PAMP (pathogen-associated molecular pattern)-induced changes in plasma membrane compartmentalization reveal novel components of plant immunity
Authors: Ralph Panstruga
Sharon A Kessler
Gildas Bourdais
Ueli Grossniklaus
Justine Lorek
Hiroko Shimosato-Asano
Nana F Keinath
Sylwia Kierszniowska
Silke Robatzek
Waltraud X Schulze
Journal: Journal of Biological Chemistry (2010)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Plasma membrane compartmentalization; immune signaling; plant plasma membrane; flagellin; quantitative mass spectrometric analysis
Affiliations: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.

Abstract:  Plasma membrane compartmentalization spatiotemporally regulates cell-autonomous immune signaling in animal cells. To elucidate immediate early protein dynamics at the plant plasma membrane in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin (flg22) we employed quantitative mass spectrometric analysis on detergent-resistant membranes (DRMs) of Arabidopsis thaliana suspension cells. This approach revealed rapid and profound changes in DRM protein composition following PAMP treatment, prominently affecting proton ATPases and receptor-like kinases, including the flagellin receptor FLS2. We employed reverse genetics to address a potential contribution of a subset of these proteins in flg22-triggered cellular responses. Mutants of three candidates (DET3, AHA1, FER) exhibited a conspicuous defect in the PAMP-triggered accumulation of reactive oxygen species. In addition, these mutants showed altered mitogen-activated protein kinase (MAPK) activation, a defect in PAMP-triggered stomatal closure as well as altered bacterial infection phenotypes, which revealed three novel players in elicitor-dependent oxidative burst control and innate immunity. Our data provide evidence for dynamic elicitor-induced changes in the membrane compartmentalization of PAMP signaling components. 

Retracted: No

PubPeer comments:
 In Fig.3, four lanes of the Coomassie. Several identical features are recognizable within each lane and keep same distance between one another. Circles highlight dark dots, white arrows highlight bright dots, all of those are identical among the four lanes. Black arrows point to a peculiar vertical discontinuity between bands and the background. Loading control of Figure 3 is of crucial importance for the validity of Figure 3 itself and for the whole article. Could the authors please provide the original Coomassie gel? ![file] , 
 Our reply is as follows: We disagree with the statement: u201cLoading control of Figure 3 is of crucial importance for u2026 the whole articleu201d. This Figure is useful since it provides an independent validation of our proteomic data. However, the main findings of our work are based on the careful proteomic analysis conducted. We further think that the Coomassie-stained membrane (please note that this is not a Coomassie-stained gel!) in its published (cropped) form exhibits enough characteristic details such as spots, (vertical) stripes and other marks in the individual lanes to exclude any putative image manipulation. It can be seen already in the published version that the lanes were not assembled (i.e. u201ccut and put togetheru201d) but, in fact, represent the individual lanes of a single gel (or blot, respectively). This impression is further corroborated by the full-size version of the Coomassie-stained membrane, which we provide below and that lacks any signs of image manipulation. We believe that the vertical stripes that are visible between some lanes and also in part within lanes are artefacts of the membrane surface structure, the blotting procedure or - most likely- the scanning process. Ralph Panstruga (corresponding author, on the behalf of all co-authors) ![file] , 
 I did not see the similarities at first, but after a close inspection, I will have to agree with Aneurus Inconstans. Looking at the Coomassie membrane provided by Dr. Panstruga in #2, which spans a larger area than the photo in the Figure 3 in the paper, I see even more similarities. There are obvious differences between the lanes, such as stripes and dots, but there are also several dots and lighter areas that I see across three or all four of the left four lanes. I have highlighted those in the left lane by boxes of various shapes and colors, and copied those boxes (preserving their respective orientation) onto the other three lanes. They line up exactly with the same features, although the whole set is sometimes a bit higher or lower positioned on the photo. In particular, there is a set of 2 light dots in the fattest band, which I marked with an orange ellipse, that is strikingly present in all four lanes. There is also a slanted light stripe in the bottom part of the lane, marked by me with a dark red rounded box, which I see in three of the four lanes. Another light dot in the top of the lane, marked with a purple star is at the exact same position respective to the other features. It had a dark dot in the fourth lane, but the light dot is there as well. Maybe the authors could take away our concerns by posting a high-resolution photo of the whole blot (edges included) somewhere e.g. on Imgur? ![file] , 
 #3 I agree with the analysis in post #3. Moreover, in contrast to the author s arguments in post #2, there are visible discontinuities in the background between several of the lanes, which would not be expected for a continuous blot. These are especially prominent at both the left and right sides of lane 1 (as previously mentioned in #1), as well as to the right of lane 4, while also being partially evident at the right edge of lane 3. These are marked with green arrows in the enhanced-contrast version below (click to enlarge). In addition, the wide gap between lanes 4 and 5 also contains several areas in which background features appear to be duplicated. Certainly, as mentioned in #3, retrieving the original highest-quality image of the full blot could help shed light on these concerns. ![file] , 
 We appreciate that some features of the Coomassie-stained membrane indeed look unusual. We will do all we can to shed light on this issue, e.g. by searching for the original documentation of these experiments. Please take into consideration that these experiments were performed more than ten years ago and we will need some time to browse through old e-mail conversations, data files and lab books, as far as they are still avaialable. , 
 Something else to consider in support of the original post is that the DSM and DRM lanes should be different in protein content but, if overlaid, should contain all or most of the bands in the total protein extract (e.g. fig 2b Borner et al 2005 http: www.plantphysiol.org content 137 1 104. However, they look more or less the same. This suggests to me that at least 2 of the 4 lanes corresponding to DSM and DRM fractions are not what they claim to be. , 
 #5 After more than a year, could Dr. Panstruga please provide an update on the status of their investigation? , 
 #7 The authors have decided to contact the respective journal (JBC) for further consultation. ,