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


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.



Ambrós, S., El-Mohtar, C., Ruiz-Ruiz, S., Peña, L., Guerri, J., Dawson, W. O., & Moreno, P. (2011). Agroinoculation of Citrus tristeza virus Causes Systemic Infection and Symptoms in the Presumed Nonhost Nicotiana benthamiana. Molecular Plant-Microbe Interactions,24(10), 1119-1131. doi:10.1094/mpmi-05-11-0110

Atallah, O. O., Kang, S., El-Mohtar, C. A., Shilts, T., Bergua, M., & Folimonova, S. Y. (2016). A 5′-proximal region of the Citrus tristeza virus genome encoding two leader proteases is involved in virus superinfection exclusion. Virology,489, 108-115. doi:10.1016/j.virol.2015.12.008

Bar-Joseph, M., Marcus, R. & Lee, R.F. (1989). The continuous challenge of Citrus tristeza virus control. Annual Review of Phytopathology,27, 291-316.

Bergua, M., Kang, S. & Folimonova, S. Y.. (2016). Understanding superinfection exclusion by complex populations of Citrus tristeza virus. Virology,499, 331-339. doi:10.1016/j.virol.2016.10.001

El-Mohtar, C., & Dawson, W. O. (2014). Exploring the limits of vector construction based on Citrus tristeza virus. Virology,448, 274-283. doi:10.1016/j.virol.2013.10.017

Folimonova, S. Y. (2012). Superinfection Exclusion Is an Active Virus-Controlled Function That Requires a Specific Viral Protein. Journal of Virology,86(10), 5554-5561. doi:10.1128/jvi.00310-12

Folimonova, S. Y. (2013). Developing an understanding of cross-protection by Citrus tristeza virus. Frontiers in Microbiology, 4(76). doi: 10.3389/fmicb.2013.00076

Kang, S., Bak, A., Kim, O., & Folimonova, S. Y. (2015). Membrane association of a nonconserved viral protein confers virus ability to extend its host range. Virology,482, 208-217. doi:10.1016/j.virol.2015.03.047

Lee, R. F., & Keremane, M. L. (2013). Mild strain cross protection of tristeza: a review of research to protect against decline on sour orange in Florida. Frontiers in Microbiology,4(256). doi: 10.3389.fmicb.2013.00259

Moreno, P., Ambrós, S., Albiach-Marti, M. R., Guerri, J. & Pena, L. (2008), Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Molecular Plant Pathology,9, 251-268. doi:10.1111/j.1364-3703.2007.00455.x

Schindelin, J., Arganda-Carreras, I. & Frise, E. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods,9(7), 676-682. doi: 10.1038/nmeth.2019