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PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis.

Zhang B, Tremousaygue D, Denancé N, van Esse HP, Hörger AC, Dabos P, Goffner D, Thomma BP, van der Hoorn RA, Tuominen H - Plant J. (2014)

Bottom Line: Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum.The stabilization of XCP2 by PRN2 was also confirmed in planta.Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: Umeå Plant Science Centre (UPSC), Department of Plant Physiology, Umeå University, 901 87, Umeå, Sweden.

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PRN2 stabilizes XCP2 by inhibiting its autolysis. (a) PRN2 effect on papain-like cysteine protease (PLCP) activities in a competitive assay. PLCP activity was profiled with DCG-04 over 120 min in protein extracts of N. benthamiana leaves transiently overexpressing XCP2 after incubation in the presence or absence of recombinant PRN2 (rPRN2) or E64. Streptavidin–HRP detection shows one major band of biotinylated PLCPs migrating around 30 kD for XCP2. The rPRN2 panel shows labeling of 25-kD polypeptides representing native PLCPs from N. benthamiana in negative controls with no overexpressed proteins. (b) The stability of recombinant XCP2 (rXCP2) during a 120-min time course in the presence or absence of E64, as detected by Coomassie Brilliant Blue staining (CBB). (c) Results of in vitro protease enzymatic assays of rXCP2 in the presence or absence of rPRN2, BSA or E64. Fluorescence emission indicates rXCP2 activity towards a casein substrate labeled with a fluorescent probe. Blank refers to a negative control without any added recombinant proteins. The asterisk indicates significant difference (P < 0.001) between the rXCP2 activities with and without rPRN2. (d) Enzyme progress curves of rXCP2 activity after incubation in the presence or absence of rPRN2 or E64. Fluorescence emission indicates rXCP2 activity towards the casein substrate labeled with a fluorescent probe. The arrow indicates the time point when rXCP2 activity was abolished without added rPRN2. (e) Lineweaver–Burk reciprocal plot of rXCP2 activity in the presence or absence of rPRN2. The reciprocal reaction velocity (1/V) is shown as a function of reciprocal substrate concentration [1/(S)] for rXCP2 activity when rXCP2 was incubated with serial concentrations of the casein substrate together with three different concentrations of PRN2, followed by protease enzymatic assay. The y-intercept is the same for the three rPRN2 concentrations, indicating that the Vmax (maximum reaction velocity) is not affected by rPRN2. The assays were performed three times yielding similar results.
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fig04: PRN2 stabilizes XCP2 by inhibiting its autolysis. (a) PRN2 effect on papain-like cysteine protease (PLCP) activities in a competitive assay. PLCP activity was profiled with DCG-04 over 120 min in protein extracts of N. benthamiana leaves transiently overexpressing XCP2 after incubation in the presence or absence of recombinant PRN2 (rPRN2) or E64. Streptavidin–HRP detection shows one major band of biotinylated PLCPs migrating around 30 kD for XCP2. The rPRN2 panel shows labeling of 25-kD polypeptides representing native PLCPs from N. benthamiana in negative controls with no overexpressed proteins. (b) The stability of recombinant XCP2 (rXCP2) during a 120-min time course in the presence or absence of E64, as detected by Coomassie Brilliant Blue staining (CBB). (c) Results of in vitro protease enzymatic assays of rXCP2 in the presence or absence of rPRN2, BSA or E64. Fluorescence emission indicates rXCP2 activity towards a casein substrate labeled with a fluorescent probe. Blank refers to a negative control without any added recombinant proteins. The asterisk indicates significant difference (P < 0.001) between the rXCP2 activities with and without rPRN2. (d) Enzyme progress curves of rXCP2 activity after incubation in the presence or absence of rPRN2 or E64. Fluorescence emission indicates rXCP2 activity towards the casein substrate labeled with a fluorescent probe. The arrow indicates the time point when rXCP2 activity was abolished without added rPRN2. (e) Lineweaver–Burk reciprocal plot of rXCP2 activity in the presence or absence of rPRN2. The reciprocal reaction velocity (1/V) is shown as a function of reciprocal substrate concentration [1/(S)] for rXCP2 activity when rXCP2 was incubated with serial concentrations of the casein substrate together with three different concentrations of PRN2, followed by protease enzymatic assay. The y-intercept is the same for the three rPRN2 concentrations, indicating that the Vmax (maximum reaction velocity) is not affected by rPRN2. The assays were performed three times yielding similar results.

Mentions: A competitive DCG-04 labeling assay, in which DCG-04 probe was added to XCP2-overexpressing N. benthamiana leaf extracts simultaneously with rPRN2 provided indications of PRN2's action mechanism. In this assay, XCP2 activity was surprisingly suppressed in the presence of PRN2 at the start of the time course, but recovered to the same or somewhat higher activity level at the end of the time course experiment (Figure4a). This suggests that PRN2 inhibits XCP2 activity, but the inhibition is reversible and eventually results in stabilization of XCP2.


PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis.

Zhang B, Tremousaygue D, Denancé N, van Esse HP, Hörger AC, Dabos P, Goffner D, Thomma BP, van der Hoorn RA, Tuominen H - Plant J. (2014)

PRN2 stabilizes XCP2 by inhibiting its autolysis. (a) PRN2 effect on papain-like cysteine protease (PLCP) activities in a competitive assay. PLCP activity was profiled with DCG-04 over 120 min in protein extracts of N. benthamiana leaves transiently overexpressing XCP2 after incubation in the presence or absence of recombinant PRN2 (rPRN2) or E64. Streptavidin–HRP detection shows one major band of biotinylated PLCPs migrating around 30 kD for XCP2. The rPRN2 panel shows labeling of 25-kD polypeptides representing native PLCPs from N. benthamiana in negative controls with no overexpressed proteins. (b) The stability of recombinant XCP2 (rXCP2) during a 120-min time course in the presence or absence of E64, as detected by Coomassie Brilliant Blue staining (CBB). (c) Results of in vitro protease enzymatic assays of rXCP2 in the presence or absence of rPRN2, BSA or E64. Fluorescence emission indicates rXCP2 activity towards a casein substrate labeled with a fluorescent probe. Blank refers to a negative control without any added recombinant proteins. The asterisk indicates significant difference (P < 0.001) between the rXCP2 activities with and without rPRN2. (d) Enzyme progress curves of rXCP2 activity after incubation in the presence or absence of rPRN2 or E64. Fluorescence emission indicates rXCP2 activity towards the casein substrate labeled with a fluorescent probe. The arrow indicates the time point when rXCP2 activity was abolished without added rPRN2. (e) Lineweaver–Burk reciprocal plot of rXCP2 activity in the presence or absence of rPRN2. The reciprocal reaction velocity (1/V) is shown as a function of reciprocal substrate concentration [1/(S)] for rXCP2 activity when rXCP2 was incubated with serial concentrations of the casein substrate together with three different concentrations of PRN2, followed by protease enzymatic assay. The y-intercept is the same for the three rPRN2 concentrations, indicating that the Vmax (maximum reaction velocity) is not affected by rPRN2. The assays were performed three times yielding similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321228&req=5

fig04: PRN2 stabilizes XCP2 by inhibiting its autolysis. (a) PRN2 effect on papain-like cysteine protease (PLCP) activities in a competitive assay. PLCP activity was profiled with DCG-04 over 120 min in protein extracts of N. benthamiana leaves transiently overexpressing XCP2 after incubation in the presence or absence of recombinant PRN2 (rPRN2) or E64. Streptavidin–HRP detection shows one major band of biotinylated PLCPs migrating around 30 kD for XCP2. The rPRN2 panel shows labeling of 25-kD polypeptides representing native PLCPs from N. benthamiana in negative controls with no overexpressed proteins. (b) The stability of recombinant XCP2 (rXCP2) during a 120-min time course in the presence or absence of E64, as detected by Coomassie Brilliant Blue staining (CBB). (c) Results of in vitro protease enzymatic assays of rXCP2 in the presence or absence of rPRN2, BSA or E64. Fluorescence emission indicates rXCP2 activity towards a casein substrate labeled with a fluorescent probe. Blank refers to a negative control without any added recombinant proteins. The asterisk indicates significant difference (P < 0.001) between the rXCP2 activities with and without rPRN2. (d) Enzyme progress curves of rXCP2 activity after incubation in the presence or absence of rPRN2 or E64. Fluorescence emission indicates rXCP2 activity towards the casein substrate labeled with a fluorescent probe. The arrow indicates the time point when rXCP2 activity was abolished without added rPRN2. (e) Lineweaver–Burk reciprocal plot of rXCP2 activity in the presence or absence of rPRN2. The reciprocal reaction velocity (1/V) is shown as a function of reciprocal substrate concentration [1/(S)] for rXCP2 activity when rXCP2 was incubated with serial concentrations of the casein substrate together with three different concentrations of PRN2, followed by protease enzymatic assay. The y-intercept is the same for the three rPRN2 concentrations, indicating that the Vmax (maximum reaction velocity) is not affected by rPRN2. The assays were performed three times yielding similar results.
Mentions: A competitive DCG-04 labeling assay, in which DCG-04 probe was added to XCP2-overexpressing N. benthamiana leaf extracts simultaneously with rPRN2 provided indications of PRN2's action mechanism. In this assay, XCP2 activity was surprisingly suppressed in the presence of PRN2 at the start of the time course, but recovered to the same or somewhat higher activity level at the end of the time course experiment (Figure4a). This suggests that PRN2 inhibits XCP2 activity, but the inhibition is reversible and eventually results in stabilization of XCP2.

Bottom Line: Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum.The stabilization of XCP2 by PRN2 was also confirmed in planta.Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: Umeå Plant Science Centre (UPSC), Department of Plant Physiology, Umeå University, 901 87, Umeå, Sweden.

Show MeSH
Related in: MedlinePlus