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. 2016 Apr 4;5:e13568. doi: 10.7554/eLife.13568

Figure 1. 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 DC3000, and bacteria number was determined 2 days later (mean ± SD; n ≥ 6; p<0.05, Student’s t-test; different letters indicate significant difference). (B) xlg2/3 and agb1 are similarly compromised in flg22-induced ROS burst. Leaves of the indicated genotypes were examined for flg22-induced ROS production, and peak RLU values are shown (mean ± SD; n ≥ 6; p<0.05, Student’s t-test; different letters indicate significant difference). (C) Flg22 treatment disrupts XLG2-AGB1 interaction. Cluc-XLG2 and AGB1-HA-Nluc constructs are transiently expressed in Nb leaves, relative luminescence unit (RLU) was measured 2 days later. Cluc-CPR5 and BAK1-HA-Nluc were used as negative control (mean ± SD; n ≥ 6). (D) Flg22-induced RbohD phosphorylation is impaired in agb1. FLAG-RbohD and/or AGB1-HA constructs were expressed under control of the 35S promoter in WT or agb1 protoplasts. The FLAG-RbohD protein was affinity purified and subject to anti-FLAG and anti-pSer39 immuoblot analyses. Numbers indicate arbitrary units of RbohD pS39 phosphorylation calculated from densitometry measurements normalized to total FLAG-RbohD protein. Each experiment was repeated three times, and data of one representative experiment are shown.

DOI: http://dx.doi.org/10.7554/eLife.13568.003

Figure 1—source data 1. Raw data and exact p value of Figure 1A, B and Figure 1—figure supplement 1.
DOI: http://dx.doi.org/10.7554/eLife.13568.004
elife-13568-fig1-data1.xlsx (15KB, xlsx)
DOI: 10.7554/eLife.13568.004

Figure 1.

Figure 1—figure supplement 1. Flg22-induced ROS burst is compromised in xlg2 plants.

Figure 1—figure supplement 1.

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 flg22-induced ROS burst. RLU represent peak value of ROS production after flg22 treatment (mean ± SD; n ≥ 6; representative data from three independent experiments are shown).
DOI: http://dx.doi.org/10.7554/eLife.13568.005
Figure 1—figure supplement 2. XLG2/3 and AGB1, but not XLG1, are transcriptionally induced by flg22.

Figure 1—figure supplement 2.

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.
DOI: http://dx.doi.org/10.7554/eLife.13568.006
Figure 1—figure supplement 3. XLG2/3 interact with AGB1 through both N and C termini.

Figure 1—figure supplement 3.

(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 activity was recorded 2 days later (mean ± SD; n≥6; representative data from three independent experiments are shown). (B) Co-IP assay for XLG2/3-AGB1 interaction in protoplasts. Three independent experiments were performed with similar results. (C) Both N and C termini of XLG2 interact with AGB1. XLG2NT-FLAG contains amino acids 1–458 whereas XLG2CT-FLAG contains amino acids 459–861. The indicated constructs were expressed in WT protoplasts, immunoprecipitated with agarose-conjugated FLAG antibody, and the immune complex was subject to immunoblot with specific antibodies.
DOI: http://dx.doi.org/10.7554/eLife.13568.007