Investigating the functions of the RIN4 protein complex during plant innate immune responses

Abstract

Pathogen recognition by the plant innate immune system invokes a sophisticated signal transduction network that culminates in disease resistance. The Arabidopsis protein RIN4 is a well-known regulator of plant immunity. However, the molecular mechanisms by which RIN4 controls multiple immune responses have remained elusive. in our recently published study, we purified components of the RIN4 protein complex from A. thaliana and identified several novel RIN4-associated proteins.¹ we found that one class of RIN4-associated proteins, the plasma membrane H⁺-ATPases AHA1 and AHA2, play a crucial role in resisting pathogen invasion. Plants use RIN4 to regulate H⁺-ATPase activity during immune responses, thereby controlling stomatal apertures during pathogen attack. Stomata were previously identified as active regulators of plant immune responses during pathogen invasion, but how the plant innate immune system coordinates this response was unknown.^2,3 Our investigations have revealed a novel function of rin4 during pathogenesis. Here, we discuss the rin4-AHA1/2 interaction and highlight additional RIN4-associated proteins (RAPs) as well as speculate on their potential roles in plant innate immunity.

RIN4 is an Important Negative Regulator in Plant Innate Immunity

In order to resist pathogen infection, plants evolved a multilayered innate immune system to actively monitor and defend against pathogen attack.^4,5 In essence, there are two primary branches of plant innate immunity. One branch recognizes conserved pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin or fungal chitin, resulting in PAMP-triggered immunity (PTI).^4,5 PTI acts to inhibit initial pathogen colonization and entry. A second line of defense utilizes disease resistance (R) proteins that recognize cognate pathogen effector proteins delivered inside plant cells during infection, resulting in effector-triggered immunity (ETI). In the absence of R proteins, these effector proteins contribute to pathogen virulence by disrupting plant innate immune signaling pathways.⁶ However, in the presence of a cognate R protein, specific effectors are recognized, leading to ETI-based defense signaling and disease resistance.

RIN4 is a well-studied Arabidopsis protein that is targeted by multiple bacterial effectors and controls aspects of both PTI and ETI-based responses to the bacterial speck pathogen Pseudomonas syringe pv. tomato.^7–10 Overexpression of RIN4 reduces PTI responses in Arabidopsis while rin4 mutant lines exhibit enhanced PTI responses.⁷ Although these data are consistent with RIN4 being a negative regulator of PTI, the mechanisms by which this regulation occurs have been difficult to uncover. In addition to regulating PTI, RIN4 also acts to negatively regulate ETI. RIN4 associates with and is monitored for effector-induced perturbations by at least two R proteins: RPS2 and RPM1. RIN4 proteolytic cleavage by the P. syringae effector protease AvrRpt2 is recognized by RPS².^8,10,11 Delivery of the P. syringae effectors AvrB or AvrRpm1 induce RIN4 phosphorylation and this specific modification of RIN4 activates RPM1.⁹ Activation of either RPS2 or RPM1 results in rapid initiation of ETI defense signaling, restricting further pathogen colonization of plant leaves. RIN4’s association with these R proteins in the absence of the cognate effectors acts to limit inappropriate ETI signaling. The identification of RIN4 as a negative regulator of both PTI and ETI uniquely positions this protein as a point of convergence of both branches of the plant innate immune system.

Purification of RIN4 Interacting Proteins

Upon pathogen perception, defense signal transduction is achieved via dynamic adjustments in the cellular proteome that in turn coordinate appropriate immune responses. Protein complex formation and function is an important emerging concept in the coordination of immune system signaling. In order to begin to elucidate RIN4 complex constituents, we purified and identified components of the RIN4 protein complex in the absence of pathogen perception.¹ While many studies have used heterologous yeast two-hybrid screening or various TAP (tandem affinity purification)-tagging approaches in planta to identify novel interacting proteins, we chose a different approach to isolate RIN4 and associated proteins. In our in planta precipitation experiments, we used affinity-purified RIN4 antibodies to capture proteins under native expression conditions. In addition, we used as few steps as possible during washing and elution steps in order to preserve complex integrity during precipitation. RIN4 expression is relatively high in plant cells, and the protein can be easily detected by immunoblotting, which was a contributing factor in the success of our approach. We immunoprecipitated RIN4 from leaf total protein extracts and used high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to directly probe our samples for RIN4-asscoiated proteins. A rin4 knockout line was used as a negative control for non-specific binding. In total, six novel RIN4-associated proteins were reproducibly identified in the experimental sample but never in the negative control (Table 1). Most of these proteins are predicted to localize to the plasma membrane, which is where RIN4 resides, further supporting the validity of our approach. A more detailed discussion of several identified proteins is described below.

Table 1.

RIN4-associated proteins (RAPs) and their interactions with RIN4

Protein	Gene identifier	Accession number (IPI)	rps2 RPS2:HA (1)	rps2 RPS2:HA (2)	rps2 RPS2:HA (3)
RIN4	At3g25070	IPI00517440	7	9	8
RPS2	At4g26090	IPI00547830	0	3	2
AHA1 or AHA2	At2g18960	IPI00526113	13	8	4
	At4g30190
ERD4	At1g30360	IPI00526219	5	4	2
Remorin	At3g61260	IPI00539947	2	3	2
MATH domain	At3g28220	IPI00528031	2	6	12
Jacalin domain	At1g52000	IPI00531879	2	2	2
Jacalin domain	At3g16420	IPI00543838	2	6	6

Counts are the sum of three biological replications (except RPS2, identified in only two replications). Protein identification required p < 0.05 (MOWSE algorithm), minimum 2 peptides. The number of unique peptides identified for each protein is listed. none of these proteins were identified in the negative control (rps2-101clrin4 knockout line). ^aSource: reference 1, used by permission.

PM H⁺-ATPases AHA1 and AHA2

Plasma membrane (PM) H⁺-ATPases are integral membrane proteins that function as proton pumps and are responsible for the creation of H⁺ gradients across the plasma membrane.¹² There are 11 PM H⁺-ATPase isoforms encoded by AHA genes and their expression patterns are differentially distributed in the plant.¹³ AHA1 and AHA2, the only PM H⁺-ATPases identified by mass spectrometry, are primarily expressed in guard cells at the RNA level and have been shown to regulate guard cell membrane electrochemical potential.¹⁴ Hyperpolarization of the plasma membrane is achieved via activation of PM H⁺-ATPases and this gradient is responsible for ion uptake and subsequent water flow into the cell. Thus, activation of the PM H⁺-ATPases causes guard cells to swell, and the resulting increase in turgor induces stomatal opening. Conversely, inactivation of PM H⁺-ATPases results in depolarization of the membrane and leads to stomatal closure.

Recently, it has been demonstrated that plants actively regulate stomatal apertures during pathogen attack.² Upon application of bacteria or purified PAMPs, plants will quickly close their stomata within one hour. However, virulent P. syringae bacteria are able to induce stomatal re-opening within three hours, thus facilitating bacterial entry into leaf apoplasts.² Interestingly, the phytotoxin fusicoccin from the fungal pathogen Fusicoccum amygdali is a well-known activator of the PM H⁺-ATPases and induces the opening of stomata.^15–18 Multiple other pathogenic microbes can manipulate stomatal apertures, indicating that modulation of stomata is a common pathogenic mechanism.^3,19–21 Given the body of evidence for AHA control of stomata and the recent data implicating stomata as regulators of pathogen entry into leaf tissue, we decided to pursue the RIN4-AHA association in more detail.

In our recent publication,¹ we verified the RIN4-AHA1/2 interaction in yeast and in planta using BiFC (Bimolecular Fluorescence Complementation). Our yeast two-hybrid data indicates that RIN4 associates with the cytosolic C-terminal regulatory domain of AHA1 and AHA2 proteins. Constitutively active AHA1 lines (ost2-1D and ost2-2D) were examined.¹⁴ These gain-of-function mutants have an open stomata phenotype and we found that these lines are more susceptible to spray inoculation of bacteria onto the leaf surface than wild-type plants.¹ Importantly, these mutants support similar levels of bacterial growth as wild-type plants when bacteria are syringe-infiltrated directly into the leaf apoplast. This suggested that the open stomata phenotype of AHA1 constitutively active mutants facilitates bacterial entry into leaves via unregulated, open stomata. These data indicate that proper regulation of PM H⁺-ATPases is critical for plants to regulate stomatal apertures during pathogen attack.

PM H⁺-ATPase activity in RIN4-overexpression and rin4 knockout lines is altered. Overexpression of RIN4 in Arabidopsis resulted in increased PM H⁺-ATPase activity while rin4 knockout lines displayed reduced PM H⁺-ATPase activity as compared with wild-type plants.¹ We also found that recombinant RIN4 protein can stimulate PM H⁺-ATPase activity in vitro. Intriguingly, the stomata of rin4 mutants cannot be reopened by virulent Pst DC3000, which normally causes the stomata of wild type plants to re-open by 3 hours.¹ These results suggest that RIN4 is a positive regulator of the PM H⁺-ATPases and plants use RIN4 to modulate stomatal apertures during pathogen attack. These findings are beginning to uncover the molecular basis for RIN4 regulation of PTI at the level of pathogen invasion.

Although our investigations have led to the discovery of a novel way in which plants regulate stomatal apertures during pathogen invasion, the exact molecular mechanisms RIN4 employs to stimulate PM H⁺-ATPase activity is still unclear. Plant PM H⁺-ATPases possess an extended C-terminus that acts as a negative regulator of the pump.²² Phosphorylation is one of the main mechanisms by which the PM H⁺-ATPase is regulated and depending upon the phosphorylation site, can induce both activation and inactivation.^23–25 One of the best-understood mechanisms of regulation involves phosphorylation of the penultimate threonine residue. Phosphorylation of this residue recruits the binding of 14-3-3 proteins which induces a conformational change, displacing the autoinhibitory C-terminal domain and activating the PM H⁺-ATPase.²² Interestingly, RIN4 can directly interact with the C-terminal region of both AHA1 and AHA2.¹ RIN4 is also phosphorylated during ETI and may be differentially phosphorylated during PTI.^9,26 Upon PAMP perception, RIN4 may be post-translationally modified or dissociate from AHA1 and AHA2. It is possible that differential phosphorylation of RIN4 may also control its association with AHA1 and AHA2. It will be important to investigate the mechanism(s) RIN4 uses to regulate PM H⁺-ATPase activity.

MATH Domain Protein

We also identified a Meprin and TRAF-Homology (MATH) domain protein (At3g28220) as a RIN4-associated protein by mass spectrometry (Table 1). This interaction was confirmed by yeast two-hybrid analysis (data not shown). MATH domains are characterized by a conserved 180 amino acid region that is present in mammalian meprins (a family of extracellular metalloproteases) and TRAF proteins [tumor necrosis factor (TNF) Receptor Associated Factor].²⁷ The MATH domain is a fold of seven antiparallel β-helices which is involved in protein-protein interactions and substrate recognition. A large family of proteins containing MATH domains in plants also possess a BTB domain, which acts as a substrate adapter for ubiquitin ligase E3 complexes.²⁸ The At3g28220 gene we identified contains two MATH domains separated by a short linker, but does not possess a BTB domain. Our preliminary analyses indicate that At3g28220 mRNA expression is modulated in response to pathogen perception (data not shown), and it will be interesting to uncover the functions of this protein and other MATH domain proteins in plant innate immune responses.

Remorin

A remorin, At3g61260, was also identified as being RIN4- associated in our proteomic analysis (Table 1). Remorins are plant-specific proteins that share some physical properties with viral movement proteins due to the presence of a hydrophobic N-terminal region. The C-terminal remorin domain is present in all remorin proteins and consists of multiple coiled-coiled domains.²⁹ The remorin identified in our recent publication¹ is a canonical remorin possessing both an N-terminal and a C-terminal PFAM domain (Remorin_N: PF03766; Remorin_C: PF03763). Although remorins are hydrophilic in composition, they are consistently found to be associated with plasma membrane fractions and are enriched in lipid rafts.^29,30 Despite the prevalence of remorins in vascular plants, there was no genetic or biochemical dissection of its function until earlier this year. Excitingly, Raffaele and colleagues³⁰ found that a remorin from tomato is enriched in lipid rafts and also specifically associates with plasmodesmata, a key channel for viral cell-tocell movement. This tomato remorin can directly interact with a viral movement protein and is likely involved in interfering with cell-to-cell movement of viral proteins during infection.³⁰ A recent proteomic study has also identified the same remorin (At3g61260) from Arabidopsis as upregulated upon RPM1 activation by inducible expression of the P. syringae effector AvrRpm1 in the Col-0 ecotype.³¹ As described above, delivery of AvrRpm1 induces RIN4 phosphorylation which results in RPM1 activation.⁹ It is tempting to speculate that this remorin may be involved in RPM1-mediated resistance or may be a virulence target of the AvrRpm1 effector. Future investigations as to why RIN4 associates with this remorin will be particularly interesting.

Conclusions and Future Directions

Protein complex formation and function are critical aspects of plant innate immunity. The Arabidopsis protein RIN4 is an important negative regulator of plant defense responses and we have successfully identified novel RIN4-associated proteins. These investigations are providing a more complete picture of how the plant innate immune system functions. Exactly how RIN4 is manipulated to the pathogen’s advantage remains to be determined, but evidence into RIN4’s regulation of PTI indicates that it may be a bona fide virulence target of bacterial effectors.

We hypothesize that RIN4 exists in compositionally and functionally distinct protein complexes within the cell. As described above, RIN4 post-translational modification by the pathogen effectors AvrRpt2, AvrB and AvrRpm1 is monitored by the R proteins RPS2 and RPM1. In addition, RIN4 interacts with NDR1, a positive regulator of immune signaling mediated by R proteins with an N-terminal coiled-coiled domain that includes both RPS2 and RPM1.^32–34 Experimental evidence in support of distinct RIN4 protein complexes has been uncovered using co-immunoprecipitation experiments.^9,10,34 Many questions arise from these observations. How do multiple RIN4 complexes function to control both PTI and ETI-based responses? How are these complexes assembled and regulated? Finally, what dynamic changes in RIN4 protein complex composition and localization occur during activation of immune signaling? It will be exciting to determine the role of this fascinating protein during plant immune responses to different pathogens and in different plant species.

Abbreviations

RIN4

RPM1-interacting protein 4

RAP

RIN4-associated protein

PTI

PAMP-triggered immunity

ETI

effector-triggered immunity

MS

mass spectrometry

PM

plasma membrane