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

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.