In this thesis work I broadly present research that investigates the biology of coinfection interactions that occur in mixed infections of poleroviruses, umbraviruses, and tombusvirus-like associated RNAs (tlaRNAs), which in nature form particularly unique asymmetrically obligate virus disease complexes. I also present work detailing the identification of emergent poleroviruses, umbraviruses, and tlaRNAs specifically associated with the Carrot motley dwarf (CMD) disease complex, as well as work on the identification and biological characterization of an emergent polerovirus—not currently known to be associated with such viral disease complexes as are described in this work—that was originally identified in Korea in a barley (Hordeum vulgare) plant sample exhibiting symptoms of yellow dwarf disease. Virus disease complexes composed of a polerovirus, umbravirus, and/or tlaRNAs are widely known to cause greatly enhanced symptom development in various economically important plant hosts and are sometimes associated with increased virus accumulation of one or more of the coinfecting viruses. There are several coinfection interactions known to occur in such disease complexes, such as the ability of poleroviruses to support systemic movement of tlaRNAs—which on their own are completely immobile—within a plant host, and to also support aphid transmission of both tlaRNAs and umbraviruses—neither of which is independently aphid transmissible—by way of transcapsidation of the genomic RNAs of these viruses in polerovirus capsid proteins. Additionally, while poleroviruses are known to be phloem limited and cannot move between cells not associated with the phloem, it has been found that coinfection with an umbravirus can help a coinfecting polerovirus escape this phloem limitation and be able to move amongst mesophyll cells and as a result, in some instances, also become mechanically transmissible, a function of which poleroviruses are not independently capable. What is less known is how umbraviruses and tlaRNAs interact with one another in these disease complexes.
In Chapter 2 of this work, I used aphid inoculation of the polerovirus Turnip yellows virus (TuYV) along with agroinoculation of infectious clones of the CMD associated umbravirus Carrot mottle virus (CMoV), CMD tlaRNAs gamma and sigma, and the TuYV tlaRNA ST9 to combine these viruses in different ways in the model host plant Nicotiana benthamiana, in order to observe the biological consequences of different coinfection pairings with a focus on symptom development, systemic movement of the tlaRNAs, virus accumulation, and altered modes of transmission, i.e. aphid or mechanical. In terms of symptom development, I found that all coinfections that included CMoV, except for coinfections of CMoV with tlaRNA gamma, resulted in greatly enhanced symptom development, while all other coinfections were asymptomatic, suggesting that in this experimental system CMoV acted as the driver of symptom development. In terms of virus accumulation, while several different coinfection combinations resulted in variable increases of each of the coinfecting viruses—as determined by RT-qPCR—the most dramatic accumulation increases were observed for TuYV and CMoV in co-infections that also included tlaRNA ST9, showing this tlaRNA strongly upregulates the accumulation of TuYV and CMoV by some as yet unknown mechanism.
The most notable findings of this work, however, were the interactions that occurred between CMoV and the tlaRNAs. It was found that not only could CMoV support systemic movement of all of the tlaRNAs used in this study, although with variable efficiencies—tlaRNAs sigma and ST9 moved systemically in coinfections with CMoV 100% of the time, whereas gamma only moved 40% of the—time, but CMoV could also impart mechanical transmissibility to tlaRNAs sigma and ST9, but not tlaRNA gamma, indicating different tlaRNAs differ in their capacity for various coinfection interactions. Given the near complete lack of studies investigating interactions between umbraviruses and tlaRNAs, these results were particularly exciting. It was also intriguing to find that in triple infections of TuYV and CMoV with either tlaRNA sigma or ST9, the efficiency with which TuYV could be co-mechanically transmitted with CMoV greatly increased (54% and 77%, respectively) relative to the rate at which TuYV was co-mechanically transmitted from plants co-infected with only CMoV (11%), suggesting coinfection with tlaRNAs sigma or ST9 facilitates interaction between CMoV and TuYV in some way. All together the results of this study highlight the variability of coinfection interactions that can occur between poleroviruses, umbraviruses, and tlaRNAs and demonstrate that some of these interactions can be rather non-specific—despite TuYV and tlaRNA ST9 having not been found in natural co-infections with the CMD associated umbravirus CMoV, these viruses nonetheless were able to interact with one another.
In Chapter 3 of this work, I further highlight the plasticity of these disease complexes by identifying emergent poleroviruses, umbraviruses, and tlaRNAs in parsley, carrot, and cilantro samples exhibiting typical symptoms of CMD; in many samples combinations of these viruses not previously known to occur were observed. The viruses historically associated with CMD include the polerovirus Carrot red leaf virus (CRLV), the umbraviruses CMoV and carrot mottle mimic virus (CMoMV), and a multitude of tlaRNAs including a8, a25, alpha, beta, gamma, and sigma. In addition to identifying these recognized CMD associated viruses, we found three emergent poleroviruses (two recently reported poleroviruses and one that appears to be a novel recombinant polerovirus), two emergent umbraviruses that were recently reported as novel species, however according to our data we believe these to actually be highly divergent strains of CMoV, as well a newly described tlaRNA. Most of these emergent viruses have not previously been reported to occur in the United States, nor have they been formerly recognized to be associated with CMD. The finding of these viruses in varying combinations with each other and the previously known CMD associated viruses highlights the modularity of these disease complexes, which has important epidemiological implications.
Lastly, in Chapter 4 I describe the identification of an emergent polerovirus—barley virus G (BVG)—that had not previously been reported in the U.S.. Since its initial discovery, BVG has been found in multiple other countries and species of monocot plants, however all reports on this virus were limited to detection by RT-PCR and sequencing based assays. To begin to understand the biology of this virus, I constructed an infectious BVG clone, which I used to establish an infection in N. benthamiana by agroinoculation. From the BVG agroinoculated N. benthamiana plants I was able to partially purify infectious BVG virions which I fed to three different species of aphids, which allowed me to identify two competent insect vectors—one efficient (the corn aphid, Rhopalosiphom maidis) and one inefficient (the bird cherry-oat aphid, R. padi)—of this virus. I subsequently used the newly identified efficient aphid vector to perform a small host range study to determine the effects of BVG on symptom development in plant species in which it has been previously described, as well as a couple additional species in which it has not. Altogether the work presented here expands on what we already know about interactions between poleroviruses, umbraviruses, and tlaRNAs in virus disease complexes comprised of these viruses and the intriguing specificities and non-specificities of these interactions. It also highlights the utility of RT-PCR based diagnostic assays in combination with high throughput sequencing technology for the identification of emergent viruses.
