Fig. 3
Primary structure, secondary structure and phylogenetic analysis of MeE3L and/or its protein product. Sequence alignment and agarose gel resolution of MeE3L partial gene and partial transcript respectively show a 53 bp insertion mutation in the lengthier susceptible T200 homolog that is absent in the reference AM560-2, model cv.60444 and tolerant TME3 homologs. Computational prediction of secondary, molecular and zinc-binding structures of MeE3L homologs shows significant differences between T200 structure and the other structures. N → C. Phylogenetic analysis shows significant evolutionary distance between susceptible T200 MeE3L and other plant MeE3L homologs. a Genomic nucleotide sequence alignment showing insertion mutation between nucleotides 397–398 and 422–423 in the susceptible T200 MeE3L homolog. b Agarose gel resolution of the PCR-amplified susceptible T200 and tolerant TME3 partial transcripts of MeE3L (c) Amino acid sequence alignment showing premature stop mutation at amino acid residue 141 in susceptible T200 MeE3L protein homolog (d) The reference AM560-2 MeE3L amino acid sequence. Asterisks denote stop codons in susceptible T200 (amino acid residue 141) and reference AM560-2 / model cv.60444 / tolerant TME3 (amino acid residue 200) homologs respectively. Red letters denote the first susceptible T200 MeE3L stop mutation at amino acid residue 141. Underlined letters denote the sequence adhering to the RING finger domain consensus sequence [CX2CX(9–39)CX(1–3)HX(2–3)CX2CX(4–48)CX2X]. (ei) Reference AM560-2 MeE3L homolog predicted secondary structure (eii) Reference AM560-2 MeE3L homolog predicted tertiary molecular structure (eiii) Zinc binding in RING domain of reference AM560-2 MeE3L (eiv) Model cv.60444 MeE3L homolog predicted secondary structure (ev) Model cv.60444 MeE3L predicted tertiary molecular structure (evi) Zinc binding in RING domain of model cv.60444 MeE3L (evii) Susceptible T200 MeE3L homolog predicted secondary structure (eviii) Susceptible T200 MeE3L predicted tertiary molecular structure (eix) Predicted ligand binding structure of susceptible T200 MeE3L (ex) Tolerant TME3 MeE3L homolog predicted secondary structure (exi) Tolerant TME3 MeE3L predicted tertiary molecular structure (exii) Zinc binding in RING domain of tolerant TME3 MeE3L [Predictions were run on the I-TASSER On-line Server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/; [97]] (f) Evolutionary analysis of plant MeE3L homologs using Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model in MEGA X [40]. Bootstrap support was calculated from 1000 replicates. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site