Key words. Acceptor photobleaching, anisotropy, fluorescence lifetime imaging microscopy (FLIM), fluorescence resonance energy transfer (FRET), homo-FRET. SummaryThe phenomenon of resonance energy transfer first described by Theodor Förster presents the opportunity of retrieving information on molecular proximity, orientation and conformation on the nanometre scale from (living) samples with conventional fluorescence microscopes (or even macroscopic devices). During the past 10 years Förster (or fluorescence) resonance energy transfer (FRET) microscopy has been revolutionized by the vast progress in fluorescent protein and in situ fluorescent labelling technology as well as by the commercial availability of advanced quantitative microscopy instrumentation. FRET microscopy is now routinely used in modern cell biology research. This short review will guide the reader through the most established FRET microscopy techniques, their inherent strengths and limitations, potential pitfalls, and assist the reader in making an educated choice on the FRET microscopy method most suited for their specific application. FRET basicsFRET is the physical phenomenon whereby energy is transferred from an excited fluorophore, called the donor (D), to a nearby chromophore, called the acceptor (A), by non-radiative dipole-dipole coupling (through space). FRET only occurs when D and A are in close proximity (nanometre range), when there is sufficient spectral overlap between donor emission and acceptor absorption and when the acceptor transition dipole moment is not perpendicular to the electric field of the dipole field of the donor. The amount of energy transfer, usually expressed as the FRET efficiency (E), is defined Correspondence to: T.W

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pellesoluble kinases. The lysin motif domain-containing receptorlike kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension ␣-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/ threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.Higher plants show a remarkable expansion of receptor-like kinases (RLKs) 4 ; for example, there are over 400 RLK genes in Arabidopsis thaliana (1), which have diverged to fulfill different physiological roles. These genes all encode proteins with the same basic structure as follows: an extracellular region (containing various domains that presumably bind different types of ligands) followed by a single transmembrane-spanning segment and an intracellular region containing a Ser/Thr kinaselike domain. Phylogenetic analysis has shown that the kinase domain is monophyletic and shares a common evolutionary origin with the human IRAK and Drosophila Pelle group of soluble Ser/Thr kinases. These kinases are quite closely related to the animal receptor Tyr kinase and the Raf kinase families (1).The structure of the human IRAK-4 kinase has been solved and has revealed several novel features, including a Tyr gatekeeper in the catalytic pocket, an N-terminal helix B, and regulation by activation loop phosphorylation that resembles Tyr kinases (2, 3). IRAK-4 contains an Arg before the catalytic Asp (an RD kinase), which interacts with a phosphorylated residue in the activation loop. It shares about 38% sequence identity with another family member, IRAK-1, which does not contain the Arg resi...

Receptor(-like) kinases with Lysin Motif (LysM) domains in their extracellular region play crucial roles during plant interactions with microorganisms; e.g. Arabidopsis thaliana CERK1 activates innate immunity upon perception of fungal chitin/chitooligosaccharides, whereas Medicago truncatula NFP and LYK3 mediate signalling upon perception of bacterial lipo-chitooligosaccharides, termed Nod factors, during the establishment of mutualism with nitrogen-fixing rhizobia. However, little is still known about the exact activation and signalling mechanisms of MtNFP and MtLYK3. We aimed at investigating putative molecular interactions of MtNFP and MtLYK3 produced in Nicotiana benthamiana. Surprisingly, heterologous co-production of these proteins resulted in an induction of defence-like responses, which included defence-related gene expression, accumulation of phenolic compounds, and cell death. Similar defence-like responses were observed upon production of AtCERK1 in N. benthamiana leaves. Production of either MtNFP or MtLYK3 alone or their co-production with other unrelated receptor(-like) kinases did not induce cell death in N. benthamiana, indicating that a functional interaction between these LysM receptor-like kinases is required for triggering this response. Importantly, structure-function studies revealed that the MtNFP intracellular region, specific features of the MtLYK3 intracellular region (including several putative phosphorylation sites), and MtLYK3 and AtCERK1 kinase activity were indispensable for cell death induction, thereby mimicking the structural requirements of nodulation or chitin-induced signalling. The observed similarity of N. benthamiana response to MtNFP and MtLYK3 co-production and AtCERK1 production suggests the existence of parallels between Nod factor-induced and chitin-induced signalling mediated by the respective LysM receptor(-like) kinases. Notably, the conserved structural requirements for MtNFP and MtLYK3 biological activity in M. truncatula (nodulation) and in N. benthamiana (cell death induction) indicates the relevance of the latter system for studies on these, and potentially other symbiotic LysM receptor-like kinases.

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.