Fig. 6 | Nature Communications

Fig. 6

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

Fig. 6

UmPit2 cleavage releases the inhibitory portion. a MS analysis of UmPit2 with F-24. 30 min incubation of UmPit2 with F-24 in the presence of inhibitor mix without E-64 was performed and reaction was stopped by adding TCA. Only peptides released during incubation and remaining in the supernatant were analyzed. The UmPit2 sequence without signal peptide including the PID14 motif (red amino acids) is shown. Black lines above the sequence represent the peptides found in the analysis. Red lines represent peptides found in the sample preincubated with E-64. The orange line represents a peptide that was reproducibly not identified in the MS analysis and might serve as a docking site. Putative cleavage sites based on peptide coverage after PLCP recognition of Val, Arg, Leu; Ile or Phe at the P2 position (blue amino acids) are shown as blue dotted lines. This figure shows the average of three MS runs of three biological replicates. b–d Surface modeling of UmPit2 without signal peptide based on ITASSER prediction analysis (http://zhanglab.ccmb.med.umich.edu/I-TASSER). The PID14 region is labeled in red. The majority of UmPID14 residues are surface exposed (front view, b). K44 and R47 form a distinctive hook-like structure (back view, c). L45 is surface localized whereas F51 resides inside UmPit2. The putative docking site with the sequence RRWWFG is shown in orange sticks and the majority of residues are surfaced localized. e UmPID14 inhibits PLCPs more efficiently than UmPit2. The activity of PLCPs in apoplastic fluids of maize leaves treated with salicylic acid (SA) was determined using the fluorogenic substrate Z-Phe-Arg-AMC. Inhibitory profiles of UmPID14 (black) and UmPit2 (red) in a concentration range against PLCP activity were compared. Error bars show the standard error of the mean calculated for three independent biological replicates. IC50 values were calculated as the mean of three biological replicates using a nonlinear curve fit. IC50 values are statistically significant at the 0.05 level (p = 0.0167) represented by an asterisk. f MV202 time-course labeling of SA-treated apoplastic fluids (AF). Labeling of maize AFs was performed using 0.2 µM MV202 in a one-pot reaction and samples were collected at different time points. The main two signals observed correspond mainly to CP1 and CP2, previously characterized by ref. 21. Samples were analyzed by gel electrophoresis and monitored using a rhodamine filter. After analysis samples were stained with SyproRuby and used as loading control. g UmPit2 competition experiment. SA-treated maize AFs were preincubated in a one-pot reaction with 20 µM UmPit2 and at different time points were collected and then labeled with 0.2 µM MV202 for 1 h. E-64 was used as a control. Asterisk represents a background signal. Samples were analyzed as described in (f). h UmPID14 competition experiment. SA-treated maize AFs were preincubated at different time points with 20 µM UmPID14 and then labeled for 1 h with 0.2 µM MV202. E-64 was used as a control and asterisk represents a background signal. Samples were analyzed as described in (f)

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