Written by Victoria Fox
OPS Diagnostics LLC
The isolation of RNA from plants is important for investigating plant health, whether it is symbiotic biomes or bacterial/viral infections. Within this group, plant RNA viruses cause significant losses in agricultural crops worldwide, affecting the yield and quality. Problems associated with RNA viruses have become compounded with the emergence of novel viruses, mixed infections with disease synergism, and impacts from global warming (Tatineni and Hein, 2023). To assay and monitor efficiently, rapid, and easy isolation of RNA from the plant is desirable. Within the agricultural realm, a simple method for isolating nucleic acids from both the host and pathogen, followed by quantification, can demonstrate the presence of a pathogen, and be used to determine the extent of the infection (Gachon and Saindrenan, 2004). For instance, the ability to rapidly assess such host/pathogen levels could be useful in determining the application of pesticides (McCartney et al., 2003).
One goal of OPS Diagnostics is the development of the Synergy™ RNA Plant RNA Extraction Kit isolation chemistry to rapidly isolate plant viral RNA for real-time RT-PCR assays. Synergy™ is a phenol and chloroform-free chemistry that relies on bead beating to separate contaminates from the nucleic acids. Therefore, traditional, and toxic protocols using phenol and chloroform extraction are not needed. Its use is already well established in rapid DNA isolation for detection purposes.
A model was designed that validates the use of SYNERGY™ 2.0 Plant RNA Extraction Kit to isolate viral RNA from pinto bean plants infected with tobacco mosaic virus (TMV). By isolating viral RNA using Synergy™ 2.0 and quantifying it using real-time RT- PCR we were able to detect relative levels of RNA for TMV allowing us to differentiate between healthy and diseased plants.
Pinto Bean (Phaseolus vulgaris) plants were grown from seeds and inoculated, after 10 days, with the tobacco mosaic virus (TMV) by damaging the leaves by gently rubbing the leaf with diatomaceous earth and inoculating the damaged area with virus stocks. After 7 to 10 days, inoculated leaves show signs of yellowing and necrotic spots. Leaves showing signs of TMV infection were collected for isolation. The experiments were designed to first test the effectiveness of isolating RNA with Synergy™ 2.0, which was assessed by using RT-qPCR. The subsequent experiments involved isolating the viral RNA from fresh and frozen pinto bean leaf samples.
Synergy™ 2.0 Plant RNA Extraction Kit and protocol were used for all isolations. The SYNERGY™ 2.0 Plant RNA Extraction Kit utilizes a proprietary method to rapidly isolate high-quality RNA from plant tissue samples. Leaf samples (50mg) and Plant Homogenization Buffer (500µl), with the addition of dithiothreitol (DTT), are placed in the Homogenization Tubes and disrupted with a high velocity bead beater (e.g., HT Mini™ or HT 6™).
SYNERGY™'s unique bead beating chemistry, combined with the utilization of DTT, replaces RNA isolation procedures requiring 2-mercaptoethanol (βME) and phenol and chloroform extraction. Each Homogenization Tube contains a grinding matrix that acts in conjunction with the Plant Homogenization Buffer to liberate RNA and capture contaminants from the sample. Synergy™ 2.0 employs a reverse phase chromatographic process of capturing the impurities while letting the RNA stay in solution. The cell debris and PCR inhibitors bind to the grinding matrix and are then removed by centrifugation, leaving the liberated RNA in the supernatant. RNA is further purified by capturing on a silica spin column, eluting, and then DNase treatment. Total extraction time was less than 30 minutes. The RNA was quantified by fluorescence and amplified via real-time RT-PCR and real-time PCR using the iTaq Universal Probes One - Step RT-qPCR. For real-time RT - PCR, the tobacco mosaic virus (TMV) RNA was detected using Taqman with FAM targeting the RNA polymerase gene, NCBI accession number NC_001367. (Table 1).
| Organisms | Primer Sequences | Conditions |
| TMV Forward | 5'-CAGACTATCGTCACCGCAAA-3' | 50°C for 10 minutes. One cycle of 95°C for 3 minutes. 40 cycles of 95°C for 10 seconds to denature, 50.7°C for 30 seconds to anneal. |
| TMV Reverse | 5'-CACGACGTTCATTGGAAACAC-3' | |
| TMV Probe (FAM-ZEN) | 5'-/56-FAM/ATTGCTGCA /ZEN/TGTTTGGCCTCGATG/3IABkFQ/-3' |
A model system was successfully tested using the SYNERGY™ 2.0 Kit chemistry to detect tobacco mosaic virus (TMV) in pinto bean leaves. Real-time PCR was performed to 1) ensure that the isolated RNA could be amplified, and 2) detect and differentiate between healthy and diseased plants. Threshold values for the target genes, in all instances, were more than ten cycles from background, demonstrating high specificity of the PCR (Table 3). Thus, this data showed that Synergy™ 2.0 chemistry is also capable of being utilized to detect plant viruses as well as the potential severity of the infection.
| Source of DNA | Threshold Cycle (CT) |
| Healthy Plant DNA | UND |
| Healthy Plant RNA | UND |
| Healthy Plant Total Nucleic Acids | 38.472 |
| TMV Infected DNA | UND |
| TMV Infected RNA | 27.678 |
| TMV Infected Total Nucleic Acids | 26.800 |
| Water | UND |
One healthy total nucleic sample had a Ct of 38.472 however, this is 12 cycles later than the tobacco mosaic virus (TMV) samples. The TMV RNA and total nucleic acid (TNA) samples returned Ct's of 26.768 and 26.800 respectively. Thus, indicating the ability to isolate, detect, and amplify RNA plant viruses. Variability in the RNA harvested from plants is highly dependent upon the sample and its proximity to young tissue as well as how the virus progresses throughout the plant.
This mockup of a plant/viral system shows that the isolation of RNA using Synergy™ 2.0 chemistry is useful for detecting and monitoring diseased plants. To validate this approach, more plant/viral systems will be tested using the Synergy™ 2.0 Plant RNA Extraction protocol.
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Figure 1: Isolation of Pinto Bean plant leaves infected with tobacco mosaic virus (TMV) with Synergy™ 2.0. QPCR was run using iTaq and TMV RNA polymerase PCR primers and FAM as a probe.
This short study concluded that viral RNA can be effectively isolated, detected, and amplified from plants using SYNERGY™ 2.0 Plant RNA Extraction Kit. As stated above this work needs to be expanded to cover more species of plants as well as multiple viruses.
Gachon, C and Saindrenan, P. 2004. Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea. Plant Physiology and Biochemistry 42: 367-371.
McCartney, H., Foster, S. J., Fraaije, B. A, and Ward, E. 2003. Molecular diagnostics for fungal plant pathogens. Pest Management Science 59: 129-142.
Satyanarayana Tatineni and Gary L. Hein, et al. "Plant Viruses of Agricultural Importance: Current and Future Perspectives of Virus Disease Management Strategies." Phytopathology®, 27 Feb. 2023, apsjournals.apsnet.org/doi/10.1094/PHYTO-05-22-0167-RVW.