Developing a Nicotiana benthamiana – Citrus tristeza virus model system to study superinfection exclusion

  • Mariana Suguieda University of Florida
  • Sung-Hwan Kang
  • Svetlana Yuryevna Folimonova
Keywords: superinfection exclusion, Citrus tristeza virus, Nicotiana benthamiana

Abstract

Citrus tristeza (CTV) is the causal agent of economically devasting diseases that affect  global citrus production. CTV has demonstrated superinfection exclusion (SIE), a phenomenon where a preexisting viral infection excludes a secondary infection by the same or closely related virus. This phenomenon has been used to develop cross-protection measures against CTV but there have been erratic results with this approach. Understanding of SIE by CTV on the molecular level has been challenging, especially due to time-consuming trials of over a year when citrus plants are used in the experiments. The purpose of this study was to develop a model system to study SIE by CTV by using a more amenable species, Nicotiana benthamiana, rather than citrus plants. cDNA constructs of CTV containing extra genes of the green or red fluorescent proteins (GFP/RFP) were engineered and transformed into Agrobacterium tumefaciens to visualize virus movement and SIE within the N. benthamiana plants by using the agro-infiltration approach for the virus constructs delivery.  The constructs carrying the full-length wild-type virus with the additional GFP- or RFP-encoding gene and the GFP-tagged hybrid constructs with deletion in the p33 gene were infiltrated with the agro-suspension and visualized under a microscope. The images captured were analyzed for peaks in grayscale values in the green or red channels along a line. The constructs used in the study behaved similarly in N. benthamiana and Citrus macrophylla but the trial time was significantly shortened with the N. benthamiana plants. Thus, N. benthamiana is a suitable alternative for studying SIE by CTV with the constructs in this study.

References

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Published
2020-02-19