MOS6 and TN13 in plant immunity

ABSTRACT

The Arabidopsis nuclear transport receptor IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for constitutive defense responses of the auto-immune mutant snc1 (suppressor of npr1-1, constitutive 1) and contributes to basal disease resistance, suggesting a role in nuclear import of defense-regulatory cargo proteins. We recently showed that MOS6 selectively interacts with TN13, a TIR-NBS protein involved in basal resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the effectors AvrPto and AvrPtoB. Consistent with a predicted N-terminal transmembrane domain, TN13 localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE) where it interacts with MOS6 in a transient expression assay. Here, we propose a model that summarizes the subcellular localization, association and function of TN13 and MOS6 in plant defense signaling.

Plants rely on two classes of immune receptors to perceive the presence of pathogen-derived molecules and trigger downstream defense signaling. The first class consists of plasma membrane-localized pattern recognition receptors (PRRs) that directly recognize pathogen-associated molecular patterns (PAMPs) via extracellular ligand recognition domains and activate PAMP-triggered immunity (PTI).¹ Recognition of PAMPs by PRRs is usually sufficient for defense against non-adapted pathogens. Host-adapted virulent pathogens however are able to suppress PTI by secretion of effector molecules, leading to effector-triggered susceptibility (ETS) of the infested host plants.² In such compatible interactions, PRRs still confer weak immune responses that contribute to the ability of susceptible plants to reduce the disease severity of virulent pathogens, and is referred to as basal defense or basal resistance.²

Intracellular nucleotide binding site-leucine rich repeat proteins (NBS-LRRs or NLRs) represent the second class of plant immune receptors that perceive pathogen-derived effector molecules inside host cells.³ Effector recognition occurs either by direct association, or indirectly by sensing effector modifications of a guarded host protein that can have a direct function in host defense (termed guardee) or be a non-functional mimic of an intended effector target (termed decoy).^2,4 Canonical NLRs are characterized by a common domain organization with a central NBS, C-terminal LRRs and a varying N-terminal Toll/Interleukin-1 receptor (TIR) or coiled-coil (CC) domain.^3,5 Recognition of effectors by TIR-NBS-LRRs (TNLs) or CC-NBS-LRRs (CNLs) typically induces a strong immune response termed effector-triggered immunity (ETI), albeit a defense reaction in form of a PTI-like “weak ETI” has also been proposed for the detection of evolutionary ancient effectors and may contribute to basal resistance.^2,6 Beside full-length TNLs and CNLs, plant genomes also encode truncated NLR proteins that lack one or two of the canonical domains, including TN proteins lacking an LRR domain, TIR-only and TIR-unknown site/domain (TX) proteins,^7-9 some of which have a reported function in immunity.^10-13 In addition, it has become evident that some NLRs can form homomeric complexes or heteromeric associations with other canonical or truncated partner NLRs. In heteromeric NLR complexes, the two partner NLRs cooperate functionally in effector detection (termed sensor NLRs) and initiation of downstream ETI signaling (termed executor NLRs), and are often encoded by genes that are physically linked on the chromosome.^14,15 Both effector- and PAMP-triggered immune responses are highly dynamic processes and involve multiple cellular compartments and organelles. In particular, the translocation of NLRs, signal transducers and transcriptional regulators into the nucleus has emerged as an essential regulatory mechanism for host cell transcriptional reprogramming and defense activation.^16-20

Nuclear import of proteins >40–60 kDa generally requires nuclear transport receptors (NTRs) of the importin-α/β class.^21,22 Importin-α proteins act as adapters that directly bind the nuclear localization signal (NLS) of cargo proteins and connect the importin-α/cargo complex to importin-β in the cytoplasm. Importin-β subsequently facilitates passage of the ternary import complexes through nuclear pore complexes (NPCs) that span the double membrane of the nuclear envelope (NE).^23,24 In Arabidopsis, IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine predicted α-importins.²⁵ MOS6 is required genetically for auto-immunity of the deregulated TNL mutant snc1 (suppressor of npr1-1, constitutive 1) and contributes to basal resistance to the oomycete Hyaloperonospora arabidopsidis Noco2 and the mildly virulent bacterium Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the effectors AvrPto/AvrPtoB, suggesting that MOS6 shuttles defense-regulatory cargo proteins into the nucleus.^25-27

In a recent effort to identify and characterize cargo proteins and interaction partners of MOS6 with relevance to plant immunity, we uncovered the truncated NLR protein TIR-NBS13 (TN13) that selectively binds to MOS6 in planta, but not to its closest homolog IMPORTIN-α6, and is required for basal resistance to Pst DC3000 ΔAvrPto/AvrPtoB.²⁸ TN13 has a predicted N-terminal transmembrane (TM) domain and accordingly, TN13 localizes to the endoplasmic reticulum (ER) and the NE, where it also interacts with MOS6 in a transient interaction assay in Nicotiana benthamiana.²⁸

Here, we present our current working model that summarizes the function, subcellular localization and association of TN13 and MOS6 in plant immunity (Fig. 1): In unchallenged tissues, TN13 localizes to the ER membrane and the NE through its N-terminal TM domain, where it associates with MOS6 in a preformed complex at the cytoplasmic side of the ER via its C-terminal bipartite NLS. Upon pathogen stimulus, TN13 is released from the ER membrane by a hypothetical protease. Accordingly, putative cleavage sites for cysteine-, metallo- and serine-proteases are predicted in the primary amino acid sequence of TN13, including the region between the predicted TM and TIR domains (https://prosper.erc.monash.edu.au).²⁹ As TN13 binds to the Pst effector HopY1 in a yeast two-hybrid assay,⁹ and TN13 is required for basal resistance to Pst DC3000 ΔAvrPto/AvrPtoB,²⁸ the interaction of TN13 with HopY1 (possibly as part of a heteromeric NLR complex), or activation of a basal defense pathway are two potential stimuli that trigger release of TN13 from the ER membrane by proteolytic cleavage. Release of the TN13-MOS6 complex from the ER membrane might allow access of another MOS6 molecule to the second NLS at the N-terminus of TN13 and accelerate nuclear import by MOS6 and importin-β. The formation of a preformed TN13-MOS6 nuclear import complex at the ER membrane provides the opportunity of rapid stimulus-induced transport into the nucleus, where TN13 may activate defense responses.

Figure 1.

MOS6 and TN13 in plant immunity. (A) Schematic representation of the predicted protein domain structure of TN13. Domains were predicted as previously described.²⁸ NBS, nucleotide binding site; NLS, bipartite nuclear localization signal; TIR, Toll/Interleukin-1 Receptor homology domain; TM, transmembrane domain. (B) Hypothetical model summarizing the function, subcellular localization and association of TN13 and MOS6 in plant immunity. TN13 localizes to the ER membrane and the nuclear envelope through its N-terminal TM domain, where it associates with MOS6 in a preformed complex at the cytoplasmic side of the ER via its C-terminal bipartite NLS. Upon pathogen stimulus the TN13-MOS6 complex is released from the ER membrane allowing access of MOS6 to the second NLS at the N-terminus of TN13 to promote nuclear import together with Importin-β (IMP-β). Inside the nucleus TN13 could activate defense responses. Whether the Pseudomonas effector HopY1, which was shown to associate with TN13 in a yeast two-hybrid assay,⁹ or activation of a basal defense pathway triggers changes in the subcellular localization of TN13 remains to be established. ER, endoplasmic reticulum; PM, plasma membrane; PRR, pattern recognition receptor; TF, transcription factor; TTSS, type III secretion system.

Our current research efforts probe the proposed model and aim to understand the spatiotemporal localization and association dynamics of TN13 in Arabidopsis responding to pathogen challenge. By analyzing biochemical and genetic interactors of TN13 we aim to gain further insights into the specific function of TN13 in plant cellular defense signaling.

Funding Statement

This work was supported by the Deutsche Forschungsgemeinschaft (grants WI3208/4-2 & IRTG2172: PRoTECT).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.