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

Figure 3. 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 analyses. (B) AGG1/2 are required for BIK1 stability. NP::BIK1-HA was introduced into agg1/2 by crossing, homozygous plants in F3 generation were subject to immunoblot analyses. (C) XLG2/3 are required for BIK1 accumulation. NP::BIK1-HA, 35S::BIK1-HA and NP::BAK1-HA plasmids were transiently expressed in WT and xlg2/3 protoplasts, and accumulation of BIK1 and BAK1 was determined by immunoblot analyses. (D) AGB1 regulates BIK1 accumulation through the proteasome pathway. One-week-old NP::BIK1-HA seedlings of WT (Col-0) or agb1 background were pretreated with DMSO (-) or 100 μM proteasome inhibitor PS341(+) for 8 hr before total protein was isolated for immunoblot analysis. (E) The agb1 extract shows accelerated degradation of BIK1 in vitro (F) The xlg2 xlg3 extract shows accelerated degradation of BIK1. Total extracts from WT (Col-0), agb1 and xlg2 xlg3 seedlings were incubated with HIS-BIK1 protein at 22°C for the indicated times, and equal amounts of sample were analyzed using anti-HIS immunoblot. Each experiment was repeated at least three times, and data from one representative experiment are shown.

DOI: http://dx.doi.org/10.7554/eLife.13568.012

Figure 3.

Figure 3—figure supplement 1. G proteins are required for BIK1 stability.

Figure 3—figure supplement 1.

(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 transcript levels. (B) Accumulation of BIK1 expressed from a transgene under control of the constitutive 35S promoter was similarly compromised in the agb1-2 mutant. 35S::BIK1-HA transgenic lines in WT and agb1-2 background with similar BIK1 transcript levels were identified by Semi-qPCR and used for detection of BIK1 accumulation. (C) Accumulation of BIK1 is not compromised in the gpa1-3 mutant. NP::BIK1-HA was introduced into gpa1-3 by crossing, and BIK1 accumulation was detected by anti-HA immunoblot. Three independent experiments were performed with similar results.
DOI: http://dx.doi.org/10.7554/eLife.13568.013
Figure 3—figure supplement 2. AGB1 regulates BIK1 stability through proteasome pathway.

Figure 3—figure supplement 2.

(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 inhibitor MG132 (+) for 8 hr, BIK1 stability was detected by anti-HA immunoblot. (B) Treatment with PS341 inhibits BIK1 degradation in vitro. Total extracts from WT plants pretreated with DMSO or 100 μM PS341 were incubated with the HIS-BIK1 recombinant protein, and equal amounts of sample were withdrawn at the indicated times for anti-HIS immunoblot assays. Three independent experiments were performed with similar results.
DOI: http://dx.doi.org/10.7554/eLife.13568.014
Figure 3—figure supplement 3. AGB1 and XLG2/3 attenuate PBL20 degradation.

Figure 3—figure supplement 3.

(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 protein, and equal amounts of sample were withdrawn at the indicated times for anti-HIS immunoblot analyses. Two independent experiments were performed with similar results.
DOI: http://dx.doi.org/10.7554/eLife.13568.015