Figure 2.

Mutation at nucleotide positions 42 and 64 of PSTVd-I attenuated symptom in tomato plants. Tomato plants inoculated with low molecular weight (LMW)-RNA of PSTVd-D_wt, PSTVd-I_wt, and mutants of PSTVd-I (PSTVd-I:C42U, PSTVd-I:64U and, PSTVd-I:C42U/64U) to examine the role of specific nucleotides in symptom attenuation. (A) Plants inoculated with LMW-RNA of PSTVd-I_wt exhibited leaf malformation at 3-wpi and severe dwarfing symptoms at 5-wpi whereas LMW-RNA of PSTVd-D inoculated plants resembled that of mock-inoculated control even at 5 wpi. Although plants inoculated with LMW-RNA of PSTVd-I:C42U and PSTVd-I:64U exhibited mild leaf malformation at 5-wpi, none of the plants inoculated with LMW-RNA of PSTVd-I:C42U/64U show any visible symptoms. (B) A schematic diagram of the comparison of the 9 internodes and accumulated height of the mock-inoculated control plants, and those infected with PSTVd-D_wt, PSTVd-I:C42U/64U, PSTVd-I:64U, PSTVd-I:C42U, and PSTVd-I_wt at 5-wpi. Different letters (a to d) on the top of the bars indicate statistical significance with a p-value of 0.05 (t-test). Mean values are based on three replicates. (C) Comparative analysis of the accumulation of viroid RNAs in infected tomato plants in time course (1- to 5-wpi) by Northern blot hybridization. Accumulation of viroid RNA was calculated by measuring the band intensity in ChemiDoc-XRS by Quantity One software and normalized by the mean value of PSTVd-I_wt at 2-wpi as 1.0. A t-test was performed with a p-value of 0.05. * and ** on the top of the bars represent a significant difference of <0.05 and <0.01, respectively. Mean values are based on three biological and three technical replicates. In the figure, green bars indicate PSTVd-D_wt inoculated plants, yellow-colored bars indicate PSTVd-I:C42U/64U inoculated plants, orange-colored bars indicate PSTVd-I:C42U inoculated plants, red bars indicate PSTVd-I:64U inoculated plants, dark red colored bars indicate PSTVd-I_wt inoculated plants, respectively.