Stack heterotrimeric G proteins and MAPK cascades on a RACK

Summary

Heterotrimeric G proteins are molecular switches that relay intracellular signaling in eukaryotes. Recent studies in plant immunity provide a link between heterotrimeric G proteins and a MAPK cascade via the RACK1 scaffolding proteins. Research also points to a potential regulation of G proteins by cell surface receptors.

The heterotrimeric G protein complex composed of Gα, Gβ, and Gγ subunits serves as one of the key signal transducers in eukaryotes. In animals, heterotrimeric G proteins are activated by G protein-coupled receptors (GPCRs) that perceive various extracellular signals. In the absence of a stimulus, the GDP-bound Gα monomer and Gβγ dimer form an inactive heterotrimer in complex with a GPCR. Ligand binding to the GPCR triggers the exchange of GDP with GTP in Gα and subsequent dissociation of Gα from Gβγ, both of which further interact with a subset of effector proteins to activate downstream signaling cascades (Urano and Jones, 2014).

G protein signaling in plants is distinct from that in animals in terms of the composition of the G protein complexes, activation mechanisms, and biological functions. Compared to a large repertoire of G protein components in animals, Arabidopsis has one Gα (GPA1), one Gβ (AGB1) and three Gγ (AGG1, AGG2 and AGG3) (Urano and Jones, 2014). The signaling mechanism of plant G proteins also appears to be different from that in animals. Arabidopsis GPA1 spontaneously releases GDP and binds GTP in vitro, suggesting that plant G proteins could be self-activated without GPCRs. In addition, plants do not have classical GPCRs (Urano and Jones, 2014).

Plant G protein signaling has been implicated in many physiological responses, including plant immunity. In particular, Arabidopsis agb1 mutants are susceptible to many necrotrophic fungal pathogens (Urano and Jones, 2014). Recent studies have provided a mechanistic understanding of Arabidopsis G proteins in regulating plant immune signaling, and hinted at a potential novel mechanism of G protein activation by cell surface receptors (Cheng et al., 2015; Liu et al., 2013; Maruta et al., 2015).

Plants recognize microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) and trigger a series of convergent defense responses including activation of mitogen-activated protein kinases (MAPKs) and production of reactive oxygen species (ROS) (Bigeard et al., 2015). However, the molecular link between PRRs and MAPK activation remains elusive. Work from Cheng et al. shows that in Arabidopsis, pathogen-secreted proteases activate a MAPK cascade lined to heterotrimeric G proteins via the scaffold protein Receptor for Activated C Kinase 1 (RACK1) (Cheng et al., 2015). Identification of the scaffolding role of RACK1 that brings together G proteins and a MAPK cascade not only reveals a novel signaling pathway activated by pathogen elicitors, but also provides new insights for the regulation and specificity of MAPK activation.

Using a β-glucuronidase (GUS) reporter driven by the pathogen-inducible CYP71A12 promoter, Cheng et al. identified a type II-secreted protease IV encoded by the prpL gene from Pseudomonas aeruginosa, a bacterial pathogen with a broad host range from plants to animals and humans, as an elicitor of plant immunity. Similar to many MAMPs, such as bacterial flagellin, PrpL and its closest homologue ArgC protease from Xanthomonas campestris activate two pathogen-responsive MAPKs, MPK3 and MPK6, an ROS burst, and defense genes in Arabidopsis. Because the protease activity of PrpL/ArgC is indispensable to trigger defense responses, it is likely that an unidentified host target cleaved by protease IV, not the protease itself, is perceived by Arabidopsis PRR(s) and activates immune signaling (Cheng et al., 2015). Alternatively, the proteases may directly act on PRRs and the PRR cleavage products induce host immunity.

In search of components required for PrpL/ArgC-triggered immunity, Cheng et al. found that G proteins are involved. Mutation of Gα (GPA1) but not Gβ (AGB1) impaired the PrpL-triggered ROS burst, whereas mutations in both Gα and Gβ, but not in either single gene, reduced PrpL/ArgC- triggered activation of MAPKs, suggesting that Gα and Gβ are important players with distinct as well as overlapping functions in PrpL/ArgC-triggered immunity likely upstream of the MAPK cascade (Cheng et al., 2015). The result is reminiscent of a previous report showing that rice OsGα is important for sphingolipid elicitor-induced OsMPK6 activation. However, the precise rice G protein signaling may be distinct, as the small GTPase OsRac1 directly interacts with OsMPK6 and likely associates with heterotrimeric G proteins and MAPKs (Lieberherr et al., 2005).

Independently, Arabidopsis G proteins have been implicated in plant immunity mediated by multiple receptor-like kinases (RLKs) including FLS2, EFR and CERK1, which recognize bacterial flagellin, elongation factor-Tu (EF-Tu) and fungal chitin, respectively (Liu et al., 2013). However, in contrast to the PrpL/ArgC proteases, flagellin, EF-Tu or chitin-induced ROS burst was reduced in a gβ single or gγ1/gγ2 double mutant but not a gα mutant, whereas MPK3/MPK6 activation was not affected in any of the G protein mutants (Liu et al., 2013), indicating that Gβγ are important players in relaying signaling triggered by these MAMPs, but likely independently or downstream of the MAPK cascades. These data suggest that G proteins function differently in protease-mediated signaling compared to signaling via canonical RLKs such as FLS2, EFR and CERK1.

In addition to the canonical Gα subunit, Arabidopsis also contains three Gα-like extra-large G proteins (XLG1, XLG2 and XLG3), which were recently found to complex with the Gβγ dimer. Furthermore, an xlg2 single mutant or xlg2 xlg3 double mutant resembles gβ and gγ mutants in pathogen resistance and the flagellin/EF-Tu-mediated ROS burst. It is possible that XLGs couple with the Gβγ dimer in mediating plant immunity triggered by these MAMPs (Maruta et al., 2015). Apparently, XLGs add another layer of diversity and specificity of plant G protein signaling, which may account for the differential requirement of G protein subunits in diverse signaling pathways.

Another important discovery by Cheng et al. is that RACK1 proteins (RACK1A, RACK1B and RACK1C) function as MAPK scaffold proteins and link upstream G proteins to a downstream MAPK cascade in PrpL/ArgC protease-triggered immunity (Cheng et al., 2015) (Fig. 1). MAPK scaffold proteins regulate signal specificity, amplitude and duration in yeast and animals (Su et al., 2015). However, the requirement and activation mechanisms of MAPK scaffold proteins remained unknown in plants. Cheng et al. show that RACK1 interacts with Gβ and all three tiers of a pathogen-responsive MAPK cascade consisting of MEKK1, MKK4/MKK5 and MPK3/MPK6 in the absence of PrpL/ArgC proteases. Upon protease treatment, the activated MAPK cascade is released from the complex to execute its cellular functions. Knocking-down of all three rack1 genes diminished PrpL/ArgC-triggered MPK3/MPK6 activation and defense gene induction. In contrast, flagellin-induced immune responses and immunity were not affected by silencing of rack1 genes, suggesting that RACK1 proteins specifically function in the PrpL/ArgC-triggered but not flagellin-triggered pathway (Cheng et al., 2015). Although scaffold proteins have been well studied in yeast and animals, RACK1 is the first identified MAPK scaffold protein in plants (Su et al., 2015). In rice, OsRACK1A regulates immune responses via interaction with the small GTPase OsRac1 (Nakashima et al., 2008). As mentioned above, the OsRac1 immune complex also contains OsMPK6 and heterotrimeric G proteins (Lieberherr et al., 2005). It remains unknown whether there is a direct interaction between OsRACK1A and OsMPK6. MAPK cascades function as a convergent point downstream of multiple PRRs. The discovery of RACK1 as a scaffold protein in PrpL/ArgC protease-, but not flagellin-triggered immunity, explains the signaling specificity of MAPK activation. A similar mechanism may account for the regulation of diverse functions of Arabidopsis MAPK cascades in different biological processes.

Figure 1. Heterotrimeric G protein signaling in plant immunity.

Recognition of bacterial PrpL/ArgC proteases or their cleavage products by host sensory proteins activates a heterotrimeric G protein complex composed of Gα, Gβ and Gγ, which in turn activates a MAPK cascade consisting of MEKK1/MTKs-MKK4/5-MPK3/6. RACK1 functions as a scaffold protein linking the G protein complex to the MAPK cascade in PrpL/ArgC-triggered immune signaling. Recognition of bacterial flagellin by the cell surface-resident FLS2-BAK1 complex activates a heterotrimeric G protein complex composed of XLGs, Gβ and Gγ and MAPK cascades separately. The XLGs/Gβγ complex is not required for flagellin-mediated MAPK activation. MTK: MAPKKK.

Apparently, heterotrimeric G proteins regulate both MAPK-dependent and MAPK-independent signaling pathways in Arabidopsis immunity (Fig. 1). The XLGs/Gβγ complex mediates immune responses downstream of multiple RLKs likely independently of MAPK cascades (Liu et al., 2013; Maruta et al., 2015), whereas the canonical Gα/Gβγ complex regulates MAPK activation and other immune responses induced by PrpL/ArgC proteases (Cheng et al., 2015). Identification of PrpL/ArgC protease host targets that trigger immune responses should provide new insights into the mechanisms by which plant heterotrimeric G proteins can be activated, especially during immune responses. Perhaps because plants lack classical GPCRs, they have evolved a large number of cell surface-resident RLKs and receptor-like proteins (RLPs) that perceive different extracellular and intracellular stimuli in regulating development and immunity. It has been postulated that plant RLKs or RLPs may function as counterparts of animal GPCRs (Bommert et al., 2013). Maize Gα functions in RLK CLAVATA1-mediated shoot meristem development via direct interaction with CLAVATA2, a RLP in the CLAVATA receptor complex (Bommert et al., 2013). In addition, Arabidopsis heterotrimeric G proteins directly interact with some PRR RLKs, such as CERK1 and BAK1, a coreceptor of several different PRRs, suggesting that heterotrimeric G proteins can relay plant immune signaling directly from PRR complexes (Aranda-Sicilia et al., 2015) (Fig. 1). It remains to be investigated whether and how RLK complexes modulate heterotrimeric G protein activity and how heterotrimeric G proteins determine signaling specificity.