Fig. 5 | Nature Communications

Fig. 5

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

Fig. 5

PLCPs cleave UmPit2 in the apoplast. a Input used to perform inhibitory test of Pit2 stability. Heterologous expressed UmPit2 and maize AF at different time points during the experiment. b Addition of E-64 stabilizes Pit2 degradation. Four inhibitors for main classes of proteases: E-64 for cysteine proteases, DCI for serine hydrolases, EDTA for metalloproteases and Pepstatin A for aspartic proteases were preincubated with maize AF for 10 min. Pit2 degradation was analyzed over time (0, 15 and 30 min) using SyproRuby staining. c Inhibition of cysteine proteases stabilizes Pit2 in the apoplast. Maize AF was coincubated with a mix of inhibitors containing DCI [10 µM], EDTA [1 mM] and Pepstatin A [10 µM] (Inh. Mix) with or without E-64 [10 µM]. As a control the noninhibitor sample containing DMSO is shown. d Fractionation of SA-treated apoplastic fluid by anion-exchange chromatography. Top panel shows elution conditions and the profile of eluted proteins using a salt gradient. Low panel shows PLCPs activity measurements of fractions with E-64 or DMSO as a control using the fluoro-substrate Z-Phe-Arg-AMC. Fraction 24 (F-24, red labeled) showed the highest PLCP activity and was then used for further experiments. e PLCPs enrichment of F-24 cleaves Pit2. F-24 was preincubated with 10 µM E-64 or DMSO followed by addition of purified UmPit2. Samples were incubated for 0, 15 and 30 min and analyzed by gel electrophoresis using SyproRuby. f UmPit2 is processed at similar rates as an endogenous maize substrate. UmPit2 as well as the endogenous maize PLCP substrate Prozip11 were heterologous expressed in E-coli. 3.2 µM proteins were coincubated with F-24 pretreated with inhibitor mix (Inh. mix) in the presence or absence of E-64. Processing of Prozip11 and UmPit2 has been monitored over time. Samples were analyzed by gel electrophoresis and stained with SyproRuby for visualization. As a control purified proteins as well as F-24 were loaded on gel (right gel). DMSO dimethyl sulfoxide, DCI 3,4-dichloroisocoumarin

Back to article page