Investigation of virus-induced defence modulations during plant-virus interactions

Abstract

Turnip mosaic virus (TuMV) is a single stranded RNA (sRNAs) virus belonging to the genus Potyvirus. Turnip mosaic virus has a wide host range including several important crop plants, making it economically significant. This study investigates the relation between a strain of TuMV and two model plant systems; Arabidopsis thaliana and Nicotiana benthamiana, focusing on the viral sequence, host gene expression and defence mechanisms, and viral derived small RNAs (viRNAs). Turnip mosaic virus has been well studied and characterised with 183 complete genome nucleotide sequences available in GenBank (5 June 2018). The TuMV isolate used in this study was sequenced and submitted to GenBank, as well as the original isolate collected in 1994, under the names TuMV-QLD1b and TuMV-QLD1a, respectively (accession numbers KX641465 and KX641466). The original TuMV isolate was PCR sequenced while the 2015 isolate was sequenced by deep RNA sequencing. A comparison between the two sequences showed minor variations with 18 single-nucleotide-polymorphisms (SNPs). Another aspect of the study involved the sequencing of a Cucumber mosaic virus (CMV) isolate (strain K) as well as an Australian Cauliflower mosaic virus (CaMV) isolate belonging to the genus Cucumovirus and Caulimovirus respectively. This strain of CaMV represents the first Australian isolate to be fully sequenced. Both the CMV and the CaMV isolates were used to study plant defence pathways in Chapter 4 of this thesis. The sequence of both isolates were published and make up Chapter 2.Chapter 3 investigates the early defence response of A. thaliana 6, 24 and 48 hours after TuMV inoculation. Marker gene expression results suggest that the virus upregulated the jasmonic acid (JA) pathway 24 hours after infection. This is significant, as viruses are classified as biotrophic pathogens which usually upregulate the salicylic acid (SA) pathway leading to hyper-sensitive response and programmed cell death, preventing the virus from spreading. It is hypothesised that the upregulation of the JA pathway may favour the virus allowing it to establish more easily and systemically infect its host.The JA and other defence pathways are further researched through studying the interactions between a mediator mutant and four different viruses; TuMV, CMV, CaMV and Alternanthera mosaic virus (AltMV). The mediator complex consists of several subunits and is highly conserved among eukaryotes as it regulates transcription. Previous studies have shown that med18 plants are more resistant to Fusarium oxysporum which was also found to upregulate the JA pathway in WT plants. Results show a similar trend with viral infected med18 plants having less viral RNA than WT plants 14 days after infection, though most were not significant due to large variations of viral load between individual plants. However, med18 plants infected with CMV had significantly less viral load than WT plants.Some of phenotypic symptoms caused by viral infection may be a secondary effect of RNA silencing. It is hypothesised that viRNAs can interfere with the plant’s regulations and development causing phenotypic symptoms. Similar to previous studies, small RNA sequencing of virus infected A. thaliana and N. benthamiana suggested that there was a significant increase in the number of small RNAs (sRNAs), specifically those of 21 nucleotides (nt) in length. This length would also suggest that these are produced through a specific biogenesis pathway. Studies have reported that certain areas of a virus genome, called “hotspots”, are more prone to being acted upon by the plant’s sRNAs biogenesis machinery. Chapter 5 results suggest this to be true, with certain regions producing more sRNAs and causing certain viRNAs to be more abundant.When comparing the most abundant viRNAs to host genes we identified possible targets based on complementarity. As a high degree of complementarity is believed to be required for endogenous small interfering RNAs (siRNAs) directed silencing it was hypothesized that highly abundant and complementary viRNAs had the potential to target and inhibit certain genes. The dual-luciferase report system was used to attempt to quickly validate possible viRNA targeting of host transcript sequences. The quantitative nature of this assay allowed us to determine whether viRNAs interacted with the target transcript based on the expression ratio. A mutated target sequence was included as a control to confirm viRNA interaction. Many targets from both A. thaliana and N. benthamiana were tested, though only one target sequence interaction was repeatedly confirmed using this assay system. The N. benthamiana gene 3160g02007 was confirmed to be targeted by TuMV as there was a clear decrease in the dual LUC expression ratio in the presence of the virus. The expression was restored when the target was mutated. Furthermore, no decrease was apparent when plants were not infected.