Sho Ikeda

To examine the role of RNA silencing in plant defenses against viroids, a Dicer-like 2 and 4 (DCL2&4)–double knockdown transgenic tomato plant line, 72E, was created. The expression of endogenous SlDCL2s and SlDCL4 in line 72E decreased to about a half that of the empty cassette line, EC. When challenged with potato spindle tuber viroid (PSTVd), line 72E showed significantly higher levels of PSTVd accumulation early in the course of the infection and lethal systemic necrosis late in the infection. The size distribution of PSTVd-derived small RNAs was significantly different with the number of RNAs of 21 and 22 nucleotides (nt) in line 72E, at approximately 66.7% and 5% of those in line EC, respectively. Conversely, the numbers of 24 nt species increased by 1100%. Furthermore, expression of the stress-responsive microRNA species miR398 and miR398a-3p increased 770% and 868% in the PSTVd-infected line 72E compared with the PSTVd-infected EC. At the same time, the expression of cytosolic and chloroplast-localized Cu/Zn-superoxide dismutase 1 and 2 (SOD1 and SOD2) and the copper chaperon for SOD (CCS1) mRNAs, potential targets of miR398 or 398a-3p, decreased significantly in the PSTVd-infected line 72E leaves, showing necrosis. In concert with miR398 and 398a-3p, SODs control the detoxification of reactive oxygen species (ROS) generated in cells. Since high levels of ROS production were observed in PSTVd-infected line 72E plants, it is likely that the lack of full dicer-likes (DCL) activity in these plants made them unable to control excessive ROS production after PSTVd infection, as disruption in the ability of miR398 and miR398a-3p to regulate SODs resulted in the development of lethal systemic necrosis.

To examine the role of RNA silencing in defense against viroid, a Dicer-like 2 and 4 (DCL2&4)—double knockdown transgenic tomato line 72E was created. The expression of endogenous DCL2 and DCL4 in line 72E decreased to about a half of the empty cassette line EC. When challenged with potato spindle tuber viroid (PSTVd), 72E allowed significantly higher level of PSTVd accumulation early in infection and showed lethal systemic necrosis. The size distribution of PSTVd-derived small RNA was significantly changed: the numbers of 21 and 22 nucleotides (nt) species in line 72E was approximately 66.7% and 5% of those in line EC, respectively. Conversely, the numbers of 24-nt species increased by 1100%. Furthermore, expression of miR398a-3p and miR398 increased 770–868% in the PSTVd-infected 72E, compared to the PSTVd-infected EC. In parallel, superoxide dismutase (SOD1) in PSTVd-infected 72E showed higher expression levels. In concert with miR398a-3p, SOD1 controls detoxification of reactive oxygen species (ROS) generated in cells. Since high levels of ROS production and its scavenging activity were observed in PSTVd-infected 72E, the lack of full-activity of DCLs was thought to have made the plant incapable to control excessive ROS production and thus resulted in to develop lethal systemic necrosis.