Roses, the universal symbol of love and affection, are one of the most popular ornamental flowering shrubs used by landscapers and home gardeners and account for almost half of the billion-dollar ornamental plant market. The growing prevalence of rose rosette disease poses a significant threat to these industries. This lethal disease is caused by the Rose rosette emaravirus (RRV) and transmitted by the tiny eriophyid mite, Phyllocoptes fructiphilus. Infection by RRV results in prolific growth of clustered and bunched plant shoots (witches’ broom), malformed flowers and leaves, malformed shoots and enlarged stems and abundant leaf growth and thorniness. This excessive growth depletes the plant’s energy, eventually causing death.
Emerging and Devastating Plant Viruses of the Genus Emaravirus
RRV is a single-stranded, segmented, negative-sense RNA virus belonging to the genus Emaravirus, a relatively new genus that was established in 2012. These emerging viruses can be devastating to trees, herbaceous woody plants and vines. At Texas A&M University, Dr. Jeanmarie Verchot’s lab is working to better characterize and understand these new viruses. In addition to threatening roses, these viruses cause damage to important agriculture crops such as wheat and pigeon peas. They also endanger sensitive ecosystems when they infect plants specialized to a particular habitat, as is the case with Palo verde broom virus infection of palo verde trees of the Sonoran Desert (1).
Currently there are no treatment for plants infected with Emaravirsus. As the number of lethal plant diseases attributed them grow, it is important that we better understand their infection cycle (viral replication, movement, and virus-host interactions). This knowledge is paramount for developing disease-fighting techniques.
What We Know About Rose Rosette Emaravirus
RRV has seven segments, named RNA1 through RNA7. RNA1 encodes RNA-dependent RNA polymerase (RdRp), RNA 2 encodes the precursor glycoprotein (pre-GP) and RNA3 encodes the nucleocapsid (N). RNA 4 encodes the putative movement protein and the products of RNA5–RNA7 have not been characterized.
A Reverse Genetic System for Studying Virus Replication and Assembly
In Dr. Verchot’s lab, PhD student Cesar Urrutia is studying viral replication and encapsidation of the Rose rosette emaravirus using a reverse genetic minireplicon system (1). Reverse genetic systems allow researchers to generate full-length or truncated virus RNA genomes using cDNA templates. Minireplicon-based reverse genetic systems use plasmid vectors to create viral RNA analogs that contain only what is needed for virus replication and gene expression, in which some, or all, of the virus open reading frames are replaced with reporter genes such as green fluorescent protein (GFP). For negative-strand RNA viruses such as RRV, the essential pieces needed for replication are RdRp and N.
The minireplicons graduate student Cesar Urrutia used in his research contained the different RNA1 and RNA3 antigenomic cDNA sequences along with a modified version of RNA5 where the GFP reporter sequence replaced the open reading frame (ORF) segment. Using these, he was able to agroinfiltrate plasmids with different combinations of RNA1, RNA3 and the RNA5/GFP into the leaves of a rose plant (N. benthamiana) and visually detect GFP to confirm the presence of the minireplicons.
Importance of Sensitive RNA Isolation for Detecting RRV Replication
To understand how sequence changes effect the viral replication of the different RNA1 and RNA3 sequences, the lab needed to detect viral levels in the tissue. Detecting replication of negative strand RNA viruses such as RRV requires detecting the minus strand genomes using RT-PCR or RT-qPCR. This is challenging for RRV because the RNA is present at very low levels. Dr. Verchot’s lab has had great success using the Maxwell® 16 LEV Simply RNA Tissue Kit and a Maxwell® Instrument for isolating enough of the low-abundance RRV RNA to effectively detect it using their RT-PCR assays. Using this method, they were able to identify two changes to the reference genome sequence for RdRp and one change to the sequence for N that negatively impacted viral replication.
Moving Our Understanding of RRV and Other Emaraviruses Forward
There are several members of the growing Emaravirus genus that pose a significant threat to economically important plant species including roses. Reverse genetic, minireplicon systems such as the ones used by the scientists at Texas A&M offer valuable tools to further our understanding of these viruses. Understanding the replication cycle of these viruses will ultimately help us identify ways to combat the diseases they cause.
- Urrutia, C.D. (2022) Advancing the ROSE Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor. Viruses 14, 836
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