Autophagy induction in tobacco leaves infected by potato virus YO and its putative roles

Autophagy plays a critical role in the innate immune response of plants to pathogen infection. In the present study, we examined autophagy induced by potato virus Y ordinary strain (PVYO) infection in tobacco (Nicotiana benthamiana). Enzyme-linked immunosorbent assays revealed that the number of virus particles in the plant peaked at 2 weeks post-inoculation and then gradually decreased.Additionally, the amount of virus increased significantly in the 3rd and 4th leaves distal to the inoculated leaf and decreased slightly in the 5th leaf. Within 2 weeks of PVYO inoculation, the tobacco leaves showed typical symptoms of Potyvirus inoculation, including mottling, yellowing, a mosaic pattern, and necrotic tissue changes at the inoculated site. Based on an ultrastructural analysis of the PVYO-infected tobacco leaves, virus aggregates appeared as longitudinal and transverse arrays and pinwheels, which are typical of Potyvirus inoculation. Moreover, PVYO infection caused changes in the number, size, and shape of chloroplasts, whereas the number of plastogranules increased markedly. Furthermore, double-membrane autophagosome-like vacuoles, including electron-dense materials, laminated structures, and cellular organelles, were found. The induction of autophagy after the PVYO infection of tobacco leaves was further confirmed by the expression of lipidated microtubule-associated protein 1 light chain 3 (LC3)-II, an autophagy marker and p62, an autophagy adaptor protein. The LC3-II levels increased daily over the 4-week period. Although virus inoculation was performed systemically on the basal leaves of the plants, LC3-II was expressed throughout the leaves and the expression was higher in leaves distal to the inoculated leaf. Moreover, PVYO infection caused the activation of stress-activated protein kinases/c-Jun N-terminal kinases. Therefore, PVYO infection-induced autophagy was positively correlated with the virus content, suggesting that autophagy induction following PVYO infection is involved in the anti-pathogen response of the host.