Suayib Üstün

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C5: Functional characterisation of bacterial type III effectors targeting host cell vesicle trafficking

Principal investigator
Frederik Börnke

Mentor
Andreas Burkovski


PhD exam: 20.09.2013

XopJ targets the proteasome to interfere with SA-mediated defence responses

The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) requires type III effector proteins (T3Es) for virulence. After translocation into the host cell T3Es are thought to interact with components of host immunity to suppress defence responses. Common effector target processes and structures in both animal and plant host cells include the cytoskeleton, defence and hormonal signalling, ubiquitination, gene expression, and vesicle trafficking. Target proteins of many T3E remain mostly unknown. Thus, the functional analysis of effector proteins represents an essential tool to understand virulence strategies and defence mechanisms of eukaryotic host cells, while commonalities across kingdoms should also be expected.
One of the T3Es suppressing defense mechanisms is XopJ from the plant pathogenic bacteria Xanthomonas campestris pv. vesicatoria (Xcv). XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Our results show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity, dependent on the catalytic activity and localisation of XopJ. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA) and changes in SA- and senescence-associated gene expression. Furthermore, necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants silenced for NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ-dependent phenotypes. Taken together, our findings suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence responses to attenuate the onset of necrosis.

 

Figure A and B: Visualization of the XopJ-RPT6 interaction in planta by BiFC. Confocal microscopy images show tobacco leaf epidermal cells transiently expressing constructs encoding the fusion proteins indicated. Autofluorescence of chloroplasts are indicated in red and fluorescence of Venus is displayed in yellow. Close up image in B shows that the fluorescence of Venus aligns with the plasmamembrane. Bars = 20 µm.
Figure C and D: The proteasome inhibitor MG132 prevents development of necrosis in Xcv ΔxopJ infected pepper leaves. (C) Xcv ΔxopJ with 100µM MG132 or 1% EtOH (control) were inoculated at a bacterial density of 2 x 108 cfu ml-1 into leaves of pepper ECW plants. Plant reactions were photographed at 3 d post infection (dpi). (D) MG132 treatment leads to a reduced ion leakage in pepper in the absence of XopJ. Ion leakage was measured in pepper plants infected with Xcv, Xcv ΔxopJ with MG132 or 1% EtOH. Conductivity was measured at the time point indicated. Data represent the mean SD (n = 3). Significant differences are indicated by asterisks (*** P < 0.001) and were calculated using Student’s t-test.

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Publications

Üstün, S., Bartetzko, V. and Börnke, F. (2013). The Xanthomonas campestris Type III Effector XopJ Targets the Host Cell Proteasome to Suppress Salicylic-Acid Mediated Plant Defence. PLoS Pathog 9: e1003427.

Üstün, S., Müller, P., Palmisano, R., Hensel, M. and Börnke, F. (2012). SseF, a type III effector protein from the mammalian pathogen Salmonella enterica, requires resistance-gene-mediated signalling to activate cell death in the model plant Nicotiana benthamiana. New Phytol 194(4), 1046-1060.

Arsova, B., Hoja, U., Wimmelbacher, M., Greiner, E., Üstün, S., Melzer, M., Petersen, K., Lein, W., Börnke, F. (2010). Plastidial Thioredoxin z Interacts with two Fructokinase-like Proteins in a Thiol-Dependent Manner: Evidence for an essential Role in Chloroplast Development in Arabidopsis and Nicotiana benthamiana. Plant Cell 22, 1498-1515.

 

Presentations

July 2013 5th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Unravelling type-III effector functions in plants
Talk
     
April 2013 Plant Immunity: Pathways and Translation (D5), Keystone Symposia, Big Sky, Montana, USA
Xanthomonas campestris pv. vesicatoria Type III Effector XopJ targets the Host Cell Proteasome to suppress Salicylic acid-mediated Immunity
Poster
     
July 2012 XV International Congress of Molecular Plant-Microbe Interaction, Kyoto, Japan
Xanthomonas campestris pv. vesicatoria Type III Effector XopJ targets the Host Cell Proteasome to suppress Salicylic acid-mediated Immunity.
Poster
     
July 2012 4th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Unravelling Type III Effector functions in planta
Talk
     
July 2012 4th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Xanthomonas campestris Type III Effector XopJ targets the Host Cell Proteasome to suppress Plant Defence
Poster
     
October 2011 1st Plant Salmonella Interaction Workshop, Paris, France
From humans to plants: Salmonella effector protein SseF requires resistance-gene mediated signalling to elict hypersensitive response in Nicotiana benthamiana
Talk
     
October 2011 First International SFB 796 Conference: Mechanisms of viral host cell manipulations: from plants to humans, Bamberg, Germany
SseF, a type III effector protein from the mammalian pathogen Salmonella enterica, requires resistance-gene mediated signaling to activate cell death in the model plant Nicotiana benthamiana
Poster
     
September 2011 Botanikertagung, Berlin, Germany
SseF, a type III effector protein from Salmonella enterica, requires resistance-gene mediated signaling to activate cell death in Nicotiana benthamiana
Poster
     
July 2011 3rd Annual Retreat "Erlangen School of Molecular Communication", Kloster Banz, Bad Staffelstein, Germany
Functional Analysis of Salmonella type III effector function in plant cells”
Talk
     
July 2011 3nd Annual Retreat "Erlangen School of Molecular Communication", Kloster Banz, Bad Staffelstein, Germany
SseF, a type III effector protein from Salmonella enterica, requires resistance-gene mediated signaling to activate cell death in Nicotiana benthamiana
Poster
     
May 2011 International Meeting-Communication in plants and their responses to the environment", Halle, Germany
SseF, a type III effector protein from Salmonella enterica, requires resistance-gene mediated signaling to activate cell death in Nicotiana benthamiana
Poster
     
April 2011 2nd Annual Spring Meeting of the SFB 796, Erlangen, Germany
Functional Analysis of Salmonella type III effector function in plant cells”
Talk
     
February 2011 24. Tagung Molekularbiologie der Pflanzen, Dabringhausen, Germany
Crossing the line: Does SseF, a type III effector protein from the mammalian pathogen Salmonella enterica, target a conserved eukaryotic protein that is guarded by a R-protein in Nicotiana benthamiana?”
Poster
     
September 2010 2nd Annual Retreat "Erlangen School of Molecular Communication", Kloster Banz, Bad Staffelstein, Germany
Regions of the Salmonella type III effector protein SseF able to elicit HR in Nicotiana benthamiana lie within the region controlling virulence function in mammalian cells”
Talk