Intra-host structure of multi-genotype populations of a plant RNA virus assessed by a novel RNA in situ hybridization approach
Text by Svetlana Folimonova and María Bergua
Citrus tristeza virus (CTV) represents a peculiar, somewhat unique, virus model. It has been known since long time that field populations of CTV are often composed of different genotypes or strains of the virus. As we demonstrated in our recent study, this results from the fact that superinfection exclusion between variants of CTV follows a specific rule: only virus variants of the same strain exclude each other, while no exclusion occurs between variants belonging to different CTV strains permitting their co-existence in the same plant (Folimonova et al., 2010). On the other hand, little was known about the intra-host structure of such complex virus populations and, for instance, whether individual genotypes spatially segregate within the infected tissues or they are well mixed and are able to co-infect the same cells. A novel RNA in situ hybridization (ISH) approach employed in this work allowed us to address these questions.
Some time ago we engineered a green fluorescent protein-tagged virus using a cDNA clone of a CTV variant of the T36 strain and used it to study virus distribution and movement within citrus hosts (Folimonova et al., 2008). The lack of the infectious clones of other CTV strains limited, however, further studies of the interaction and distribution of CTV variants representing heterologous strains upon their co-infection. While searching for a technique that enables differential labeling of individual variants of CTV simultaneously present in a plant, we came across the RNAscope ISH assay (Advanced Cell Diagnostics, Inc.). The manufacturer claimed that this novel method allows specific detection and separation of two RNA targets sharing up to 90% of sequence identity. This seemed exciting!
Using the assistance from Advanced Cell Diagnostics, we designed probes that target the RNA genomes of two distinct CTV genotypes, T36 and T68. The probes were tested in the ISH assay using tissue samples from singly or doubly infected citrus plants. One of the challenging parts was to optimize the RNAscope protocol for virus-infected plant tissue. The final results, however, were remarkable. Highly specific labeling showing no cross-reaction between a probe targeting one virus variant and RNA molecules produced by another variant allowed us to demonstrate that in doubly infected plants the two virus variants appeared to be well mixed and often co-infected the same cells. According to our knowledge, this is the first report of simultaneous in situ visualization of two distinct strains of a plant virus.
Simultaneous visualization of Citrus tristeza virus (CTV) T36 (blue stain) and T68-1 (red stain) RNA in cross sections from petioles of a Citrus macrophylla plant coinfected with T36 and T68-1 isolates using a multiplex RNAscope in situ hybridization assay. Both CTV variants were found occupying the same cells (black stain).
Introducing the authors
At the left: María Bergua, she is a former postdoctoral researcher in Svetlana Folimonova’s laboratory at the University of Florida. Currently María is a postdoctoral researcher at the University of California, Department of Plant Pathology, Davis, CA, USA.
At the right: Svetlana Folimonova, Assistant Professor at the University of Florida, Department of Plant Pathology, Gainesville, FL, USA.