Quantification of Pea enation mosaic virus 1 and 2 during infection of Pisum sativum by one step real-time RT-PCR

- Normalized quantitative real-time one-step reverse transcription PCR method to assess PEMV1 and 2 replication in pea plants with two inoculum conditions.
- Compared two different housekeeping genes for normalization of viral accumulation data.
- Accumulation of both viral RNAs was similar whether actin or β-tubulin mRNAs were used as reference transcripts.
- PEMV1 replication dynamics were changed by inoculum concentration when compared to PEMV2.

Pea enation mosaic virus 1 (PEMV1) and Pea enation mosaic virus 2 (PEMV2) are two viruses in an obligate symbiosis that cause pea enation mosaic disease mainly in plants in the Fabaceae family. This virus system is a valuable model to investigate plant virus replication, movement and vector transmission. Thus, here we describe growth conditions, virus detection methods, and virus accumulation behavior. To measure the accumulation and movement of PEMV1 and PEMV2 in plants during the course of infection, we developed a quantitative real-time one-step reverse transcription PCR procedure using the SYBR-green® technology. Viral primers were designed that anneal to conserved but distinct regions in the RNA-dependent RNA polymerase gene of each virus. Moreover, the normalization of viral accumulation was performed to correct for sample-to-sample variation by designing primers to two different Pisum sativum housekeeping genes: actin and β-tubulin. Transcript levels for these housekeeping genes did not change significantly in response to PEMV infection. Conditions were established for maximum PCR efficiency for each gene, and quantification using QuBit® technology. Both viruses reached maximum accumulation around 21 days post-inoculation of pea plants. These results provide valuable tools and knowledge to allow reproducible studies of this emerging model virus system virus complex.