This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Viruses are the second greatest contributors to yield losses of agricultural crops after fungi. ‘Potato spraing’ disease caused by Potato Mop-Top Virus (PMTV) results in qualitative economic losses. The virus and its protist vector cannot be controlled by agrochemicals, and there are no sources of resistance to PMTV. Previously, phylogenetic analysis identified three major genotypes for PMTV RNA-CP. Genomic RNA-CP of one of these genotypes appeared to be longer than that of the rest of genotypes. The difference in length is due to the presence of a tandem repeat of the “coremin”-like motif in the 3´-untranslated region of RNA-CP. “Coremin” motif is required for stalling Xrn1, a major exoribonuclease involved in the RNA metabolism and, thus, leads to accumulation of exoribonuclease-resistant RNAs (xrRNAs). Folded RNA elements that block processive cellular exoribonuclease RNA decay machinery to yield biologically active viral non-coding RNAs are produced by many RNA viruses including plant viruses. Whereas xrRNAs contribute to pathogenicity of human and animal viruses, the role of xrRNAs in plant virus infectious remains elusive. The aim of my summer student project in Eugene Savenkov’s lab at The Swedish University of Agricultural Sciences (SLU) was to gain insight into the role of xrRNA in suppression of RNA silencing – one the major resistance mechanisms against plant viruses – using TCV-sGFP complementation system.
The main focus of the project was to employ a complementation assay suitable in identifying silencing suppression activity for even weak viral suppressors of RNA silencing (VSRs). The identification of VSR activity is achieved using an assay based on complementation of cell-to-cell movement of Turnip Crinkle Virus (TCV), the genome of which was modified by swapping a capsid protein (CP) gene of the virus with a reporter gene for Green Fluorescent Protein (GFP). The cell-to-cell movement of TCV in host plants requires expression of its CP, a well-charactersed VSR. Thus, in the absence of CP, TCV-GFP is not able to move efficiently to neighboring cells and is confined to single cells due to induction of antiviral silencing and spread of the virus derived short interfering RNAs into neighboring cells, which immunise them against incoming virus.
XrRNA was amplified from the complete RNA-CP of Peruvian isolate J20, and then inserted in pCB plasmid via In-fusion cloning and transformed into Escherichia coli. After confirming the construct by sequencing, the plasmid was transformed into Agrobacterium tumefaciens prior to agroinfiltration into N. benthamiana leaves to overexpress xrRNAs. However, before doing these experiments, I wanted to test whether RNA-CP of the Peruvian isolate J20 of PMTV, which produce xrRNA, is able to complement VSR-deficient TCV-GFP. I also used a potyviral HC-Pro, the strong suppressor of RNA silencing, as a positive control, infiltration of leaves with A. tumefaciens carrying an empty plasmid (EP) as a negative control, and full-length RNA-CP construct of the Swedish isolate (Sw) of PMTV (which does not have a “coremin”-like motif) as another control.
The results show that Sw RNA-CP, J20 RNA-CP, and J20 xrRNA constructs could have a weak ability to complement TCV-sGFP movement (9, 5 and 4 foci, respectively, consisting of more than four cells). In average the area of GFP fluorescence in square micrometers (µm²) were 20,977µm² (Sw RNA-CP), 40,011 µm² (J20 RNA-CP) and 47,288 µm² (J20 xrRNA) respectively, whereas in case of the EP control, I mostly observed foci consisting of one cell with an average fluorescent area of foci of 6,222 µm². The results suggest that the tested constructs might suppress RNA silencing although rather weakly. The results will be validated further employing another set of experiments and different experimental approaches. Mutagenesis will be performed as well to identify xrRNA structural elements needed for suppression of RNA silencing.
Anderson Stiward González Rivera
I.U Colegio Mayor de Antioquia, Colombia
