Fig. 7 | Nature Communications

Fig. 7

From: A fungal substrate mimicking molecule suppresses plant immunity via an inter-kingdom conserved motif

Fig. 7

PID14 is conserved in bacteria and fungi and its function depends on two amino acids. a Pit2 evolutionary relationships of sequences found in bacteria and fungi. Using blastp and the Pit2 sequence from U. maydis as template, 11 homologous sequences were found in bacteria and fungi. Their evolutionary history was inferred using the Minimum Evolution method. The optimal tree with the sum of branch length = 6.46244084 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) is shown next to the branches. b PID14 is the most conserved motif in Pit2 sequences. The 11 Pit2 sequences were used for a search of sequence motifs using MEME. Extracted sequences that are part of the motif are shown. c Conserved microbial inhibitor of proteases (cMIP). Icelogo representing the most conserved amino acids made of 11 sequences found in bacteria, yeast and smut fungi containing the cMIP motif using MEME. cMIP appears to be similar to UmPID14 except there is one more amino acid at the end of the motif. d Inhibitory profile of bacterial and fungi cMIPs against papain. The activity of papain was determined using the fluorogenic substrate Z-Phe-Arg-AMC. Values without inhibitor were set to 100% activity. UmPID14 (gray), StrepPID14 (green) and cMIP (orange) inhibition of papain was monitored in a concentration range between 0 and 64 µM. Log inhibitor concentrations were plotted against papain activity. Error bars represent the standard deviation of three replicates. Shown is a representative plot of five independent replicates showing similar results. e UmPit2 structural model representing the PID14 motif (red) and the localization of the most conserved amino acids RW (green) found in cMIP. UmPID14 and cMIP sequence comparison. In red dissimilar amino acids and in green the conserved RW. e UmPit2 structural model representing the PID14 motif (red) and the localization of the most conserved amino acids RW (green) found in MEME. f Disease rating of maize seedlings at 12 dpi infected with U. maydis SG200 strain, ΔPit2 mutant and complemented ΔPit2 strains with UmPit2 (ΔPit2-UmPit2) and the mutant strain ΔPit2-UmPit2R48AW49A, carrying R48A and W49A point mutations