Membrane microdomain may be a platform for immune signaling

Abstract

Arabidopsis RPS2 is a typical disease resistance (R) protein with nucleotide-binding leucine-rich repeats (NB-LRR). Previously, we reported that RPS2 is physically associated with some Arabidopsis hypersensitive induced reaction (AtHIR) proteins, which are enriched in membrane microdomains. Biochemical and genetic analyses suggested that members of the AtHIR gene family have a function in RPS2-mediated immune signaling. Here, we provide evidence that the pattern recognition receptor (PRR) FLS2 is also physically associated with AtHIR2 in a N. benthamiana transient expression system. We thus speculate that PM microdomains provide a platform for both types of immune receptors, R proteins and PRRs, and that the activation of the receptors is facilitated by AtHIR proteins.

Pattern-Triggered Immunity and Effector-Triggered Immunity

Plants contain two distinct types of immune receptors. The first type, the PRR, recognizes microbe-associated molecular patterns (MAMPs) and initiates pattern-triggered immunity (PTI) upon recognition.^1-3 Arabidopsis FLS2 is a PRR, which recognizes a part of bacterial flagellin.^4,5 The second type, the R protein, recognizes pathogen effectors and initiates effector-triggered immunity (ETI),² which is also known as gene-for-gene resistance.⁶ The majority of R proteins belong to the NB-LRR class, which can be divided into two subclasses: the coiled coil (CC)-NB-LRR subclass and the Toll and interleukin-1 region (TIR)-NB-LRR subclass.⁷ Arabidopsis CC-NB-LRR R proteins RPM1,⁸ RPS2^9,10 and RPS5¹¹ are all associated with the PM.^12-14 Our recent finding that all three R proteins are physically associated with FLS2 suggests interplay between ETI and PTI signaling at a very early stage.¹⁵

Arabidopsis Hypersensitive Induced Reaction (AtHIR) Proteins are Members of RPS2 Protein Complexes

Through a biotinylated affinity tag-based approach,¹⁶ we identified a list of proteins as candidate members of RPS2 immune receptor protein complexes.¹⁷ Among them are two hypersensitive induced reaction (HIR) proteins (also known as band 7 proteins), AtHIR1 (At1g69840) and AtHIR2 (At3g01290). HIR genes from different plant species are known to be highly inducible upon pathogen challenge or during the hypersensitive response (HR), which is a rapid plant cell death during ETI.^18-21 A pepper HIR gene, CaHIR1, was found to be capable of inducing cell death when ectopically expressed in tobacco and Arabidopsis, and this phenotype was suppressed by its interactor CaLRR1, a small leucine-rich repeat protein.²⁰ A physical association between OsHIR1 and OsLRR1 was also reported.²² Ectopic expression of OsLRR1 in Arabidopsis greatly enhanced resistance to Pseudomonas syringae pv tomato DC3000 (Pst DC3000),²² while CaLRR1 had the opposite effect.²⁰ Despite the physical association with LRR1 proteins, it remains unclear how CaHIR1 or OsHIR1 are involved in PTI or ETI. In Arabidopsis, there are four AtHIR genes which are highly homologous to each other. The Arabidopsis gene most closely related to pepper CaHIR1 is AtHIR4 (At5g62740). In a recent report, we confirmed physical association between the AtHIR proteins and RPS2 in both Arabidopsis and N. benthamiana by pulldown and FRET analyses.²³ The physical association suggests that AtHIR proteins may play a role in RPS2-mediated ETI, which is supported by the genetic evidence that knocking out either AtHIR2 or AtHIR3 compromised RPS2-mediated ETI.²³ It is yet to be tested whether AtHIR genes are involved in the RPS2-mediated HR response. Since HIR genes are generally conserved in plants,^24,25 it will be interesting to test whether they participate in ETI mediated by other R proteins and/or in PTI.

AtHIR Proteins may be Important Structural Proteins for Plasma Membrane Microdomains

Protein localization often provides useful clues about function, which seems to be the case for AtHIR proteins. HIR proteins contain a SPFH domain which is conserved in both plants and animals.^25,26 An important function of SPFH domain-containing proteins appears to be assisting formation of microdomains in a variety of cell membranes, providing platforms for multiple biological processes.²⁶ We demonstrated that all four AtHIR proteins are localized to the PM in Arabidopsis.²³ Importantly, we confirmed that at least some AtHIR proteins are highly enriched in detergent resistant microdomains (DRMs), consistent with previous reports.^27-29 FRET analysis suggests that the RPS2-AtHIR1 protein association occurs in some punctate regions in the PM.²³ Interestingly, CaHIR1 also has a punctate membrane localization pattern.²⁴ Thus, both DRM fractionation and imaging results imply that AtHIR proteins are localized in membrane microdomains. In addition, all AtHIR proteins can form homo-oligomers as well as hetero-oligomers,²³ which not only points to a potential functional redundancy among the family members but also provides a structural basis for their function. When overexpressed in N. benthamiana or Arabidopsis, AtHIR1, AtHIR2 and AtHIR4 can form very stable SDS-, 2-mercaptoethanol- and heat-resistant oligomers.²³ The apparent very stable oligomer formation among AtHIR proteins suggests that AtHIR proteins may be important scaffold proteins for organizing membrane microdomains and/or recruiting other proteins to membrane microdomains.

Membrane Lateral Compartmentalization in Immune Signaling

Three recent proteomic studies suggest that plant membrane microdomains and their dynamics may play important roles in the plant immune response.^29-31 These studies not only identified many plant immune signaling proteins in DRMs, such as known and putative ETI and PTI receptors, but also demonstrated significant changes in the protein composition in DRMs upon elicitation by pathogen elicitors, such as chitin, cryptogein or flg22. For example, a study using Arabidopsis suspension cells revealed that 64 proteins showed significant enrichment in the DRM fraction 15 min after flg22 treatment.²⁹ Among them were FLS2 and three AtHIR proteins (AtHIR1, AtHIR2 and AtHIR3). This observation matches nicely with our finding that these three AtHIR genes were all significantly induced by flg22.²³ Since FLS2 can form a protein complex with RPS2,¹⁵ we wondered whether FLS2 also physically associates with HIR proteins. We thus tested a possible physical association between FLS2 and AtHIR2, which is the most abundant AtHIR protein in Arabidopsis.²³ Myc-tagged AtHIR2 protein could indeed be pulled down by HPB-tagged FLS2 after expression of both proteins in N. benthamiana (Fig. 1). Agrobacterium-mediated transient expression in N. benthamiana is likely a PTI-activated condition. It is thus plausible that AtHIR proteins, upon pathogen challenge, may recruit ETI receptor RPS2 and PTI receptor FLS2 to membrane microdomains.

Figure 1. AtHIR2-Myc was pulled down by FLS2-HPB after expression in N. benthamiana. Both HIR2-Myc and FLS2-HPB were expressed under their own promoters in N. benthamiana leaves through Agrobacterium-mediated transient expression, and subsequently a streptavidin bead-based pulldown assay was performed. N. benthamiana leaves expressing only AtHIR2-Myc were used as a negative control. Both pulldown samples and input samples were analyzed by immunoblotting using anti-HA and anti-Myc antibodies. No cross-linking was used. The washing buffers used contained 1% Nonidet P-40, 0.5% sodium deoxycholate and 150 and 50 mM NaCl.²³ SDS-, 2-mercaptoethanol- and heat-resistant oligomers of AtHIR2 proteins are indicated with asterisks. This experiment was performed twice with similar results.

Many questions remain to be answered, such as: (1) Is the physical association between AtHIR proteins RPS2 or FLS2 through direct protein-protein interaction? (2) Do these proteins form a large protein complex or multiple different protein complexes? (3) If the physical association is indirect, what are the other proteins involved and how do they contribute to making such potentially large protein complexes? In this context, it is worth pointing out similarities between AtHIR proteins and Remorin proteins in the capability of high-order oligomerization, enrichment in DRMs, differential expression upon pathogen challenges, interactions with immune receptors and the potential functional redundancy among family members.^32-36 In summary, a variety of results suggest that dynamics of PM microdomain organization upon pathogen challenge are likely important in very early immune signaling in plants.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.