Johannes Priller

Suche


B4 Re-programming of plant metabolism by type III effectors from Xanthomonas campestris

Principal investigator
Sophia Sonnewald

Mentor
Andreas Burkovski

PhD exam: 20.11.2015

Functional Characterization of the Type III Effector Protein XopB fromXanthomonas campestris pv. vesicatoria

Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease in tomato and pepper plants. The pathogenicity of Xcv depends on a type III protein secretion system (TTSS) delivering effector proteins supposed to be involved in suppression of plant defence and nutrient acquisition. Previous studies aiming to unravel the underlying molecular mechanisms focused on the compatible interaction between Xanthomonas campestris pv. vesicatoria and pepper (Capsicum annuum) by monitoring the expression of cell wall-bound invertase (cw-Inv). Cw-Inv hydrolyses sucrose into glucose and fructose providing nutrients for bacterial growth, sugar signals for induction of plant defence responses and down-regulation of photosynthesis. Comparing pepper leaves infected with X. campestris pv. vesicatoria wild-type strain (85-10) or a TTSS-deficient X. campestris pv. vesicatoria mutant strain suggested that bacterial effectors might prevent the generation of sugar-mediated signals by inhibition of cw-Inv activity. This is further supported by analysis of transgenic tomato plants with reduced expression of cw-Inv. Using X. campestris pv. vesicatoria effector mutants, three effector proteins, namely XopB, XopJ and AvrRxv, could be identified as suppressors of cw-Inv. Furthermore, recent experiments could identify other effectors involved in suppression and, interestingly, activation of cw-Inv activity. Whether there is a benefit for the pathogen due to the increased cw-Inv is still unknown.

Currently, our principal aim is the structural and functional analysis of these effector proteins. Due to the unknown molecular function of most effectors we attempt to solve crystal structures in collaboration with Y. Muller (project A3, Lehrstuhl für Biotechnik). Confocal laser scanning microscopy (CLSM) of transiently transformed Nicotiana benthamiana leaves indicates XopB as a cytosolic protein (Figure A) but additional GFP signals were observed in vesicle-like structures (Figure B). By utilising a set of organelle specific mCherry-fused marker proteins the subcellular localisation of XopB and other effector proteins in the host cell will be determined. In addition, biochemical and genetic approaches, e. g. yeast two-hybrid system, western blot analysis and transcript profiling, will be used to unravel the impact of bacterial effector proteins on host cell’s metabolism.

Figure: (A) Subcellular localisation of XopB:GFP in N. benthamiana. Agrobacterium strains containing xopB:GFP were infiltrated into N. benthamiana leaves. Samples were taken 48 h postinoculation and lower epidermal cell were analysed using confocal laser scanning microscopy. The fusion protein is located in the cytosol, indicated by green fluorescent Hecht’s filaments (white arrows), the green fluorescent cell edge and the green fluorescent nucleus. GFP fluorescence is shown in green, autofluorescence of chloroplasts is shown in blue. (B)  Subcellular localisation of XopB:GFP in N. benthamiana. The fusion protein shows at higher resolution vesicle-like structures. Agrobacterium strains containing xopB:GFP were infiltrated into N. benthamiana leaves. Samples were taken 48 h postinoculation and lower epidermal cell were analysed using confocal laser scanning microscopy. GFP fluorescence is shown in green, autofluorescence of chloroplasts is shown in blue.

 

Publications

Sonnewald, S., Priller, J.P.R., Schuster, J., Glickmann, E., Hajirezaei, M.-R., Siebig, S., Mudgett, M.B. and Sonnewald, U. (2012). Regulation of Cell Wall-Bound Invertase in Pepper Leaves by Xanthomonas campestris pv. vesicatoria Type Three Effectors. PLoS ONE 7(12), e51763.

 

Presentations

July 2014 6th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
“The Xanthomonas campestris pv. vesicatoria type III effector XopB supports bacterial virulence by interfering with ROS signalling”
Talk
     
July 2013 5th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Analysis of the Type-III-Effector XopB from Xanthomonas campestris pv. vesicatoria
Talk
     
February 2012 25. Tagung der Molekularbiologie der Pflanzen, Dabringhausen, Germany
Functional and structural characterization of Type III effector protein XopB from the phylopathogenic Gram-negative bacterium Xanthomonas campestris pv. vesicatoria
Poster
     
October 2011 First International SFB 796 Conference: Mechanisms of viral host cell manipulations: from plants to humans, Bamberg, Germany
Functional and structural characterization of Type III effector protein XopB from the phylopathogenic Gram-negative bacterium Xanthomonas campestris pv. vesicatoria
Poster
     
July 2011 3rd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Characterisation of Type III Effector Protein XopB of Xanthomonas campestris pv. vesicatoria regulating plant cell wall-bound invertasee
Talk and Poster
     
May 2011 International Meeting "Communication in Plants and their Responses to the Environment", Halle (Saale), Germany
Molecular Mechanisms of Information Processing in Plants
Poster
     
September 2010 2nd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Characterisation of Xanthomonas campestris pv. vesicatoria Effectors Regulating Cell wall-bound Invertase
Talk