour of the head of modified plants did not differ from the non-transgenic control plants. A low level of LNYV infection occurred in the second field trial, but not at a high enough level to identify resistant lines.
The analysis of the LNYV 3' leader and 5' trailer regions for promoter activity in transgenic plants was also conducted, to possibly allow the use of one of these regions as a LNYV-specific inducible transcription switch. The leader and trailer sequences were isolated from the LNYV genome and cloned in plant expression vectors containing the reporter β-glucuronidase (GUS). The constructs required the presence of the viral RNA-dependent-RNA polymerase to transcribe GUS mRNA, but due to the recalcitrance of the infection system no definitive results were obtained.