Identification of Reference Genes and Quantification of Gene Expression Changes in Nicotiana glutinosa Plants Infected by Subgroups I & II of Lettuce Necrotic Yellows Virus
MetadataShow full metadata
Lettuce necrotic yellows virus (LNYV) is a plant virus that has been reported to cause widespread crop losses in lettuce in Australia and New Zealand for the last 60 years. Phylogenetic analysis has determined two subgroups of the virus exist within the population, identified as subgroup I and II. It appears subgroup II has emerged more recently than subgroup I and currently has a wider geographical distribution, with subgroup I appearing to now be extinct in Australia. Limited research has been undertaken into understanding the molecular mechanisms by which the virus operates upon establishing infection within a host plant. It is not known whether the two different subgroups influence different molecular pathways which may explain the current distribution of the virus in the environment. This study was designed to determine the expression of four target genes in the host plant Nicotiana glutinosa after inoculation by subgroup I and subgroup II of LNYV. A reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay was utilised to determine the relative gene expression of the target genes CPK3, SGS3, WRKY26 and WRKY70 in response to LNYV infection. For a RT-qPCR experiment, a set of validated reference genes are necessary to act as internal controls and need to be specifically selected for studies in a particular host. Currently, no validated reference genes have been reported in the literature for N. glutinosa, so candidates were selected to determine their suitability for this purpose; Actin, EF1α, F-BOX, L23 Ntubc2, PDF2, PP2A, SAND and Ubiquitin. No full genome has been published for N. glutinosa, so molecular data from related species had to be obtained to infer the structure of these genes in order to design primers to amplify the genes in a qPCR experiment. Primers could not be designed for F-BOX, L23 and Ubiquitin, and non-specific products were amplified during amplification of WRKY26, EF1α and PDF2. The remaining candidate reference and target gene primers specifically amplified a single product and were considered suitable for testing in the gene expression study. To obtain sufficient LNYV infected biological replicates for the qPCR experiment, N. glutinosa plants were grown from seed and inoculated with LNYV subgroup I or II. Infection rates varied between 0% and 15% for subgroup 1 and was 26.6% for subgroup II after 28 days of growth. After failing to grow enough replicates to study the virus across six time points, the experimental design was amended to determine target gene expression after 28 days in both subgroups. Normalised, outlier removed qPCR data was processed using GeNorm and Normfinder algorithms and the values obtained suggested of the remaining candidate reference genes tested, SAND and Ntubc2 were suitable to be used in subsequent experiments based on their stable expression, though additional reference genes are required, and further biological replicates may be necessary to confirm this. Using these reference genes and comparing the data of the genes of interest, it was determined that CPK3, SGS3 and WRKY70 were upregulated between uninfected and subgroup I infected conditions, and CPK3 and SGS3 were downregulated between uninfected and subgroup II infected conditions, whilst WRKY70 was upregulated. Differences were identified between the subgroups, with all three genes being more highly expressed in subgroup I compared to subgroup II with an approximate 7-fold difference in WRKY70 expression, suggesting that subgroup I isolates may induce transcription and signalling pathways in hosts to a higher degree than subgroup II during infection. Though this was a small scale study it indicates that the biological impact of the different subgroups of the LNYV subgroups may differ which may have influenced the current geographical distribution of the virus. Further research could focus on identifying additional reference genes for qPCR-based studies utilising N. glutinosa, or research additional target genes to try and identify additional molecular pathways the subgroups impact.