Plant viruses are surprisingly efficient and successful pathogens with great persistence. They can evolve rapidly through mutation and genetic recombination to overcome unfavourable situations. Their potential to prevail over adverse conditions is assisted mainly by their ability to mutate frequently. Potyviruses are plant RNA viruses of great economic importance. They are plus strand RNA viruses and have the same evolving capacity as any other RNA virus. In this study we have characterised and sequenced the complete genome of a recombinant Turnip mosaic virus (TuMV) isolate, TuMV-BRS1, from Brisbane, Australia. The protein sequence alignment of the virus indicates mismatches to other isolates in HC-Pro, C1, NIb and CP.
Before the search for potential microRNAs in viral genome we studied the presence of the virus in the plant nuclei. Surprisingly, we were able to detect both strands of the virus in the nucleus. This finding proves that RNA viruses are not restricted to the cytoplasm but can move into the plant nucleus. This is also supported by the fact, reported earlier, that RNA virus polymerase has nuclear localisation signals and it is mainly present in the nucleus.
The new TuMV isolate was then screened for potential microRNA sequences and, using microRNA prediction softwares, we were able to predict several microRNA sequences in the viral genome. MicroRNAs are 21-26 nt ubiquitous regulatory molecules of RNA which control gene expression. They have the capability to modulate host gene expression through repression and activation. Several animal viruses, especially the ones belonging to the family herpesviridae, encode microRNAs which regulate cellular processes like apoptosis. These microRNAs can also specifically target the viral genome for its own regulated replication. We have for the first time discovered that plant viruses are equally capable to encode microRNAs. These microRNAs could target gene expression of the host and the virus itself. The two microRNAs, TuMV-mir-S1 and TuMV-mir-S2, were predicted, and confirmed through Northern blot hybridisation. Expression of this microRNA in several RNAi pathway Arabidopsis mutants helped us to develop a pathway for plant viral microRNA biogenesis. This pathway varies at certain steps from the microRNA biogenesis pathway known to exist in plants.
MicroRNA target prediction software suggested that this microRNA could target the stress responsive gene HVA22D in Arabidopsis. This gene has been assigned a new role after this study as it was earlier known to act only in abiotic stress conditions. Our results show that HVA22D is also crucial in cellular defence pathway against viruses. HVA22D is regulated by abscisic acid. This finding also provides further information regarding the coordinated cross-talk of biotic and abiotic stress responses in plants.
To analyse the role of plant-genome encoded microRNA in viral defence, a study was conducted to discover host microRNAs in response to TuMV infection. A microRNA microarray was used for small RNA hybridisation experiments which resulted in detection of 40 differentially expressed Arabidopsis thaliana microRNAs in response to virus infection. Host microRNAs with the highest induction during TuMV infection included ath-miR172b, ath-miR168a, ath-miR403 and ath-miR156h. The two microRNAs, ath-miR168a and ath-miR403 are known to have target binding sites in AGO1 and AGO2 sequences. Search for a predicted target sequence for miR156h resulted in the finding that this microRNA could potentially target AGO7. An increased transcript level was detected for all three AGO genes after virus infection. This increase in both the microRNAs and their respective targets reveals the likely existence of a co-regulation mechanism which has already been reported for ath-miR168a and AGO1. The induced expression of these AGO proteins could be due to their role in the plant RNAi pathway, which is a plant defence arsenal against viruses.
A search was also conducted for potential target sequences for these microRNAs in the viral genome. This search indicated a possible target binding of ath-miR172b in the NIb gene of the virus. This is the viral RNA dependant RNA polymerase which is the major viral replication protein. A GFP-fused target sequence was co-infiltrated in Nicotiana benthamiana leaves along with a construct over-expressing the ath-miR172b precursor. This transient assay resulted in observation of increased GFP expression when the ath-miR172b over-expression construct was infiltrated along with the target fusion construct. This led us to the possibility that a higher level of ath-miR172b after virus infection could regulate virus replication after binding to the viral replication gene (NIb) transcript. This is a new observation in plant virus interaction but, interestingly, such kind of interaction has already been observed in animal systems for Hepatitis C virus (HCV) replication in liver cells.
Research on microRNA-based interaction between a virus and its plant host during virus infection is essential to increase our understanding on how viruses infect plants and even use the plant’s RNAi pathways to their own benefit. It has been a matter of debate how viruses are able to infect plants in the presence of numerous host defence response. This study shows that viruses can directly target host defence response genes and down-regulate these through viral microRNAs, thus facilitating their proliferation in plants. Plants also use their microRNAs and their respective targets to combat the virus. This study is also significant because of its use in designing new strategies for developing virus resistant plants. This study reveals that targeted inhibition of specific viral microRNAs or mutation in their respective target sequences, without altering the protein sequence, could be used as a possible resistance development strategy.