Cyril Zipfel

Plant receptor kinase mediated innate immunity

Surface-localized receptor kinases as major plant PRRs

Plants lack the adaptive immunity mechanisms of jawed vertebrates, so rely solely on innate immune responses for defence. In addition, plants do not have circulating cells specialized in microbe recognition, such as macrophages. Instead, each cell is able to recognize and respond to pathogens autonomously. Plants employ a multi-layered innate immune system to fight disease. The first layer involves the perception of conserved microbial elicitors that are characteristic of a given group of microbes, known as pathogen/microbe-associated molecular patterns (P/MAMPs) by surface-localized pattern recognition receptors (PRRs). PRRs also perceive host signals released under stress conditions known as damaged-associated molecular patterns (DAMPs). Activation of PRRs results in PAMP-triggered immunity (PTI) and acts as an early warning system against invading non-self that is sufficient to fend off most microbial infections. Adapted pathogens must quell these early defence responses by escaping or suppressing PTI through the activity of secreted effector proteins. Intracellular Resistance (R) proteins, that are homologous to mammalian nucleotide-binding domain and leucine-rich repeat (NLR) proteins, detect - directly or indirectly - specific effectors and mediate the second layer of plant innate immunity, effector-triggered immunity. These events reflect the dynamic interplay between plants and microbes, where PTI probably constitutes the initial driving force in the evolutionary arms race between plants and their pathogens.

Surface-localized receptor kinases as major plant PRRs

Over the last two decades, plant scientists were at the forefront of the identification and characterization of microbial elicitors. Therefore, dozens of PAMPs from bacteria, fungi and oomycetes that activate plant immune responses are known. In contrast, the number of known plant PRRs is still very limited. Plant PRRs are either receptor kinases (RKs) or receptor-like proteins (RLPs) (Figure 1).

Figure 1: Plant PRRs

In contrast to mammals, Toll-like receptors (TLRs) do not exist in plants and no cytoplasmic PRR has been identified so far. RKs carry a ligand-binding ectodomain, a single-pass transmembrane domain and an intracellular kinase domain. Given the lack of obvious signalling domain in the cytoplasmic tail of RLPs, RLPs most likely form (ligand-induced) complexes with RKs to trigger PTI signalling.
Noteworthy, NLRs and surface-localized immune receptors (RLPs and RLKs) were first identified in plants and significant research is still ongoing on these proteins and related pathways. The combination of an intracellular kinase domain with a ligand-binding ectodomain within a single PRR or the ligand-induced association between RLPs and RKs represent plant-specific innovations, whereas metazoan PRRs couple with downstream signalling kinases through adapter domains via TIR domains, for example. This justifies the study of RK-type PRRs and subsequent phosphorylation-dependent signalling in plants, rather than in other systems.

FLS2 and EFR as model PRRs for studying RK-mediated PTI signaling

The best studied PRRs are the Arabidopsis thaliana leucine-rich repeat (LRR)-RKs FLS2 and EFR that recognize the bacterial PAMPs flagellin (or its derived synthetic peptide flg22) and elongation factor (EF)-Tu (or its derived synthetic peptide elf18), respectively (Figure 2). While EF-Tu responsiveness is restricted to the plant family of Brassicaceae, most higher plants respond to flagellin and functional FLS2 orthologs have been cloned in tomato, rice and Nicotiana benthamiana (a wild tobacco relative).

Figure 2: The LRR-RKs perceive the bacterial PAMPs flagellin (flg22) and EF-Tu (elf18), respectively.

The importance of flagellin and EF-Tu perception and subsequent PTI activation in plants is clearly demonstrated by the enhanced disease susceptibility of mutants or lines defective in FLS2 and/or EFR. Plants defective in FLS2 are more susceptible to adapted and non-adapted Pseudomonas species, while EF-Tu recognition by EFR contributes to resistance to Agrobacterium tumefaciens and Pseudomonas syringae. Notably, transgenic expression of the Arabidopsis EFR in the Solanaceae plants N. benthamiana and tomato conferred enhanced resistance to several economically important bacterial pathogens belonging to different genera (Figure 3).

Figure 3: Transgenic expression of EFR in tomato increases resistance to Ralstonia solanacearum (modified from Lacombe et al., 2010)

These results demonstrate that the inter-family transfer of PRRs is possible and provides a novel strategy to engineer broad-spectrum disease resistance in plants that is likely to be durable given the importance of PAMPs for microbial fitness.  Furthermore, this indicates that signalling cascades downstream of PRRs are conserved between plant families, predicting that work on PTI in model plants could be translated into crops.

Within seconds of PAMP binding, FLS2 and EFR form a ligand-induced complex with the regulatory LRR-RK BAK1 leading to phosphorylation of both proteins. BAK1 belongs to a five-member family of SERK proteins and is therefore also named SERK3. Additional SERKs, such as BKK1/SERK4, are recruited in a ligand-dependent manner into EFR and FLS2 protein complexes with different preferences. FLS2 (and potentially EFR) also forms a constitutive complex with the membrane-associated cytoplasmic kinases BIK1 and related PBL proteins that get phosphorylated in a BAK1-dependent manner upon flg22 binding and are positive regulators of most PTI responses downstream of FLS2 and EFR. Downstream of FLS2 and EFR receptor complexes, activation leads to several rapid responses; including bursts of Ca2+ and reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), and transcriptional reprogramming, ultimately leading to PTI (Figure 4). The importance of FLS2, EFR, BAK1, BIK1 and downstream kinases in plant immunity is exemplified by the fact that bacterial effectors target them to suppress PTI and achieve virulence.

Figure 4: FLS2/EFR-mediated PTI signalling

Despite the relevance of PTI for plant disease resistance and recent notable advances, our knowledge on PTI signalling is still very limited. The main activity of our laboratory is to decipher PTI signalling using FLS2 and EFR as paradigmatic RK-type PRRs and a combination of genetic and biochemical approaches. We are following 3 major research programmes:

Our lab aims at deciphering PAMP perception and downstream signalling leading to the establishment of PTI. This is a prerequisite to understand how plants fight microbes and how effectors function, as effector’s targets are likely key PTI components. As PTI is not directed against particular pathogens, outcomes of our research can be potentially used to generate sustainable broad-spectrum disease resistance. We use mostly the paradigmatic EFR and FLS2 perception systems as working models, but we are also currently exploring novel perception systems. Since these pattern recognition receptors (PRRs) are receptor kinases (RKs), our work will ultimately also shed light on how this important class of proteins function and how signalling specificity is achieved in RK-mediated signalling pathways.