The growth-defense pivot: crisis management in plants mediated by LRR-RK surface receptors

Abstract

Plants must adapt to their environment and require mechanisms for sensing their surroundings and responding appropriately. An expanded family of more than 200 leucine-rich repeat (LRR) receptor kinases (LRR-RKs) transduces fluctuating and often contradictory signals from the environment into changes in nuclear gene expression. Two LRR-RKs, BRASSINOSTEROID INSENSITIVE 1 (BRI1), a steroid receptor, and FLAGELLIN SENSITIVE 2 (FLS2), an innate immune receptor that recognizes bacterial flagellin, act cooperatively to partition necessary growth-defense trade-offs. BRI1 and FLS2 share common signaling components and slightly different activation mechanisms. BRI1 and FLS2 are paradigms for understanding the signaling mechanisms of LRR-containing receptors in plants.

Keywords: LRR-RKs; cell surface signaling; crosstalk; innate immunity; plant growth; trade-offs.

Figure 1. A comparison of LRR-RK kinase domains and LRR numbers

A- The kinase domains of LRR-RKs in the Arabidopsis genome were aligned by HMMER [92], a neighbor-joining phylogenetic tree was created for the kinase domains alignment using MEGA [93], and the phylogenetic tree was tested with bootstrapping. LRR-RKs discussed in the review are labeled accordingly. The kinase subfamilies are indicated along the outer edge of the alignment. B- LRRs for each LRR-RK from the kinase alignment were identified using HMMER for domain analysis with a low stringency for the LRR domain from the PFAM database PF00560 [92]. For comparison the LRRs from LRR-RLPs were identified using the PFAM database as well. The total number of LRRs for each LRR-RK and LRR-RLP were calculated to give the distribution of number of LRRs seen in these proteins. The frequency of each number of LRRs was determined by dividing each value by the total number of LRR-RKs and LRR-RLPs analyzed and then plotting the result. The validity of this method was verified using known LRR numbers based on crystal structures and analyzing several LRR-RKs by hand. LRR-RKs discussed in the review are labeled accordingly. C- Mutations in pathways that are controlled by LRR-RKs can have obvious growth and development phenotypes. Disruption of endogenous BR signaling through loss-of-function mutations in steroid biosynthesis or signaling translates into severe dwarfism. Opposite effects can be achieved by gain-of-function mutations that elevate signaling through increased biosynthesis or receptor overexpression. Abbreviations: LRR-RKs, leucine-rich repeat receptor kinases; LRR-RLPs, leucine-rich repeat receptor-like proteins; BR, brassinosteroid.

Figure 2. A model of BR signaling from the cell surface to the nucleus

A- In the absence of BR, signal-competent BRI1 is regulated by “cis” and “trans” mechanisms. The kinase domains of BAK1 and BRI1 are both inhibited by their own C-terminal tail [94]. BRI1 activity is further inhibited by an association with BKI1, a plasma membrane-associated phosphoprotein. In this configuration, BIN2, exists in an active and phosphorylated form. As a negative regulator of BR signaling, cytoplasmic and nuclear localized BIN2 inactivates BZR1 and BES1, two plant specific transcription factors. BIN2 has a dual action on BZR1/BES1: First, by phosphorylating them in the cytoplasm, BIN2 promotes their cytoplasmic retention by phosphopeptide-binding 14-3-3 proteins [95]; second, by phosphorylating them in the nucleus BIN2 blocks their DNA binding and transcriptional activities. B- Binding of BR to BRI1 triggers the Tyrosine phosphorylation of BKI1 and its subsequent dissociation from the plasma membrane, thereby allowing the recruitment of BAK1/SERK3 or SERK1. Meanwhile, released, cytosolic BKI1 interacts with and thus titrates 14-3-3 proteins away from BZR1 [95, 96], promoting the accumulation of BZR1/BES1 in the nucleus in their active forms. In the absence of ligand, BAK1/SERK1 are auto-inhibited by their C-terminal tails; this inhibition is relieved by binding to BRI1 [94]. After a series of reciprocal trans-phosphorylation events on Ser-Thr and Tyr residues, signal competent BRI1-BAK1 hetero-oligomers phosphorylate the RLCKs BSK1 and CDG1, which in turn activate BSU1; BSU1 dephosphorylates and inactivates BIN2. The inactivation of BIN2, coupled with the activities of a PP2A, allow for the accumulation of BZR1/BES1 in their dephosphorylated active forms. BZR1/BES1 can multimerize either on their own or together with other transcription factors to bind target promoters to either repress or activate the expression of hundreds of genes to optimize BR-regulated growth (see Figure 5). Once BR signals have been transduced, the leucine carboxylmethyltransferase SBI1 promotes PP2A association with the plasma membrane by methylating it [97]. Once at the membrane, PP2A triggers specific termination of BR signaling via BRI1 dephosphorylation and subsequent degradation [97]. The BL docking platform is depicted in yellow. BRI1 constitutively cycles between the plasma membrane and early endosomal compartments and can signal from either location (not shown here, [98]). BRI1 may also exist as pre-formed homo-oligomers at the cell surface [99]. For simplicity, we show BRI1 as a monomer. Phosphorylation events are indicated by the small white circles surrounded by a fluorescent red halo. The question mark highlights knowledge gaps in the signaling pathway. Abbreviations: BR, brassinosteroid; BRI1, BRASSINOSTEROID INSENSITIVE 1; BAK1, BRI1-ASSOCIATED KINASE 1; BKI1, BRI1 KINASE INHIBITOR 1; BSK1; BIN2, BRASSINOSTEROID INSENSITIVE 2; BZR1, BRASSINAZOLE RESISTANT 1 ; BES1, BRI1-EMS-suppressor 1; RLCK, receptor like cytoplasmic kinase; CDG1, Constitutive Differential Growth 1; SBI1, SUPPRESSOR OF bri1; BSU1; bri1 SUPPRESSOR 1; PP2A, PROTEIN PHOSPHATASE 2A.

Figure 3. A model of FLS2-controlled defense in Arabidopsis

A- In the absence of ligand, the signaling potential of FLS2 is kept in check by its association with KAPP [100]. In this signal-competent state, FLS2 associates with at least BIK1, BSK1 and RbohD. BIK1 is a member of the family of AvrPphB SUSCEPTIBLE1 (PBS1)-like proteins (PBLs). Inactive FLS2 cycles on and off the plasma membrane via Brefeldin A-sensitive vesicle transport [101]. PBLs belong to a small subfamily of acylated RLCKs. BAK1 also associates with BIK1 in the absence of flg22 and is sequestered away from FLS2 by BIR2. BSK1 (see Figure 2) also associates with FLS2 to positively regulate flg22-dependent signaling. B- As in BR signaling, flg22 detection triggers the very rapid formation of an FLS2-BAK1 hetero-oligomer. Flg22 binding allows the release of BAK1 from BIR2 and the dissociation or inactivation of KAPP phosphatases. The FLS2-BAK1 oligomer undergoes a series of trans-phosphorylation events and phosphorylates BIK1 at both tyrosine and serine/threonine residues. BIK1 auto-phosphorylates on multiple Tyr residues, trans-phosphorylates FLS2 and BAK1, and then dissociates from the complex. BIK1 targets at least the NADPH oxidase RBOHD for phosphorylation, activating an extracellular superoxide burst and intracellular calcium increase. Downstream of receptor activation, the MAP kinases MEKs, MKKs and MPKs and CDPKs are activated by an unknown mechanism and may contribute to induction of flg22-responsive genes. Once flg22 signals have been transduced, BSK1 partly dissociates from the receptor complex and BAK1 phosphorylates closely related PUBs, which in turn ubiquitylate FLS2 to regulate its abundance at the cell surface [100]. The flg22-docking platform is depicted in yellow in the concave side of FLS2 LRRs. Much as BRI1, FLS2 can exist as a homo-oligomer. For clarity we depict FLS2 as a monomer. Phosphorylation events are indicated by the small white circles surrounded by a fluorescent red halo. The question mark associated with dashed arrows highlights important knowledge gaps in the signaling pathway. Abbreviations: FLS2, FLAGELLIN SENSITIVE 2, KAPP, kinase-associated protein phosphatase; BIK1, BOTRYTIS INDUCED KINASE 1; BSK1; BR SIGNALING KINASE 1; RbohD, Respiratory burst oxidase homolog D; PBS1, AvrPphB SUSCEPTIBLE1; PBLs; (PBS1)-like protein; RLCKs, receptor like cytoplasmic kinases; BAK1, BRI1-ASSOCIATED KINASE 1; flg22, flagellin 22; BIR2, BAK1-interacting Receptor kinase 2; MAPK/MPKs, mitogen activated protein kinases; MEK/MKKs, mitogen activated protein kinase kinase; CDPKs, calcium-dependent protein kinases; PUBs, U-Box E3 ubiquitin ligase proteins.

Figure 4. Atomic structures of ligands, receptor and co-receptor LRR complexes

A- Comparison of BL (in blue) and flg22 (in red) atomic structures to scale. B- Top-down of BRI1 (in red) and FLS2 (in purple) ECDs in complex with their respective ligands. BL (in blue) interacts with a 70 amino-acid loop-out island domain (in yellow) between LRRs20–21 and lays on the surface of LRRs 21–25 (of 25 total LRRs). The flg22 (in red) binding platform is distributed across 14 LRRs (in yellow) of FLS2 (28 LRRs total). Flg22 establishes direct links with amino-acids that project their lateral chains from the concave side of the FLS2 LRR solenoid (LRR3–16; in yellow). The central part of flg22 interacts with the lateral chains of residues derived from FLS2 LRR2–6. FLS2 specifically recognizes the C- and N-terminal segment of flg22. C- The N-terminal capping domain of BAK1 (in black) is important for recognition of BL (in blue) and folds on top of the BRI1 steroid binding pocket (in yellow) where it establishes contacts with the BRI1 LRR 25 and with the hormone itself. Bulky amino acids distributed throughout the BAK1 ECD also establish an interaction with the BRI1 C-terminal capping domain. In complex with FLS2, the N terminus of BAK1 associates through two separate interfaces involving LRRs 18–20 and 23–26 of FLS2. Mutations of key amino acids in either of them greatly reduce the binding of BAK1 to FLS2. Abbreviations: BL, brassinolide; flg22, flagellin 22; BRI1, BRASSINOSTEROID INSENSITIVE 1; FLS2, FLAGELLIN SENSITIVE 2; ECDs, extracellular domains; LRRs, leucine-rich repeat; BAK1, BRI1-ASSOCIATED KINASE 1.

Figure 5. Network representation of BR and flg22 signaling crosstalk in the nucleus

Transcriptional regulation is the final level of complexity in the growth-defense nexus between BR signaling and MTI. BZR1 integrates BR signals to repress MTI. BZR1 forms a complex with WRKY40 to up-regulate the expression of additional WRKY transcription factors that suppress MTI. HBI1 is another transcription factor performing dual roles in promoting growth and suppressing defense. HBI1 activates an overlapping set of positive regulators of cell elongation with BZR1, and represses the expression of defense genes including some WRKYs. BZR1 and HBI1 form a positive feedback loop in which BZR1 activates PRE1 expression to block IBH1-mediated inhibition of HBI1; HBI1 subsequently activates BZR1 by promoting BR biosynthesis and signaling. However, the activation of FLS2-mediated MTI inhibits the transcription of HBI1, demonstrating an antagonistic crosstalk between BR signaling and MTI at the HBI1 node. Signaling events downstream of BRI1 and FLS2 are annotated in red and purple, respectively. The rounded green rectangle represents a nucleus. Abbreviations: BR, brassinosteroid; MTI, Microbes associated molecular patterns Trigerred Immunity, BZR1, BRASSINAZOLE RESISTANT 1 ; WRKY40 W-box binding transcritpion factor 40; HBI1, HOMOLOG OF BRASSINOSTEROID ENHANCED EXPRESSION 2 INTERACTING WITH IBH1; IBH1, ILI1-BINDING bHLH PROTEIN1 PRE1, PACLOBUTRAZOL RESISTANT1.