Virus del moteado de la parietaria (PMoV): estudio de mecanismos de interacción del virus con la planta y caracterización de aislados virales

Student thesis: Doctoral thesis

Abstract

In the first Thesis chapter , we have studied the genetic variation and evolution of parietaria mottle virus (PMoV). Phylogenetic analysis showed that the Italian isolates clustered in the clade I and the Spanish isolates clustered in the clades II, III and IV. The studied isolate GrT-1 from Greece clustered in the clade IV for the CP phylogenetic tree whereas it fell out as an individual isolate for the 2b phylogenetic tree. The nucleotide diversity was low as for other plant viruses, but higher than that for other ilarviruses. The distribution of synonymous (S) and non synonymous (N) substitutions revealed that 2b and CP were under strong purifying selection with some positions under diversifying selection. These results suggest that both genes are under evolutionary constrains probably as a consequence of the essential roles played on the virus life cycle. In addition, we have detected some events of genetic exchange, probably by reassortement of different genomic segments between PMoV Spanish isolates. A molecular and biological characterization of the PMoV isolate T32 was performed. The results obtained suggested that this PMoV isolate is a different pathotype and genotype respect to the Spanish isolated CR8 and Italian isolate Pe1. Nucleotide sequence analysis of the T32 genomic RNAs and the encoded putative proteins showed different conserved motifs. The CP of isolate T32 showed a nucleotide (cytosine) deletion that resulted in a different start codon rendering a CP which was 16 amino acids shorter than those of the Italian isolates (Pe1 and ST-1). Finally, the analysis of N and S substitutions indicated a negative or purifying selection pressure for all genomic regions. Later, the role of 3a (MP) protein in the virus cell-to-cell movement was studied. In silico analysis revealed the presence of two hydrophilic non-contiguous regions (R1 and R2) with many basic amino acids: lysines (K) and arginines (R), and a secondary structure in α-helix. Results of Electrophoretic Mobility Shift Assays (EMSA) showed that both R1 and R2 regions were able to bind RNA in an independent manner. Mutational analysis showed that K and R basic amino acids of these regions were essential for virus cell-to-cell movement. The assays carried out to determine the subcellular localization of PMoV MP reveled that the wild-type MP was located in the PDs whereas the MP mutants which the basic amino acids were removed, lost total or partially the ability to accumulate in the PDs. Assays with a recombinant construction containing the RNA 3 of Alfalfa mosaic virus (AMV) whose MP was replaced with those of PMoV showed that MP localization in the PDs was essential for the intercellular virus movement. Finally, the role played for the CP, MP and 2b proteins of PMoV in the development of infection symptoms (pathogenicity or avirulence factors) was studied. The PMoV CP induced strong symptoms of stunting, mosaic and leaf deformation in Nicotiana benthamiana plants, while PMoV MP and 2b proteins induced only leaf deformation and mosaic symptoms. The analysis to determine if PMoV CP, MP and 2b proteins act as suppressors of RNA silencing (VSRs) through a viral vector based on the Turnip crinkle virus (TCV) showed that neither CP, MP or 2b proteins were able to suppress the genic silencing mechanism. These results showed that suppression of RNA silencing pathway could not be implied on symptoms induction in N. benthamiana plants by CP, MP or 2b proteins of PMoV.
Date of Award4 Feb 2016
Original languageSpanish
SupervisorFrederic Aparicio (Director), Luis Galipienso (Director) & Charlotte Poschenrieder (Tutor)

Keywords

  • Pellitory
  • Virus
  • ARN

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