Arabidopsis heterotrimeric G proteins regulate immunity by directly coupling to the FLS2 receptor
- Chinese Academy of Sciences, China
- University of British Columbia, Canada
- National Institute of Biological Sciences, China
Figures
Figure 1 with 3 supplements
G proteins are required for FLS2-mediated immunity.
(A) XLG2/3 and AGB1 play overlapping but not identical roles in disease resistance to Pst. Plants of indicated genotypes were infiltrated with H2O and flg22 1 day before infiltration with P. syringae…
-
Figure 1—source data 1
Raw data and exact p value of Figure 1A, B and Figure 1—figure supplement 1.
- https://doi.org/10.7554/eLife.13568.004
Figure 1—figure supplement 1
Flg22-induced ROS burst is compromised in xlg2 plants.
Flg22-induced ROS burst is compromised in xlg2 plants. Col-0, xlg2, and xlg2 transgenic lines complemented with the XLG2 transgene under control of the XLG2 native promoter were examined for …
Figure 1—figure supplement 2
XLG2/3 and AGB1, but not XLG1, are transcriptionally induced by flg22.
qRT-PCR analyses of the indicated genes in WT plants 0 hr and 3 hr after infiltration with flg22. Representative data from three independent experiments are shown.
Figure 1—figure supplement 3
XLG2/3 interact with AGB1 through both N and C termini.
(A) Luciferase complementation assay for XLG2/3-AGB1 interactions in Nb plants. Nb leaves were infiltrated with Agrobacterium tumefaciens strains carrying the indicated constructs and luciferase …
Figure 2 with 3 supplements
Flg22 regulates interactions between G proteins and the FLS2-BIK1 receptor complex.
(A) XLG2 and AGB1 interact with BIK1 and FLS2 in Nb plants. The indicated Nluc and Cluc constructs were transiently expressed in Nb plants for luciferase complementation assay. Relative luminescence …
Figure 2—figure supplement 1
PBL20 interacts with G proteins.
(A) Identification of PBL20 as a XLG2-interacting protein. XLG2-FLAG was expressed in protoplasts, isolated by anti-FLAG immunoprecitation and subject to LC-MS/MS. No PBL20 peptides were identified …
Figure 2—figure supplement 2
XLG3 interacts with BIK1.
(A) XLG3 interacts with BIK1 in Nb plants. Agrobacteria containing the indicated constructs were infiltrated into Nb leaves, and luciferase activity was recorded 2 days later (mean ± SD; n ≥ 6). (B) …
Figure 2—figure supplement 3
XLG2 interacts with FLS2 and BIK1 primarily through the C terminus.
(A) XLG2 C terminus interacts with FLS2 in Nb plants. Agrobacteria carrying the indicated constructs were infiltrated into Nb leaves, and luciferase activity was recorded 2 days later (mean ± SD; …
Figure 3 with 3 supplements
G proteins positively regulate immunity and BIK1 stability.
(A) AGB1 is required for accumulation of BIK1, but not FLS2 and BAK1. BIK1-HA was introduced into agb1 by crossing, homozygotes of the indicated genotypes in F3 generation were used for immunoblot …
Figure 3—figure supplement 1
G proteins are required for BIK1 stability.
(A) agb1 plants are largely normal in BIK1 transcription. Total RNA was isolated from plants of the indicated genotypes, and quantitative real time PCR was carried out to determine BIK1-HA …
Figure 3—figure supplement 2
AGB1 regulates BIK1 stability through proteasome pathway.
(A) Treatment with MG132 allows accumulation of BIK1 in agb1 mutant seedlings. NP::BIK1-HA seedlings of WT (AGB1) and agb1 background were treated with DMSO (-) or 100 μM specific proteasome …
Figure 3—figure supplement 3
AGB1 and XLG2/3 attenuate PBL20 degradation.
(A) Accelerated PBL20 degradation in agb1 extracts. (B) Accelerated PBL20 degradation in xlg2 xlg3 extracts. Total extracts from WT, agb1 and xlg2/3 were incubated with the PBL20-HIS recombinant …
Figure 4
BIK1 level accounts for G protein-mediated regulation of FLS2 immunity.
(A) Transient expression of BIK1 in agb1 and xlg2/3 mutant protoplasts restores RbohD phosphorylation. FLAG-RbohD and BIK1-HA constructs are transiently expressed in protoplasts from WT (Col-0), agb1…
-
Figure 4—source data 1
Raw data and exact p value of Figure 4B—E.
- https://doi.org/10.7554/eLife.13568.017
Figure 5 with 4 supplements
Phosphorylation of XLG2 by BIK1 regulates flg22-induced ROS.
(A) Flg22-induces phosphorylation of XLG2 in the N terminus. Protoplasts expressing XLG21-203-FLAG were treated with flg22. The total protein was treated with (+) or without (-) λ protein …
-
Figure 5—source data 1
- https://doi.org/10.7554/eLife.13568.019
Figure 5—figure supplement 1
The N terminus of XLG3, but not XLG1, is phophorylated upon flg22-treatment.
WT protoplasts were transfected with XLG11-188-FLAG or XLG31-200-FLAG, treated with flg22, and protein was analyzed by anti-FLAG immunoblot.
Figure 5—figure supplement 2
Phospho-sites in XLG2 isolated from flg22-treated protoplasts.
List of phospho-peptides identified. Protoplasts prepared from WT plants were transfected with XLG2-FLAG and treated with flg22 for 10 min, affinity-purified and subjected to LC-MS/MS for …
Figure 5—figure supplement 3
Mutations that block or mimic XLG2 phosphorylation do not impact BIK1 stability and XLG2-BIK1 interaction.
WT or xlg2 xlg3 protoplasts were transfected with BIK1-HA along with WT, non-phosphorylatable (3A, 4A), phospho-mimicking (4D) forms of XLG2-FLAG constructs, treated with (+) or without (-) flg22, …
Figure 5—figure supplement 4
XLG2/3 interact with RbohD in Nb plants.
Agrobacteria containing the indicated constructs were infiltrated in to Nb leaves, and luciferase activity was recorded 2 days later (mean ± SD; n≥6; representative data from 2 independent …
Figure 6
Model for G protein-coupled FLS2 signaling.
In the pre-activation state, the heterotrimeric G proteins composed of XLG2/3, AGB1, and AGG1/2 interact with the FLS2-BIK1 complex. Stimulation by flg22 induces BAK1-FLS2 interaction and activation …
