CMV- and PVY-mediated RNA silencing in mixed infections in tomato

We investigated the role of RNA silencing-based defence mechanisms in tomato plants supporting a double infection with Cucumber mosaic virus (CMV) and Potato virus Y (PVY). For this purpose we generated in vitro transcripts of: (i) two CMV strains (CMV-Fny and CMV-LS); (ii) two pseudorecombinants (CMVF1LS2F3 and CMV-F1d2bF3) and (iii) the strain SON41 of PVY (PVY-SON41), expressing the strong RNA silencing suppressor HC-Pro protein. CMV-F1LS2F3 and CMV-F1d2bF3 had a different RNA-2, which influenced their ability to induce disease symptoms and to move systemically in the plant. CMV-F1LS2F3, as well as the RNA-2 donor strain CMV-LS, induced very mild symptoms in tomato, while CMV-F1d2bF3, a modified CMV-Fny that cannot translate the 2b protein, was unable to infect tomato plants systemically. The PVY-SON41 infection was symptomless in tomato. All CMV variants, when inoculated to plants at three days post-inoculation with PVY, reduced the level of PVY RNA accumulation. On the other hand, PVY affected the CMV-Fny infection increasing symptom severity and RNA accumulation. PVY effects on CMV-F1LS2F3 led to the appearance of CMVFny-like symptoms, but were not significant on the accumulation of CMV RNAs. Additionally, CMV-F1d2bF3 was able to spread systemically in PVY-infected tomato plants. Small interfering RNA (siRNA) accumulation varied in the different PVY/CMV combinations, suggesting no role of CMV 2b protein in preventing RNA silencing directed against PVY. The role of RNA silencing and silencing suppression on both the increased CMV accumulation and severity of disease phenotypes observed in mixed infections remains to be cleared.