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Beet curly top Iran virus Rep gene works as a silencing suppressor by reducing small RNA accumulation

  • Autor/in: Ebrahimi, S., O. Eini, M. Wassenegger, G. Krczal, V.V. Uslu
  • Jahr: 2022
  • Zeitschrift: Tagungsband 54. Jahrestagung des DPG Arbeitskreises Viruskrankheiten der Pflanzen. Dossenheim, Hybrid, 21.- 22.03.2022.
  • Seite/n: 20


Beet curly top Iran virus (BCTIV) is a yield-limiting becurtovirus of the Geminiviridae family. While BCTIV mobility (V3) or coat (V1) protein genes on clockwise orientation are closely related to curtoviruses, the replication (RepA, C1) and cell cycle related proteins (C2) resemble mastreviruses. In this study, we screened the silencing suppressor role of BCTIV genes in wild type (WT) and GFP expressing (16C) Nicotiana benthamiana. We revealed that V2 and Rep genes showed silencing suppressor activity when full length GFP sequence was transiently expressed in WT. V2 gene was already shown to interfere with gene silencing-3 (SGS3) activity, an essential component of secondary siRNA production and viral resistance. However, the mode of action of Rep gene in BCTIV remained almost fully elusive. Rep is a product of alternative splicing between C1 and C2 genes, leading to a novel protein with the N-terminus of the C1 and C-terminus of frameshifted the C2. Further analysis on the 16C line demonstrated that on the contrary to V2, Rep could also suppress local post transcriptional gene silencing triggered by transient expression of a sense transgene (PTGS-S), containing a truncated GFP sequence matching to the 5’ GFP of the 16C. While neither V2 nor Rep could suppress post transcriptional gene silencing induced by inverted repeats (PTGS-IR) locally, only Rep blocked systemic silencing completely. Northern blot analysis showed that while PTGS-S or PTGS-IR lead to accumulation of small RNAs (sRNAs) in the presence of V2, Rep alleviated the sRNA production substantially upon both PTGS-S and PTGS-IR. Furthermore, we structurally dissected the Rep protein into seven Domains and addressed the silencing suppressor activity of these domains in combinations. We found that only a 30-amino-acid-long domain at the very end of the C-terminus did not contribute to the silencing function, but the removal of any other domain resulted in the loss of the silencing suppressor activity of Rep. Overall, our results demonstrated for the first time that becurtovirus Rep gene has silencing suppressor activity and our findings implicated that Rep has a distinct mode of action than V2, which is yet to be fully discovered.
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