Arabidopsis heterotrimeric G proteins regulate immunity by directly coupling to the FLS2 receptor
As an intensely studied Pattern Recognition Receptor ( PRR ) in plants , FLS2 serves as an excellent model understanding plant innate immune signaling and receptor kinases in general ( Macho and Zipfel , 2014 ) . It forms a dynamic complex with the co-receptor BAK1 and the receptor-like cytoplasmic kinase BIK1 to perceive a conserved bacterial flagellar epitope , flg22 , to activate a variety of defense responses ( Chinchilla et al . , 2007; Heese et al . , 2007; Lu et al . , 2010; Zhang et al . , 2010; Sun et al . , 2013 ) . Stability of FLS2 and BIK1 is subject to regulation by ubiquitin-proteasome system and a calcium-dependent protein kinase ( Lu et al . , 2011; Monaghan et al . , 2014 ) . We and others previously showed that BIK1 directly phosphorylates the NADPH oxidase RbohD to prime flg22-induced reactive oxygen species ( ROS; Kadota et al . , 2014; Li et al . , 2014 ) . Heterotrimeric G proteins are central for signaling in animals ( McCudden et al . , 2005; Oldham et al . , 2008 ) , which contain hundreds of G Protein-Coupled Receptors ( GPCRs ) . In the pre-activation state , the GDP-bound Gα interacts with the Gβγ dimer to form a heterotrimer . Upon activation by GPCR , Gα exchanges GDP for GTP , resulting in the activation of the heterotrimer . The activated Gα and Gβγ dissociate from each other to regulate downstream effectors . Plants contain canonical Gα ( encoded by GPA1 in Arabidopsis ) , Gβ ( encoded by AGB1 in Arabidopsis ) , Gγ proteins ( encoded by AGG1 , AGG2 ) , and a non-canonical Gγ ( encoded by AGG3 in Arabidopsis ) ( Urano and Jones , 2013 ) . Plants additionally encode extra-large G proteins ( XLGs , encoded by XLG1 , XLG2 , and XLG3 in Arabidopsis ) that carry a variable N-terminal domain and a C-terminal Gα domain ( Lee and Assman , 1999; Ding et al . , 2008 ) . Recent advances indicate that the Arabidopsis XLGs are functional Gα proteins and interact with Gβγ dimers to form heterotrimers ( Zhu et al . , 2009; Maruta et al . , 2015; Chakravorty et al . , 2015 ) . Heterotrimeric G proteins play important roles in a variety of biological processes in plants , including cell division ( Ullah et al . , 2001 , 2003; Chen et al . , 2003 ) , meristem maintenance ( Bommert et al . , 2013 ) , root morphogenesis ( Ding et al . , 2008 ) , seed development and germination ( Chen et al . , 2006; Pandey et al . , 2006 ) , nitrogen assimilation ( Sun et al . , 2014 ) , and response to ABA ( Wang et al . , 2001 ) , low temperature ( Ma et al . , 2015 ) , and blue light ( Warpeha et al . , 2006 ) . Accumulating evidence indicate that heterotrimeric G proteins also play an important role in plant disease resistance against diverse pathogens ( Llorente et al . , 2005; Trusov et al . , 2006; Zhu et al , 2009; Ishikawa , 2009; Cheng et al . , 2015 ) . Recent reports indicate that XLG2 , AGB1 , and AGG1/2 mediate immune responses downstream of PRRs ( Ishikawa , 2009; Zhu et al , 2009; Liu et al . , 2013; Lorek et al . , 2013; Torres et al . , 2013; Maruta et al . , 2015 ) . XLG2 was first shown to play an important role in basal resistance to P . syringae ( Zhu et al . , 2009 ) . A recent report showed that XLG2 , but not XLG1 , is required for resistance to P . syringae and flg22-induced ROS production ( Maruta et al . , 2015 ) . AGB1 and AGG1/2 , but not AGG3 , are required for resistance to P . syringae and microbial pattern-induced ROS production ( Liu et al . , 2013; Lorek et al . , 2013; Torres et al . , 2013 ) . Furthermore , epistatic analyses indicated that AGB1 acts in the same pathway as RbohD ( Torres et al . , 2013 ) . However , it is still debated whether plants possess 7 transmembrane GPCRs ( Taddese et al . , 2014; Urano and Jones , 2014 ) . One recent report suggests that GPA1 , AGG1/2 can interact with BAK1 and the chitin-binding receptor kinase CERK1 , but not FLS2 ( Aranda-Sicilia et al . , 2015 ) . However , GPA1 does not appear to play a role in flg22-induced ROS and disease resistance to P . syringae ( Liu et al . , 2013; Torres et al . , 2013 ) . How XLG2 and AGB1 regulate PRR-mediated immunity remains elusive . In this study , we report XLG2 , AGB1 , and AGG1/2 modulates flg22-triggered immunity by directly coupling to the FLS2-BIK1 receptor complex . Prior to activation by flg22 , the G proteins attenuate the proteasome-dependent degradation of BIK1 , ensuring optimum signaling competence . After flg22 stimulation , XLG2 dissociates from AGB1 , indicating a ligand-induced dissociation of Gα from Gβγ . In addition , we provide evidence that activation by flg22 additionally leads to XLG2 phospohrylation by BIK1 , and this phosphorylation positively regulates RbohD-dependent ROS production . Together the study illustrates two distinct mechanisms underlying the G protein-mediated regulation of the FLS2 signaling .
To identify additional components of the FLS2 immune pathway , we conducted a reverse genetic screen for mutants that were compromised in flg22-induced disease resistance to Pseudomonas syringae pv tomato ( Pst ) . One mutant displayed significantly reduced resistance was xlg2 ( Figure 1A ) , confirming previous report by Zhu et al . ( 2009 ) . Further characterization indicated that the mutant is compromised in flg22-induced ROS ( Figure 1B , Figure 1—figure supplement 1 ) , confirming results reported previously ( Maruta et al . , 2015 ) . An examination of gene expression showed that XLG2 , XLG3 , and AGB1 , but not XLG1 , were induced in response to flg22 treatment ( Figure 1—figure supplement 2 ) . This is in agreement with the recent report that XLG2/3 , but not XLG1 , are required for flg22-induced responses and disease resistance to P . syringae ( Maruta et al . , 2015 ) . A comparison of xlg2 and agb1 mutant showed that the two mutants were similarly compromised in flg22-induced ROS burst and resistance against Pst , supporting that they act together to regulate FLS2 immunity . Interestingly , xlg2 plants were more susceptible to Pst than agb1 plants in the absence of flg22 treatment ( Figure 1A ) , suggesting that XLG2 plays additional role in plant immunity independent of AGB1 . As reported previously ( Maruta et al . , 2015 ) , we found that the xlg3 mutant was similar to WT in Pst resistance and flg22-induced ROS , whereas the xlg2 xlg3 double mutant was slightly more defective in Pst resistance ( Figure 1A ) and flg22-induced ROS ( Figure 1B ) , indicating that XLG2/3 play additive roles in flg22-induced immunity . Because AGG1/2 , but not AGG3 and GPA1 , are required for flg22-induced defense responses and Pst resistance ( Liu et al . , 2013; Torres et al . , 2013 ) , these results confirmed that the heterotrimeric G proteins required for FLS2 signaling and Pst resistance include the non-canonical Gα proteins XLG2/3 , Gβ protein AGB1 , and Gγ proteins AGG1/2 . 10 . 7554/eLife . 13568 . 003Figure 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 . 00310 . 7554/eLife . 13568 . 004Figure 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 . 00410 . 7554/eLife . 13568 . 005Figure 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 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 . 00510 . 7554/eLife . 13568 . 006Figure 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 00610 . 7554/eLife . 13568 . 007Figure 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 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 Previous yeast two-hybrid , yeast tri-hybrid and Bi-Fluorescence Complementation assays showed that XLG2/3 form heterotrimers with the Gβγ dimer through AGB1 ( Zhu et al . , 2009; Maruta et al . , 2015; Chakravorty et al . , 2015 ) . Consistent with this , luciferase complementation and co-immunoprecipitation assays detected XLG2-AGB1 and XLG3-AGB1 interactions in Nicotiana Benthamiana ( Nb ) plants ( Figure 1C , Figure 1—figure supplement 3A ) and Arabidopsis protoplasts ( Figure 1—figure supplement 3B–C ) . Both the N and C termini of XLG2 are sufficient for the interaction with AGB1 ( Figure 1—figure supplement 3C ) . Note that XLG2-FLAG and AGB1-HA used in co-IP assays retain their biological functions when introduced into agb1 protoplasts or xlg2 plants ( see below in Figures 1D and 5E ) . In the animal model , the heterotrimeric G protein is dynamically regulated upon ligand stimulation of GPCR . We tested whether flg22 treatment can similarly regulate XLG2-AGB1 interaction . As shown in Figure 1C , the flg22 treatment resulted in a great reduction in XLG2-AGB1 interaction in Nb plants , suggesting that Arabidopsis heterotrimeric G proteins are reminiscent of their animal counterparts in that the heterotrimer is dynamically regulated upon ligand activation of receptors . Because the heterotrimeric G protein mutants under investigation all display defects in flg22-induced ROS , which requires phosphorylation of the NADPH oxidase RbohD by the BIK1 kinase ( Kadota et al . , 2014; Li et al . , 2014 ) , we monitored the flg22-induced phosphorylation of RbohD in agb1 using antibodies that recognize the BIK1-specific S39 phosphorylation . Flg22-induced S39 phosphorylation was significantly reduced in agb1 mutant protoplasts , whereas complementation of the mutant protoplasts with AGB1 restored RbohD S39 phosphorylation ( Figure 1D ) . Consistent with a positive role of AGB1 in flg22-induced RbohD S39 phosphorylation , transient overexpression of AGB1 in WT protoplasts resulted in constitutive S39 phosphorylation in RbohD ( Figure 1D ) . Together these results suggest that the hetero-trimeric G proteins regulate early components of the FLS2 signaling pathway . To identify potential proteins regulated by XLG2 , we performed immunoprecipitation of XLG2-FLAG transiently expressed in protoplasts . LC-MS/MS analysis of the immune complex identified four peptides corresponding to PBL20 ( Figure 2—figure supplement 1A ) , a homolog of BIK1 . In contrast , immunoprecipitation of the control protein CPR5-FLAG failed to identify any PBL20 peptides . Co-IP and luciferase complementation assays confirmed that XLG2/3 indeed interacted strongly with PBL20 ( Figure 2—figure supplement 1B–C ) . AGB1 also interacted with PBL20 , albeit much weaker than did XLG2 . Because PBL20 and BIK1 belong to the same family of receptor like cytoplasmic kinases ( RLCKs ) and share similar biochemical properties ( Zhang et al . , 2010 ) , we therefore tested whether BIK1 also interacts with the G proteins . Luciferase complementation assays showed that XLG2/3 strongly interacted with BIK1 but not BAK1 or CPR5 in Nb plants ( Figure 2A , Figure 2—figure supplement 2A ) , whereas AGB1 interacted with BIK1 at a much lower level . Similarly , FLS2 also displayed strong interactions with XLG2/3 and a weaker interaction with AGB1 ( Figure 2A , Figure 2—figure supplement 2A ) . The interactions were further confirmed by Co-IP assays ( Figure 2B–C , Figure 2—figure supplement 2B ) . In the absence of flg22 treatment , XLG2/3 strongly interacted with BIK1 and FLS2 ( Figure 2B–C , Figure 2—figure supplement 2B ) . Treatment of protoplasts with flg22 led to a much weaker interaction , indicating that flg22 induces dissociation of XLG2 from the FLS2 receptor complex . The BIK1K105E-HA mutant protein , which is defective in ATP-binding and dominantly inhibits flg22-induced signaling when expressed in protoplasts ( Zhang et al . , 2010 ) , interacted with XLG2 and XLG3 irrespective of the presence or absence of flg22 , suggesting that the activation of the BIK1 kinase is required for the dissociation . 10 . 7554/eLife . 13568 . 008Figure 2 . 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 unit ( RLU ) shows the strength of protein-protein interaction ( mean ± SD; n ≥ 6 . ( B ) XLG2 interacts with BIK1 in Arabidopsis protoplasts and the interaction is dynamically regulated by flg22 . ( C ) XLG2/3 interact with FLS2 in Arabidopsis protoplasts and the interaction is dynamically regulated by flg22 . The indicated constructs were co-expressed in WT protoplasts , and Co-IP assays were performed using agarose-conjugated anti-FLAG antibody . BIK1K105E carries a mutation in the ATP-binding site . ( D ) The C terminus of XLG2 directly interacts with FLS2 kinase domain . XLG2CT-HIS ( amino acids 459–861 ) was incubated with GST or GST-FLS2KD ( FLS2 kinase domain ) for GST pull-down assay and detected by anti-HIS and anti-GST immunoblots . ( E ) XLG2 primarily interacts with non-phosphorylated BIK1 . XLG2CT-HIS was incubated with GST or GST-BIK1 that was untreated or pre-treated with λ phosphatase ( PPase ) , and GST pull-down assay was performed . ( F ) AGB1 interacts with the non-phosphorylated BIK1 . Untreated or PPase-treated BIK1-HIS was incubated with GST or GST-AGB1 , and GST pull-down assay was performed . Each experiment was repeated two ( D–F ) or three ( A–C ) times , and data of one representative experiment are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 00810 . 7554/eLife . 13568 . 009Figure 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 in the control experiment using CPR5-FLAG as bait . ( B ) PBL20 interacts strongly with XLG2 and weakly with AGB1 in Arabidopsis protoplasts . Co-IP assay was performed using Arabidopsis protoplasts transfected with the indicated constructs . Three independent experiments were performed with similar results . ( C ) PBL20 interacts with XLG2 in Nb plants . Nb leaves were infiltrated with Agrobacteria containing the indicated constructs and luciferase activity was recorded 2 days later ( mean ± SD; n≥6; representative data from two independent experiments are shown ) . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 00910 . 7554/eLife . 13568 . 010Figure 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 ) XLG3 interacts with BIK1 in protoplasts . Co-IP assay was performed using WT Arabidopsis protoplasts transfected with the indicated constructs . Representative data from three independent experiments are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 01010 . 7554/eLife . 13568 . 011Figure 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; n≥6 ) . ( B ) XLG2 C terminus interacts with BIK1 . ( C ) XLG2 C terminus interacts with FLS2 . WT Arabidopsis protoplasts were transfected with the indicated constructs , and total protein was subject to Co-IP assays . Two independent experiments were performed with similar results . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 011 We next tested XLG2 domains for interactions with BIK1 and FLS2 . Luciferase complementation and Co-IP assays showed that the C terminus of XLG2 interacted strongly with BIK1 and FLS2 , whereas the N terminus of XLG2 displayed weak interactions ( Figure 2—figure supplement 3A–C ) , indicating that XLG2 interacts with FLS2 and BIK1 primarily through its C terminus . Similar to the full-length XLG2 protein , the C terminus of XLG2 also dissociated from BIK1 when protoplasts were treated with flg22 ( Figure 2—figure supplement 3C ) . GST pull-down assays showed that the HIS-tagged C-terminal fragment of XLG2 strongly interacted with GST-tagged FLS2 kinase domain , but not GST alone ( Figure 2D ) , indicating that the C terminus of XLG2 directly interacts with FLS2 . Surprisingly , GST pull-down assay showed a weak interaction between XLG2 and BIK1 ( Figure 2E ) . Interestingly , phosphatase treatment of BIK1 enhanced its interaction with XLG2 ( Figure 2E ) , suggesting that XLG2 has greater affinity with non-phosphorylated BIK1 . We similarly tested interaction between AGB1 with untreated and de-phosphorylated BIK1 by GST pull-down assay . While the un-treated BIK1 is unable to interact with AGB1 , the de-phosphorylated form of BIK1 strongly interacted with AGB1 ( Figure 2F ) . Together the results show that the G proteins dynamically interact with the FLS2-BIK1 receptor complex during FLS2 signaling , and the findings are consistent with the observed defects in flg22-induced responses and RbohD phosphorylation in the G protein mutants . 10 . 7554/eLife . 13568 . 012Figure 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 . 01210 . 7554/eLife . 13568 . 013Figure 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 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 . 01310 . 7554/eLife . 13568 . 014Figure 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 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 . 01410 . 7554/eLife . 13568 . 015Figure 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 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 The direct interaction of XLG2/3 with the FLS2 receptor complex prompted us to examine FLS2 , BIK1 and BAK1 proteins in the G protein mutants . To examine BIK1 accumulation , we crossed agb1 with a transgenic line carrying a BIK1-HA transgene under control of the native BIK1 promoter ( Zhang et al . , 2010 ) and identified sibling transgenic lines of either agb1 or AGB1 genotype . Immunoblot analyses showed that the BIK1-HA protein accumulated to a much lower level in agb1 compared to WT ( Figure 3A ) , indicating that AGB1 is required for BIK1 accumulation in plants . In contrast , FLS2 and BAK1 protein accumulation was not affected in agb1 , indicating a specific effect on BIK1 protein . Examination of BIK1-HA transcripts indicated that the transgene is similarly expressed in agb1 and WT plants ( Figure 3—figure supplement 1A ) . To further rule out the possibility that AGB1 modulates BIK1 at transcriptional level , we transformed agb1 and WT plants with BIK1-HA transgene under the control of the 35S promoter . Again , a much lower BIK1-HA accumulation was observed in agb1 than in WT plants although the BIK1-HA transcripts accumulated to similar levels in the two lines ( Figure 3—figure supplement 1B ) . Similarly , the BIK1-HA protein accumulation was greatly reduced in agg1 agg2 double mutant plants ( Figure 3B ) , indicating that AGG1/2 are also required for BIK1 accumulation . We further asked whether XLG2 and XLG3 are required for BIK1 accumulation by transiently expressing BIK1-HA in xlg2 xlg3 mutant protoplasts . Regardless of the promoter used for BIK1-HA expression , BIK1 accumulated to lower levels in the xlg2 xlg3 double mutant than in WT protoplasts ( Figure 3C ) , indicating that XLG2/3 are also required for BIK1 accumulation . The defect in protein accumulation is specific to BIK1 , as BAK1 accumulation was not affected in xlg2 xlg3 protoplasts ( Figure 3C ) . The accumulation of BIK1-HA was not affected in gpa1 mutant plants ( Figure 3—figure supplement 1C ) , a result that is consistent with previous report that GPA1 is not required for flg22-induced ROS ( Liu et al . , 2013; Torres et al . , 2013 ) . BIK1 is known to be turned-over through the ubiquitin-proteasome pathway ( Monaghan et al . , 2014 ) . Treatment of plants with proteasome inhibitors PS341 ( Figure 3D ) and MG132 ( Figure 3—figure supplement 2A ) restored BIK1 accumulation in agb1 mutant plants , suggesting that AGB1 regulates BIK1 stability through the proteasome pathway . We further performed in vitro protein degradation assay to compare the rate of BIK1 degradation in total protein extracts from WT and mutant plants . The recombinant BIK1 was degraded in the WT extract , and this degradation was blocked by PS341 ( Figure 3—figure supplement 2B ) , recapitulating the proteasome-dependent degradation of BIK1 in plants . The rate of BIK1 degradation was more rapid in agb1 and xlg2 xlg3 extracts compared to that in the WT extract ( Figure 3E–F ) . Because XLG2 also interacted with PBL20 , we further tested whether XLG2/3 and AGB1 similarly regulate PBL20 stability . Indeed , PBL20 was also degraded in WT extracts , and this degradation was enhanced when extracts from xlg2 xlg3 or agb1 mutants were used ( Figure 3—figure supplement 3A–B ) . Together these results demonstrate that the heterotrimeric G proteins formed by XLG2/3 , AGB1 , and AGG1/2 attenuate proteasome-dependent degradation of BIK1 and PBL20 . We next tested whether the regulation of BIK1 stability contributes to the role of heterotrimeric G proteins in flg22-induced immune responses . Transient overexpression of BIK1 in xlg2 xlg3 and agb1 protoplasts largely restored the flg22-induced RbohD S39 phosphorylation ( Figure 4A ) , indicating that the heterotrimeric G proteins positively regulate RbohD phosphorylation at least in part by enhancing BIK1 stability . We next asked whether increased copy number of BIK1 could rescue flg22-induced ROS burst in agb1 mutant . Introgression of the NP::BIK1-HA transgene into agb1 by crossing significantly enhanced flg22-induced ROS production in these plants compared to agb1 plants that contained only the endogenous copy of BIK1 ( Figure 4B ) , indicating that increasing BIK1 accumulation by the transgene at least partially restored flg22-induced ROS in agb1 . Col-0 NP::BIK1-HA plants displayed higher level of ROS compared to non-transgenic WT plants ( Figure 4B ) , indicating that BIK1 protein level is rate-limiting in flg22-induced immune responses . We further tested the impact of the NP::BIK1-HA transgene on flg22-induced resistance to Pst . While the agb1 consistently supported greater amount of bacterial growth compared to WT plants following flg22 treatment , the agb1 NP::BIK1-HA plants were indistinguishable from WT ( Figure 4C ) . Col-0 NP::BIK1-HA plants supported less bacterial growth than did WT plants , further supporting the importance of BIK1 abundance in FLS2-mediated immunity . We similarly introduced the NP::BIK1-HA transgene into xlg2 xlg3 double mutant plants by transformation . The resulting transgenic lines displayed near WT ROS production ( Figure 4D ) and Pst resistance ( Figure 4E ) in response to flg22 treatment , indicating that XLG2/3 also regulate FLS2-mediated immunity by controlling BIK1 stability . 10 . 7554/eLife . 13568 . 016Figure 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 and xlg2/3 . FLAG-RbohD protein was affinity purified and detected by anti-FLAG and anti-pSer39 immuoblots . ( B ) BIK1 transgene restores flg22-induced ROS burst in agb1 . ( C ) BIK1 transgene partially restores flg22-induced resistance to Pst in agb1 . NP::BIK1-HA was introduced into agb1 by crossing , transgenic lines of agb1 or Col-0 background in the F3 generation were used for the assays . ( D ) BIK1 transgene partially restores flg22-induced ROS burst in xlg2 xlg3 mutant . ( E ) BIK1 transgene partially restores flg22-induced resistance to Pst . The NP::BIK1-HA transgene was introduced into WT ( Col-0-L32 ) and xlg2 xlg3 ( xlg2/3-L64 and xlg2/3-L51 ) plants by Agrobacterium-mediated transformation . Independent T2 transgenic lines were used for the assays . Peak relative luminescence unit ( RLU ) values were shown for ROS assays ( B and D ) and leaf bacterial populations 2 days after bacterial inoculation were shown for flg22-protection assays ( C and E ) . Bars in B-E represent mean ± SD ( n ≥ 6; p<0 . 05 , Student’s t-test; different letters indicate signiﬁcant difference ) . Each experiment was repeated two ( A ) or three ( B–E ) times , and data of one representative experiment are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 01610 . 7554/eLife . 13568 . 017Figure 4—source data 1 . Raw data and exact p value of Figure 4B—E . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 017 BIK1 is postulated to phosphorylate multiple proteins , including RbohD , in PTI ( Pattern-Triggered Immunity ) signaling ( Kadota et al . , 2014; Li et al . , 2014; Macho and Zipfel , 2014 ) . An examination of XLG2 N-terminal fragment expressed in protoplasts following flg22 treatment showed a prominent shift in protein migration in SDS-PAGE ( Figure 5A ) . The shift of protein mobility was sensitive to phosphatase treatment , which is indicative of flg22-induced phosphorylation in XLG2 . The flg22-induced phosphorylation similarly occurred to the N terminus of XLG3 , but not XLG1 ( Figure 5—figure supplement 1 ) , which is consistent with the notion that XLG2/3 but not XLG1 , are involved in flg22 signaling . To determine phospho-sites in XLG2 , we expressed full-length XLG2-FLAG in protoplasts . Following flg22 treatment , the XLG2-FLAG protein was affinity-purified and subject to LC-MS/MS analysis for phospho-peptides . We were able to recover 87% of total peptide sequence , among which we identified 23 phospho-peptides corresponding to at least 15 phospho-sites ( Figure 5—figure supplement 2 ) . Interestingly , all but two phospho-peptides were located in the N terminus . Strikingly , phospho-sites between amino acids 141–156 accounted for nearly half of the phospho-peptides . We further tested whether these are the major phospho-sites by site-directed mutagenesis . Simultaneously substituting three to four of these residues into non-phosphorylatable alanine ( XLG2S148A , S150A , S151A; XLG2S141A , S148A , S150A , S151A ) resulted in severe reduction to complete elimination in flg22-induced band shift in XLG2 N terminus ( Figure 5B ) , indicating that amino acids Ser141 , Ser148 , Ser150 and Ser151 are indeed required for overall phosphorylation of XLG2 in response to flg22 treatment . 10 . 7554/eLife . 13568 . 018Figure 5 . 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 phosphatase ( PPase ) prior to anti-FLAG immunoblot analysis . ( B ) Flg22-induced phosphorylation of XLG2 in protoplasts primarily occurs in Ser141 , Ser148 , Ser150 and Ser151 . Different mutated form of XLG21-203-FLAG constructs were transiently expressed in WT protoplast , treated with flg22 and the migration of XLG21-203-FLAG were examined by anti-FLAG immunoblot . ( C ) BIK1 phosphorylates XLG2 N terminus in vitro . XLG21-203-HIS was incubated with HIS-BIK1 and HIS-BIK1K105E in the presence of 32P-γ-ATP and analyzed by autoradiography . CBB , coomassie brilliant blue . ( D ) BIK1 phosphorylates XLG2 at Ser148 and Ser150 in vitro . XLG21-203-HIS was incubated with HIS-BIK1 and HIS-BIK1K105E in kinase reaction buffer . Protein phosphorylation was detected by anti-pSer148 and pSer150 immunoblots . ( E ) XLG2 phosphorylation is required for flg22-induced ROS . xlg2 mutant plants were transformed with WT ( NP::XLG2-L34 ) , non-phosphorylatable ( 4A-L1 and 4A-L7 ) , or phospho-mimicking ( 4D-L7 and 4D-L9 ) forms of XLG2 under control of the native XLG2 promoter . Independent T2 lines were examined for flg22-induced ROS burst and peak relative luminescence unit ( RLU ) values are shown . ( mean ± SD; n ≥ 6; p<0 . 05 , Student’s t-test; different letters indicate signiﬁcant difference ) . ( F ) XLG2 phosphorylation is required for Pst resistance . xlg2/3 double mutant plants were transformed with WT ( XLG2-L3 ) or non-phosphorylatable ( 4A-L6 and 4A-L7 ) form of XLG2 under control of the native XLG2 promoter . Independent T2 lines were inoculated with Pst , and bacterial populations in leaves were measured 3 days post inoculation . ( mean ± SD; n ≥ 6; p<0 . 05 , Student’s t-test; different letters indicate signiﬁcant difference ) . ( G ) XLG2 interacts with RbohD in Arabidopsis plants . rbohD plants were transformed with the FLAG-RbohD transgene under control of the RbohD native promoter . The resulting plants were used for Co-IP assay . Each experiment was repeated two ( C , G ) or three ( A , B , D–F ) times , and data of one representative experiment are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 01810 . 7554/eLife . 13568 . 019Figure 5—source data 1 . Raw data and exact p value of Figure 5E and F . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 01910 . 7554/eLife . 13568 . 020Figure 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 02010 . 7554/eLife . 13568 . 021Figure 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 phospho-sites identification . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 02110 . 7554/eLife . 13568 . 022Figure 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 , and total protein was subject to Co-IP assays and immunoblot analysis . Three independent experiments were performed with similar results . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 02210 . 7554/eLife . 13568 . 023Figure 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 experiments are shown ) . DOI: http://dx . doi . org/10 . 7554/eLife . 13568 . 023 We next tested whether BIK1 is able to phosphorylate XLG2 N terminus in vitro Radio-labelling assay showed that the recombinant BIK1 , but not BIK1K105E , strongly phosphorylated the N terminus of XLG2 ( Figure 5C ) . To determine whether the major phospho-sites in XLG2 were also phosphorylated by BIK1 , we raised antibodies that specifically recognize phospho-S148 and phospho-S150 . Immunoblot analysis showed that S148 and S150 were indeed phosphorylated in vitro by BIK1 , but not BIK1K105E ( Figure 5D ) . We introduced WT , non-phosphorylatable ( XLG2S141A , S148A , S150A , S151A ) and phospho-mimicking ( XLG2S141D , S148D , S150D , S151D ) forms of XLG2-FLAG transgene under control of the native XLG2 promoter into xlg2 plants and analyzed flg22-induced ROS production in the resulting transgenic lines . While the xlg2 transgenic lines carrying the WT or phospho-mimicking XLG2-FLAG transgene completely restored the flg22-induced ROS to WT level , the two lines transformed with the non-phosphorylatable XLG2-FLAG transgene only partially restored the flg22-induced ROS ( Figure 5E ) . The inability of phospho-mimicking XLG2 to constitutively activate ROS suggests that the phosphorylation of XLG2 is required , but not sufficient , for optimum activation of RbohD . We further introduced WT and the non-phosphorylatable XLG2-FLAG transgene into xlg2/3 double mutants , and inoculated independent lines with Pst . While the line carrying the WT XLG2-FLAG transgene was fully restored in resistance to Pst that was indistinguishable from the WT non-transgenic plants , the two lines transformed with the non-phosphorylatable XLG-FLAG transgene were only marginally more resistant to Pst ( Figure 5F ) . These results indicated that the phosphorylation is required , but not sufficient , for full function of XLG2 . We next asked whether the phospho-site mutants affect BIK1 stability or XLG2-BIK1 interaction . When expressed in protoplasts isolated from xlg2 xlg3 plants , the phospho-site mutants allowed similar BIK1-HA accumulation compared to protoplasts expressing the WT XLG2 protein ( Figure 5—figure supplement 3 ) . These mutants also showed normal interaction with BIK1 in the absence of flg22 and normal dissociation from BIK1 in the presence of flg22 . The results suggest that the phosphorylation of these sites affected neither BIK1 stability nor interaction with BIK1 . Because XLG2 dissociates from BIK1 following activation by flg22 ( Figure 2B ) , we reasoned that XLG2 may have additional effectors . Indeed , XLG2 interacted strongly with RbohD in Arabidopsis plants ( Figure 5G ) and Nb plants ( Figure 5—figure supplement 4 ) , providing an explanation that XLG2 phosphorylation regulates ROS burst independent of BIK1 stability . The XLG2-RbohD interaction was detected in the absence of flg22 treatment , indicating that the XLG2 constitutively interacts with RbohD .
Heterotrimeric G proteins play important role in signal transduction in plants and animals . Paradoxically , plants do not possess functional GPCRs . The maize Gα protein CT2 interacts with the receptor kinase CLV1 to control shoot meristem development ( Bommert et al . , 2013 ) . GPA1 and AGG1/2 have been shown to interact with BAK1 and CERK1 in yeast two-hybrid and Bi-Fluorescence Complementation assays , although the biological significance of this remains unknown ( Aranda-Sicilia et al . , 2015 ) . We show that XLG2 directly interacts with FLS2 and BIK1 to regulate FLS2-mediated immunity . Thus , these studies collectively support that plant receptor kinases fulfil the roles of GPCRs by directly coupling to heterotrimeric G proteins . Importantly , we show that the G proteins coupled to the FLS2 receptor complex are subject to multiple regulations during FLS2 signaling . The interaction between XLG2/3 and AGB1 , which leads to the formation of XLG2/3-AGB1-AGG1/2 heterotrimers ( Maruta et al . , 2015; Chakravorty et al . , 2015 ) , is dynamically regulated by flg22 . The flg22-induced dissociation of XLG2 from AGB1 likely reflects an increase of GTP-bound form of Gα which dissociates from the Gβγ dimer upon receptor activation . Secondly , the interactions of the G proteins with FLS2 and BIK1 are also dynamically regulated by flg22 . The flg22-induced dissociation of the G proteins from BIK1 coincides with the flg22-induced BIK1 phosphorylation , suggesting that the flg22-induced BIK1 phosphorylation triggers the dissociation of the G proteins . Indeed , de-phosphorylation of BIK1 strongly enhanced its interactions with both XLG2 and AGB1 in vitro . The decoupling of the G proteins from the receptor maybe necessary for the regulation of down-stream effectors . BIK1 is subject to proteasome-dependent degradation ( Monaghan et al . , 2014 ) . Our genetic analyses indicated that XLG2 , AGB1 and AGG1/2 are required for BIK1 stability in the pre-activation state . Loss of XLG2 and AGB1 leads to accelerated degradation of BIK1 , and this is blocked by the addition of proteasome inhibitors , indicating that the G proteins attenuating the proteasome-dependent degradation of BIK1 . FLS2 is also subject to proteasome-dependent degradation ( Lu et al . , 2011 ) . However , FLS2 accumulation is not affected by the agb1 mutation , indicating that the G proteins do not generally regulate proteasome-mediated protein degradation . These observations raise an interesting question as to whether the G proteins specifically impede the ubiquitination of BIK1 or loading of BIK1 to proteasome . Importantly , introduction of a transgenic copy of BIK1 largely restored flg22-triggered immune responses and disease resistance in xlg2 and agb1 mutants , demonstrating that a major function of the heterotrimeric G protein is to maintain the signaling competence of the FLS2-BIK1 complex in the pre-activation state . In addition to the ligand-induced dissociation of XLG2 from AGB1 , flg22 induces the phosphorylation of XLG2 in the N terminus . This phosphorylation is likely caused by the BIK1 family kinases , as two of the major phosphor-sites induced by flg22 are also phosphorylated by BIK1 in vitro . Non-phosphorylatable XLG2 mutants are compromised in flg22-induced ROS production and disease resistance to Pst , indicating that the phosphorylation plays a positive role . However , mutations in the phospho-sites did not affect XLG2-BIK1 interaction nor BIK1 stability , suggesting that the phosphorylated XLG2 regulates components other than BIK1 . Indeed , XLG2 constitutively interacts with RbohD , suggesting that the phosphorylated XLG2 further enhances ROS production by modulating RbohD . RbohD is known to be subject to multiple regulations including phosphorylation by CPK5 and BIK1 ( Dubiella et al . , 2013; Kadota et al . , 2014; Li et al . , 2014 ) and calcium binding to EF-hand ( Ogasawara et al . , 2008 ) . The results presented here show that RbohD is additionally regulated by a phosphorylated XLG2 , although the underlying mechanism remains unknown . The Arabidopsis heterotrimeric G proteins reported here are analogous to the animal counterpart in that , upon activation by receptors , dissociate from each other and activate downstream effectors . Unlike the animal heterotrimeric G proteins , however , the Arabidopsis heterotrimeric G proteins additionally regulate signaling competence of the FLS2-BIK1 complex prior to receptor activation . Taken together , our results highlight remarkable similarities and striking differences in heterotrimeric G protein-coupled receptor signaling in animals and plants as a result of independent evolution .
Arabidopsis plants used in this study include WT ( Col-0 ) , xlg2 , xlg2 xlg3 ( Ding et al . , 2008 ) , xlg3 ( SALK_107656c ) , agb1-2 ( Ullah et al . , 2003 ) , gpa1-3 ( Jones et al . , 2003 ) and agg1 agg2 mutants ( Trusov et al . , 2007 ) , and NP::BIK1-HA ( Zhang et al . , 2010 ) and NP::FLAG-RbohD transgenic lines ( Li et al . , 2014 ) . Nicotiana benthamiana plants used for luciferase-complementation assay and Arabidopsis plants used for ROS burst , protoplast preparation and bacterial infection assays were grown in soil at 23°C and 70% relative humidity with 10/14 hr day/night photoperiod for 4–5 weeks . Arabidopsis seedlings used for BIK1 stability and in vitroprotein degradation assay were grown in half Murashige-Skoog ( MS ) plates at 23°C with 16/8 hr day/night photoperiod for 7–10 days . To generate constructs for transient expression in protoplast , coding sequences of desired genes are amplified by PCR and cloned into the pUC19-35S-FLAG/HA-RBS vector . For GST and HIS fusion constructs , coding sequences were PCR-amplified and cloned into pGex 6P-1 ( for GST fusion constructs ) or pET28a ( for HIS fusion constructs ) . For complementation of xlg2 ( related with Figure 1—figure supplement 1 ) , XLG2 genomic sequence containing 2 kb native promoter and 0 . 5 kb 3’UTR was cloned into pENTY vector using pENTY Directional Cloning Kit ( Invitrigen . Carlsbad , CA ) , transferred to pFAST-G01 vector using Gateway LR Clonase ( Invitrogen ) and introduced into xlg2 mutant plants by Agrobacterium-mediated transformation . . To generate XLG2 transgenic plants containing mutated phosphosites ( Figures 5E and F ) , a fragment containing 2 kb native promoter and coding sequence of XLG2 was fused to FLAG tag at the C terminus followed by the RBS terminator and cloned into pENTY vector . Mutations were introduced into pENTY-XLG2-FLAG by site-directed mutagenesis . All forms of XLG2-FLAG were transferred to pFAST-G01 and introduced into xlg2 or xlg2 xlg3 mutant plants by Agrobacterium-mediated transformation . To generate BIK1-HA transgenic plants in agb1 , gpa1-3 and agg1 agg2 background , the NP::BIK1-HA transgene ( Zhang et al . , 2010 ) was introduced into agb1 , agg1/2 and gpa1-3 by crossing . Homozygous mutants and WT plants containing the transgene in the F3 generation were used for BIK1 stability and immune response assays . To express BIK1-HA under the 35S promoter , the cDNA of BIK1 was amplified by PCR and cloned into pCambia1300 with a 3x HA tag . Transgenic plants expressing 35S::BIK1-HA were generated by transforming WT or agb1-2 mutant plants with the pCambia1300-BIK1-HA construct . Transgenic lines in wild type and agb1-2 background with similar expression levels of the BIK1-HA transgene were identified by quantitative RT-PCR analysis . To generate BIK1-HA transgenic plants in xlg2 xlg3 background , the pCAMBIA1300-NP::BIK1-HA-RBS ( Zhang et al . , 2010 ) construct was introduced into WT and xlg2 xlg3 plants by Agrobacterium-mediated transformation . Independent lines in T2 generation were used for ROS assay and flg22-protection assay . Leaf strips form 4- to 5-week-old soil-grown Arabidopsis plants were incubated in water for 12 hr in 96-well plate before treated with luminescence detection buffer ( 20 μM luminol and 10 mg/ml horseradish peroxidase ) containing 1 μM flg22 as described ( Zhang et al . , 2007 ) . Luminescence was recorded by using GLOMAX 96 microplate luminometer ( Promega , Madison , WI ) . 4- to 5-week-old plants were pre-treated with water or 1 μM flg22 1 day before infiltration with P . syringae DC3000 at a concentration of 1×106/ml , and bacterial population was determined 2 days after bacterial inoculation . Total protein was extracted from ground transgenic Arabidopsis seedlings or protoplasts with protein extraction buffer ( 50 mM HEPES [pH 7 . 5] , 150 mM KCl , 1 mM EDTA , 0 . 5% Trition-X 100 , 1 mM DTT , proteinase inhibitor cocktail ) . Supernatants were collected by centrifugation at 13 , 000 rpm for 15 min . Total protein was separated in SDS-PAGE gel and detected by immunoblot by using the indicated antibodies . For immunoprecipitation in protoplasts , Arabidopsis protoplasts were transfected with 50–100 μg indicated plasmids , incubated overnight , and then treated with water or 1 μM flg22 for 10 min . Total protein was incubated with agarose-conjugated anti-FLAG antibody ( Sigma , St . Louis , MO ) for 4 hr , washed 6 times with extraction buffer and eluted with 3× FLAG peptide ( Sigma ) for 30 min . The immunoprecipitates were separated in a SDS-PAGE gel and detected with immunoblot . XLG2-FLAG plasmid was transfected into WT Arabidopsis protoplasts and incubated at room temperature for 14 hr . Total protein was extracted with IP buffer I ( 50 mM HEPES [pH 7 . 5] , 50 mM NaCl , 10 mM EDTA , 0 . 2% Triton X-100 , 0 . 1 mg/mL Dextran ( Sigma ) , proteinase inhibitor cocktail ) and the immunoprecipitation was performed as previously reported ( Liu et al . , 2009 ) . To identify XLG2 phosphosites , protoplasts were treated with 1 μM flg22 for 10 min before extraction . Total protein was incubated with 70 μl agarose-conjugated anti-FLAG antibody for 4 hr , washed with IP buffer II ( 50 mM HEPES [pH 7 . 5] , 50 mM NaCl , 10 mM EDTA , 0 . 1% Triton X-100 , proteinase inhibitor cocktail ) for 2 times , IP buffer III ( 50 mM HEPES [pH 7 . 5] , 150 mM NaCl , 10 mM EDTA , 0 . 1% Triton X-100 , proteinase inhibitor cocktail ) for 2 times and eluted with 3× FLAG peptide for 45 min . The immunoprecipitates were separated in 10% NuPAGE gel ( invitrogen ) and subject to Mass Spectrometric analysis as previously described ( Li et al . , 2014 ) . For GST pull-down assay , GST-BIK1 , GST-BIK1K105E , GST-FLS2KD and XLG2CT-HIS proteins were purified using the glutathione agarose beads and Ni-NTA agarose beads . 10 μg GST- and HIS-tagged proteins were incubated with 30 μl glutathione agarose beads in GST buffer ( 25 mM Tris-HCl , 100 mM NaCl , 1 mM DTT , pH 7 . 5 ) for 3 hr . The beads were washed 5–6 times with GST buffer and eluted with elution buffer ( 25 mM Tris-HCl , 100 mM NaCl , 1 mM DTT , 15 mM GSH , pH 7 . 5 ) . Samples were separated in SDS-PAGE gel and detected by anti-HIS immunoblot . For in vitro phosphorylation assay , 200 ng HIS-BIK1 or HIS-BIK1K105E was incubated with 2 μg XLG21-203-HIS protein in the kinase reaction buffer ( 25 mM Tris-HCl , 10 mM MgCl2 , 1 mM DTT , 100 mM ATP , pH 7 . 5 ) for 30 min . The phosphorylation of XLG21-203 was detected by autoradiograph ( by adding γ-P32 ATP in kinase reaction buffer ) or by immunoblot using phosphosite-speciﬁc antibodies . The coding sequences of indicated genes are cloned into pCAMBIA1300-35S-Cluc-RBS or pCAMBIA1300-35S-HA-Nluc-RBS and are introduced into Agrobacterium tumefaciens strain GV3101 . The assay was carried out as previously described ( Chen et al . , 2008 ) , Agrobacterial strains carrying the indicated constructs were infiltrated into Nb leaves . Leaf discs were taken 2 days later , incubated with 1 mM luciferin in a 96-well plate for 5–10 min , and luminescence was recorded with the GLOMAX 96 microplate luminometer . XLG2 phosphosite-speciﬁc antibodies were produced by Abmart ( China ) as previous described ( Li et al . , 2014 ) using the following peptides: In vitro protein degradation assay was performed as previous described ( Wang et al . , 2009 ) with slightly modification . 1-week-old seedlings of indicated genotype were ground in 200–300 μl extraction buffer ( 25 mM Tris-HCl [pH 7 . 5] , 10 mM NaCl , 10 mM MgCl2 , 4 mM PMSF , 5 mM DTT , and 10 mM ATP ) . Supernatants were collected by centrifugation at 14 , 000 rpm for 10 min and total protein concentration was determined by Bio-Rad protein assay and adjusted to a final concentration of 1μg protein/μl . 300 ng recombinant HIS-BIK1 or PBL20-HIS protein was incubated with 100 μl total extract at 22°C , and equal amounts of samples were withdrawn at the indicated times for anti-HIS immunoblot analysis . 4-week-old WT Arabidopsis plants were treated with flg22 for 0 hr and 3 hr and total RNA was extracted by using RNeasy Plant Mini Kit ( Qiagen , Germany ) . First-strand cDNA synthesis was performed using SuperScript III RNA transcriptase ( Invitrogen ) following manufacturer’s instructions . Real-Time PCR was performed using and specific primers and SYBR Premix Ex Taq Kit ( TaKaRa , Japan ) . Primers for Real-Time PCR: ACT8-RT-F: 5'-TGTGACAATGGTACTGGAATGG-3' ACT8-RT-R: 5'-TTGGATTGTGCTTCATCACC -3' XLG1-RT-F: 5'-TGATGGTGAGGATTGTGAATTGA-3' XLG1-RT-R: 5'-TTCCCAATCCGGTACTAACGG-3' XLG2-RT-F: 5'-ATTGCTAATGTGCCACGAGCT-3' XLG2-RT-R: 5'-ACGAGAGGTGCCACTGGGTAA-3' XLG3-RT-F: 5'-CCGGTTGTGAAATTCAAACCTG-3' XLG3-RT-R: 5'-TCCCTCTCTGTCTCTGCCTCC-3' BIK1-HA-RT-F: 5'-CAGGACAACTTGGGAAAACCG-3' BIK1-HA-RT-R: 5'-TAGGATCCTGCATAGTCCGGG-3' Primers for site-directed mutagenesis XLG2 ( S141A ) -F: 5'-ATGTACCAGAAGAAGTGAAAGCTCCTGCTGATTTTCGGTTATC-3' XLG2 ( S141A ) -R: 5'-GATAACCGAAAATCAGCAGGAGCTTTCACTTCTTCTGGTACAT-3' XLG2 ( S148A ) -F: 5'-TCCTGCTGATTTTCGGTTAGCACCATCATCACCATTGTCTG-3' XLG2 ( S148A ) -R: 5'-CAGACAATGGTGATGATGGTGCTAACCGAAAATCAGCAGGA-3' XLG2 ( S150A/S151A ) -F: 5'-GCTGATTTTCGGTTATCACCAGCAGCACCATTGTCTGCATCAGCGAGA-3' XLG2 ( S150A/S151A ) -R: 5'-TCTCGCTGATGCAGACAATGGTGCTGCTGGTGATAACCGAAAATCAGC-3' XLG2-3A ( S148A/S150A/S151A ) -F: 5'-AAAGTCCTGCTGATTTTCGGTTAGCACCAGCAGCACCATTGTCTGCATCAGCGAGA GA-3' XLG2-3A ( S148A/S150A/S151A ) -R: 5'-TCTCTCGCTGATGCAGACAATGGTGCTGCTGGTGCTAACCGAAAATCAGCAGGACT TT-3' XLG2-3D ( S148D/S150D/S151D ) -F: 5'-AAAGTCCTGCTGATTTTCGGTTAGACCCAGACGACCCATTGTCTGCATCAGCGAGA GA-3' XLG2-3D ( S148D/S150D/S151D ) -R: 5'-TCTCTCGCTGATGCAGACAATGGGTCGTCTGGGTCTAACCGAAAATCAGCAGGACT TT--3' XLG2 ( S141D ) -F: 5'-ATGTACCAGAAGAAGTGAAAGATCCTGCTGATTTTCGGTTATC-3' XLG2 ( S141D ) -F: 5'-GATAACCGAAAATCAGCAGGATCTTTCACTTCTTCTGGTACAT-3'
The Arabidopsis immune receptor FLS2 perceives bacterial flagellin epitope flg22 to activate defenses through the central cytoplasmic kinase BIK1 . The heterotrimeric G proteins composed of the non-canonical Gα protein XLG2 , the Gβ protein AGB1 , and the Gγ proteins AGG1 and AGG2 are required for FLS2-mediated immune responses through an unknown mechanism . Here we show that in the pre-activation state , XLG2 directly interacts with FLS2 and BIK1 , and it functions together with AGB1 and AGG1/2 to attenuate proteasome-mediated degradation of BIK1 , allowing optimum immune activation . Following the activation by flg22 , XLG2 dissociates from AGB1 and is phosphorylated by BIK1 in the N terminus . The phosphorylated XLG2 enhances the production of reactive oxygen species ( ROS ) likely by modulating the NADPH oxidase RbohD . The study demonstrates that the G proteins are directly coupled to the FLS2 receptor complex and regulate immune signaling through both pre-activation and post-activation mechanisms .
Living cells need to be able to detect changes in their environment and respond accordingly . This ability involves signals from outside of the cell triggering changes to the activity inside the cell . Heterotrimeric G proteins are important for this kind of signaling in a wide range of organisms . In animals and fungi , these proteins directly work with a specific class of receptor proteins called G protein-coupled receptors ( or GPCRs for short ) . Plants also have heterotrimeric G proteins , but it remains unclear whether they similiarly work with GPCRs . Plants detect invading microbes by using receptors that are completely different from GPCRs . For example , a receptor called FLS2 from the model plant Arabidopsis senses a telltale protein produced by bacteria , and then passes the signal to another protein called BIK1 to activate the plant’s defenses . Heterotrimeric G proteins are required for this process , but the underlying mechanisms remain unknown . Liang , Ding et al . now show that heterotrimeric G proteins regulate FLS2-controlled defenses by directly interacting with FLS2 and BIK1 . Heterotrimeric G proteins also enhance defenses in at least two different ways . Firstly , in the absence of an infection , heterotrimeric G proteins stabilize the BIK1 protein to ensure that it is ready to respond . Secondly , if FLS2 does detect the telltale bacterial protein , BIK1 marks one of the heterotrimeric G proteins with a phosphate group . This then allows the G protein to boost the activity of another plant enzyme that is vital for defense signaling . In the future , it will be important to work out how activation of FLS2 leads to the activation of heterotrimeric G proteins . Furthermore , heterotrimeric G proteins are likely to regulate additional plant proteins when defenses are activated , and further studies are needed to identify these proteins .