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Genomic screens identify a new phytobacterial microbe-associated molecular pattern and the cognate Arabidopsis receptor-like kinase that mediates its immune elicitation.

Mott GA, Thakur S, Smakowska E, Wang PW, Belkhadir Y, Desveaux D, Guttman DS - Genome Biol. (2016)

Bottom Line: We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides.These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses.The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Systems Biology, University of Toronto, 25 Willcocks St., Toronto, Ontario, Canada.

ABSTRACT

Background: The recognition of microbe-associated molecular patterns during infection is central to the mounting of an effective immune response. In spite of their importance, it remains difficult to identify these molecules and the host receptors required for their perception, ultimately limiting our understanding of the role of these molecules in the evolution of host-pathogen relationships.

Results: We employ a comparative genomics screen to identify six new immune eliciting peptides from the phytopathogenic bacterium Pseudomonas syringae. We then perform a reverse genetic screen to identify Arabidopsis thaliana leucine-rich repeat receptor-like kinases required for the recognition of these elicitors. We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides. From this primary screen data, we focused on the interaction between the xup25 peptide from a bacterial xanthine/uracil permease and the Arabidopsis receptor-like kinase xanthine/uracil permease sensing 1; a family XII protein closely related to two well-characterized receptor-like kinases. We show that xup25 treatment increases pathogenesis-related gene induction, callose deposition, seedling growth inhibition, and resistance to virulent bacteria, all in a xanthine/uracil permease sensing 1-dependent manner. Finally, we show that this kinase-like receptor can bind the xup25 peptide directly. These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses.

Conclusions: The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception. Further exploration of these molecules will increase our understanding of plant-pathogen interactions and the basis for host specificity.

No MeSH data available.


Related in: MedlinePlus

XPS1 binds specifically to xup25. aCoomassie stain of purified XPS1 ectodomain used in MST assays. b Quantification of binding between the XPS1 ectodomain and xup25 by label-free MST. The XPS1 ectodomain was kept at a constant concentration (0.5 μM) whereas varying peptide concentrations were added. Data points indicate the difference in normalized fluorescence (%) generated by xup25 binding to the XPS1 ectodomain. Curves are plots of xup25 concentrations against percent changes of normalized fluorescence (ΔFnorm [%] y-axis). Curve fitting was performed by using the NT affinity analysis software from Nanotemper. The EC50 value calculated at MST power of 60 % is indicated on top. The binding profile is representative of two independent assays performed with two independent protein preparations (see Additional file 9: Figure S8). The standard error of the regression fit is 2.866732
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Fig8: XPS1 binds specifically to xup25. aCoomassie stain of purified XPS1 ectodomain used in MST assays. b Quantification of binding between the XPS1 ectodomain and xup25 by label-free MST. The XPS1 ectodomain was kept at a constant concentration (0.5 μM) whereas varying peptide concentrations were added. Data points indicate the difference in normalized fluorescence (%) generated by xup25 binding to the XPS1 ectodomain. Curves are plots of xup25 concentrations against percent changes of normalized fluorescence (ΔFnorm [%] y-axis). Curve fitting was performed by using the NT affinity analysis software from Nanotemper. The EC50 value calculated at MST power of 60 % is indicated on top. The binding profile is representative of two independent assays performed with two independent protein preparations (see Additional file 9: Figure S8). The standard error of the regression fit is 2.866732

Mentions: In order to demonstrate that XPS1 and xup25 represent a cognate MAMP-PRR pair, we next tested the binding of the peptide to the ectodomain of the receptor. For this, we expressed the extracellular domain of XPS1 in insect cells and purified it to near homogeneity (Fig. 8a). Subsequently, we used label-free microscale thermophoresis (MST) [28] to study the abilities of xup25 to bind to XPS1. As shown in Fig. 8b, XPS1 was able to bind the xup25 peptide with an observed EC50 of 103 nM ± 0.048 at an MST power of 60 %. We repeatedly performed our measurements on two independent protein preparations to ensure reproducibility. Our second assay at the same MST power displayed a similar binding profile, yet showed an increased EC50 (381 nM ± 0.054). A control experiment in which we tested the ability of XPS1 to bind the flg22 peptide demonstrated the specificity of the xup25-XPS1 interaction (Additional file 8: Figure S7). Thus XPS1 can discriminate unrelated peptides and our binding assays strongly indicate that XPS1 is directly involved in the perception of xup25.Fig. 8


Genomic screens identify a new phytobacterial microbe-associated molecular pattern and the cognate Arabidopsis receptor-like kinase that mediates its immune elicitation.

Mott GA, Thakur S, Smakowska E, Wang PW, Belkhadir Y, Desveaux D, Guttman DS - Genome Biol. (2016)

XPS1 binds specifically to xup25. aCoomassie stain of purified XPS1 ectodomain used in MST assays. b Quantification of binding between the XPS1 ectodomain and xup25 by label-free MST. The XPS1 ectodomain was kept at a constant concentration (0.5 μM) whereas varying peptide concentrations were added. Data points indicate the difference in normalized fluorescence (%) generated by xup25 binding to the XPS1 ectodomain. Curves are plots of xup25 concentrations against percent changes of normalized fluorescence (ΔFnorm [%] y-axis). Curve fitting was performed by using the NT affinity analysis software from Nanotemper. The EC50 value calculated at MST power of 60 % is indicated on top. The binding profile is representative of two independent assays performed with two independent protein preparations (see Additional file 9: Figure S8). The standard error of the regression fit is 2.866732
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862170&req=5

Fig8: XPS1 binds specifically to xup25. aCoomassie stain of purified XPS1 ectodomain used in MST assays. b Quantification of binding between the XPS1 ectodomain and xup25 by label-free MST. The XPS1 ectodomain was kept at a constant concentration (0.5 μM) whereas varying peptide concentrations were added. Data points indicate the difference in normalized fluorescence (%) generated by xup25 binding to the XPS1 ectodomain. Curves are plots of xup25 concentrations against percent changes of normalized fluorescence (ΔFnorm [%] y-axis). Curve fitting was performed by using the NT affinity analysis software from Nanotemper. The EC50 value calculated at MST power of 60 % is indicated on top. The binding profile is representative of two independent assays performed with two independent protein preparations (see Additional file 9: Figure S8). The standard error of the regression fit is 2.866732
Mentions: In order to demonstrate that XPS1 and xup25 represent a cognate MAMP-PRR pair, we next tested the binding of the peptide to the ectodomain of the receptor. For this, we expressed the extracellular domain of XPS1 in insect cells and purified it to near homogeneity (Fig. 8a). Subsequently, we used label-free microscale thermophoresis (MST) [28] to study the abilities of xup25 to bind to XPS1. As shown in Fig. 8b, XPS1 was able to bind the xup25 peptide with an observed EC50 of 103 nM ± 0.048 at an MST power of 60 %. We repeatedly performed our measurements on two independent protein preparations to ensure reproducibility. Our second assay at the same MST power displayed a similar binding profile, yet showed an increased EC50 (381 nM ± 0.054). A control experiment in which we tested the ability of XPS1 to bind the flg22 peptide demonstrated the specificity of the xup25-XPS1 interaction (Additional file 8: Figure S7). Thus XPS1 can discriminate unrelated peptides and our binding assays strongly indicate that XPS1 is directly involved in the perception of xup25.Fig. 8

Bottom Line: We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides.These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses.The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Systems Biology, University of Toronto, 25 Willcocks St., Toronto, Ontario, Canada.

ABSTRACT

Background: The recognition of microbe-associated molecular patterns during infection is central to the mounting of an effective immune response. In spite of their importance, it remains difficult to identify these molecules and the host receptors required for their perception, ultimately limiting our understanding of the role of these molecules in the evolution of host-pathogen relationships.

Results: We employ a comparative genomics screen to identify six new immune eliciting peptides from the phytopathogenic bacterium Pseudomonas syringae. We then perform a reverse genetic screen to identify Arabidopsis thaliana leucine-rich repeat receptor-like kinases required for the recognition of these elicitors. We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides. From this primary screen data, we focused on the interaction between the xup25 peptide from a bacterial xanthine/uracil permease and the Arabidopsis receptor-like kinase xanthine/uracil permease sensing 1; a family XII protein closely related to two well-characterized receptor-like kinases. We show that xup25 treatment increases pathogenesis-related gene induction, callose deposition, seedling growth inhibition, and resistance to virulent bacteria, all in a xanthine/uracil permease sensing 1-dependent manner. Finally, we show that this kinase-like receptor can bind the xup25 peptide directly. These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses.

Conclusions: The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception. Further exploration of these molecules will increase our understanding of plant-pathogen interactions and the basis for host specificity.

No MeSH data available.


Related in: MedlinePlus