Reduced content of tRNA-derived 2’-<i>O</i>-methylcytosine (Cm), 2’-<i>O</i>-methyluracil (Um) and 2’-<i>O</i>-methylguanine (Gm) in <i>scs9-1</i> and <i>scs9-2</i> mutants.

<p>(<b>A</b>) Position of common modifications found in cytoplasmic tRNA in Arabidopsis and its major domains. tRNA is shown in its common secondary structure form, with circles representing nucleotides and lines representing base pairs. Position in shaded grey indicates the proposed tRNA methylation site for Trm7-like MTases in eukaryotes. Residue 34 corresponds to the wobble nucleotide. AAS (amino acid-accepting stem); DSL (dihydrouridine stem and loop); ASL (anticodon stem loop); VL (variable loop). Conventional abbreviations for nucleotide modifications are used; see the Modomics database (<a href="http://modomics.genesilico.pl/" target="_blank">http://modomics.genesilico.pl</a>). (<b>B</b>) Percent reduction of Cm, Um, and Gm content in <i>scs9-</i>1 and <i>scs9-2</i> relative to Col-0 plants as determined by HPLC analysis. Each value was relative to the amount of tRNA-derived 7 methylguanosine (m⁷G) and 1-methylguanosine (m¹G), which remained invariant in all the genotypes. Data points represent the average of three biological replicates. (<b>C</b>) Relative content of Cm and Um modified nucleosides in Col-0, <i>scs9-1</i> and <i>scs9-2</i> plants during the course of an infection wit <i>P</i>.<i>s</i>. DC3000 as determined by LC-MS. Samples were taken at 0, 24, 48, 72 and 120 h.p.i. Each value was relative to the amount of tRNA-derived 1-methylguanosine (m¹G) which remained invariant during the infection process. Data represent the mean ± SD; n = 3 replicates. Asterisks indicate statistical differences compared with its respective genotype as referred to its time 0 control (P<0.05) analyzed using a Student’s t-test. (<b>D</b>) tRNA Northern blot in mock- and <i>P</i>.<i>s</i>. DC3000-inoculated Col-0, <i>scs9-1</i>, and <i>scs9-2</i> plants. Leaf samples were collected at 3 d.p.i. The membranes were hybridized with radiolabel probes to tRNA^Phe, tRNA^Gln, tRNA^Leu, tRNA^Trp. Equal RNA loading was verified by staining with EtBr (low panel).