Coaxial interferometry for camera-based ultrasound-modulated optical tomography with paired illumination.

Ultrasound-modulated optical tomography (UOT), which combines the advantages of both light and ultrasound, is a promising imaging modality for deep-tissue high-resolution imaging. Among existing implementations, camera-based UOT gains huge advances in modulation depth through parallel detection. However, limited by the long exposure time and the slow framerate of modern cameras, the measurement of UOT signals always requires holographic methods with additional reference beams. This requirement increases system complexity and is susceptible to environmental disturbances. To overcome this challenge, we develop coaxial interferometry for camera-based UOT in this work. Such a coaxial scheme is enabled by employing paired illumination with slightly different optical frequencies. To measure the UOT signal, the conventional phase-stepping method in holography can be directly transplanted into coaxial interferometry. Specifically, we performed both numerical investigations and experimental validations for camera-based UOT under the proposed coaxial scheme. One-dimensional imaging for an absorptive target buried inside a scattering medium was demonstrated. With coaxial interferometry, this work presents an effective way to reduce system complexity and cope with environmental disturbances for camera-based UOT.

ERECTA family signaling constrains CLAVATA3 and WUSCHEL to the center of the shoot apical meristem.

The shoot apical meristem (SAM) is a reservoir of stem cells that gives rise to all post-embryonic above-ground plant organs. The size of the SAM remains stable over time owing to a precise balance of stem cell replenishment versus cell incorporation into organ primordia. The WUSCHEL (WUS)/CLAVATA (CLV) negative feedback loop is central to SAM size regulation. Its correct function depends on accurate spatial expression of WUS and CLV3 A signaling pathway, consisting of ERECTA family (ERf) receptors and EPIDERMAL PATTERNING FACTOR LIKE (EPFL) ligands, restricts SAM width and promotes leaf initiation. Although ERf receptors are expressed throughout the SAM, EPFL ligands are expressed in its periphery. Our genetic analysis of Arabidopsis demonstrated that ERfs and CLV3 synergistically regulate the size of the SAM, and wus is epistatic to ERf genes. Furthermore, activation of ERf signaling with exogenous EPFLs resulted in a rapid decrease of CLV3 and WUS expression. ERf-EPFL signaling inhibits expression of WUS and CLV3 in the periphery of the SAM, confining them to the center. These findings establish the molecular mechanism for stem cell positioning along the radial axis.

Structural Biology of NOD-Like Receptors.

The nucleotide-binding domain (NBD) and leucine-rich repeat (LRR) containing (NLR) proteins are a large family of intracellular immune receptors conserved in both animals and plants. Mammalian NLRs function as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) or host-derived danger associated molecular patterns (DAMPs). PAMP or DAMP perception activates NLRs which consequently recruit pro-caspase-1 directly or indirectly. These sequential events result in formation of large multimeric protein complexes termed inflammasomes that mediate caspase-1 activation for pyroptosis and cytokine secretion. Recent structural and biochemical studies provide significant insights into the acting mechanisms of NLR proteins. In this chapter, we review and discuss these studies concerning autoinhibition, ligand recognition, activation of NLRs, and assembly of NLR inflammasomes.

Structural basis for specific flagellin recognition by the NLR protein NAIP5.

The nucleotide-binding domain- and leucine-rich repeat (LRR)-containing proteins (NLRs) function as intracellular immune receptors to detect the presence of pathogen- or host-derived signals. The mechanisms of how NLRs sense their ligands remain elusive. Here we report the structure of a bacterial flagellin derivative in complex with the NLR proteins NAIP5 and NLRC4 determined by cryo-electron microscopy at 4.28 Å resolution. The structure revealed that the flagellin derivative forms two parallel helices interacting with multiple domains including BIR1 and LRR of NAIP5. Binding to NAIP5 results in a nearly complete burial of the flagellin derivative, thus stabilizing the active conformation of NAIP5. The extreme C-terminal side of the flagellin is anchored to a sterically constrained binding pocket of NAIP5, which likely acts as a structural determinant for discrimination of different bacterial flagellins by NAIP5, a notion further supported by biochemical data. Taken together, our results shed light on the molecular mechanisms underlying NLR ligand perception.

Functional and Structural Characterization of a Receptor-Like Kinase Involved in Germination and Cell Expansion in Arabidopsis.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) are widespread in different plant species and play important roles in growth and development. Germination inhibition is vital for the completion of seed maturation and cell expansion is a fundamental cellular process driving plant growth. Here, we report genetic and structural characterizations of a functionally uncharacterized LRR-RLK, named GRACE (Germination Repression and Cell Expansion receptor-like kinase). Overexpression of GRACE in Arabidopsis exhibited delayed germination, enlarged cotyledons, rosette leaves and stubbier petioles. Conversely, these phenotypes were reversed in the T-DNA insertion knock-down mutant grace-1 plants. A crystal structure of the extracellular domain of GRACE (GRACE-LRR) determined at the resolution of 3.0 Å revealed that GRACE-LRR assumed a right-handed super-helical structure with an island domain (ID). Structural comparison showed that structure of the ID in GRACE-LRR is strikingly different from those observed in other LRR-RLKs. This structural observation implies that GRACE might perceive a new ligand for signaling. Collectively, our data support roles of GRACE in repressing seed germination and promoting cell expansion of Arabidopsis, presumably by perception of unknown ligand(s).

A receptor-like protein acts as a specificity switch for the regulation of stomatal development.

Stomata are microscopic openings that allow for the exchange of gases between plants and the environment. In Arabidopsis, stomatal patterning is specified by the ERECTA family (ERf) receptor kinases (RKs), the receptor-like protein (RLP) TOO MANY MOUTHS (TMM), and EPIDERMAL PATTERNING FACTOR (EPF) peptides. Here we show that TMM and ER or ER-LIKE1 (ERL1) form constitutive complexes, which recognize EPF1 and EPF2, but the single ERfs do not. TMM interaction with ERL1 creates a binding pocket for recognition of EPF1 and EPF2, indicating that the constitutive TMM-ERf complexes function as the receptors of EPF1 and EPF2. EPFL9 competes with EPF1 and EPF2 for binding to the ERf-TMM complex. EPFL4 and EPFL6, however, are recognized by the single ERfs without the requirement of TMM. In contrast to EPF1,2, the interaction of EPFL4,6 with an ERf is greatly reduced in the presence of TMM. Taken together, our data demonstrate that TMM dictates the specificity of ERfs for the perception of different EPFs, thus functioning as a specificity switch for the regulation of the activities of ERfs.

Structural insights into ligand recognition and activation of plant receptor kinases.

The large family of membrane-localized receptor kinases (RKs) has important roles in many aspects of plant physiology. RKs function to perceive external signals, leading to RK activation and downstream signaling. Progress has been recently made in structural elucidation of the mechanisms underlying ligand recognition and activation of RKs. These structural studies mainly on leucine-rich repeat RKs (LRR-RKs) support the idea that ligand-induced dimerization is an essential step for RK activation, though the modes for dimerization vary. Here we review the structural knowledge with an emphasis on the ligand recognition and activation mechanisms that are likely conserved in a subfamily of LRR-RKs.

Structural basis for specific self-incompatibility response in Brassica.

Self-incompatibility (SI) is a widespread mechanism in flowering plants which prevents self-fertilization and inbreeding. In Brassica, recognition of the highly polymorphic S-locus cysteine-rich protein (SCR; or S-locus protein 11) by the similarly polymorphic S-locus receptor kinase (SRK) dictates the SI specificity. Here, we report the crystal structure of the extracellular domain of SRK9 (eSRK9) in complex with SCR9 from Brassica rapa. SCR9 binding induces eSRK9 homodimerization, forming a 2:2 eSRK:SCR heterotetramer with a shape like the letter "A". Specific recognition of SCR9 is mediated through three hyper-variable (hv) regions of eSRK9. Each SCR9 simultaneously interacts with hvI and one-half of hvII from one eSRK9 monomer and the other half of hvII from the second eSRK9 monomer, playing a major role in mediating SRK9 homodimerization without involving interaction between the two SCR9 molecules. Single mutations of residues critical for the eSRK9-SCR9 interaction disrupt their binding in vitro. Our study rationalizes a body of data on specific recognition of SCR by SRK and provides a structural template for understanding the co-evolution between SRK and SCR.

Signature motif-guided identification of receptors for peptide hormones essential for root meristem growth.

Peptide-mediated cell-to-cell signaling has crucial roles in coordination and definition of cellular functions in plants. Peptide-receptor matching is important for understanding the mechanisms underlying peptide-mediated signaling. Here we report the structure-guided identification of root meristem growth factor (RGF) receptors important for plant development. An assay based on a signature ligand recognition motif (Arg-x-Arg) conserved in a subfamily of leucine-rich repeat receptor kinases (LRR-RKs) identified the functionally uncharacterized LRR-RK At4g26540 as a receptor of RGF1 (RGFR1). We further solved the crystal structure of RGF1 in complex with the LRR domain of RGFR1 at a resolution of 2.6 Å, which reveals that the Arg-x-Gly-Gly (RxGG) motif is responsible for specific recognition of the sulfate group of RGF1 by RGFR1. Based on the RxGG motif, we identified additional four RGFRs. Participation of the five RGFRs in RGF-induced signaling is supported by biochemical and genetic data. We also offer evidence showing that SERKs function as co-receptors for RGFs. Taken together, our study identifies RGF receptors and co-receptors that can link RGF signals with their downstream components and provides a proof of principle for structure-based matching of LRR-RKs with their peptide ligands.

Allosteric receptor activation by the plant peptide hormone phytosulfokine.

Phytosulfokine (PSK) is a disulfated pentapeptide that has a ubiquitous role in plant growth and development. PSK is perceived by its receptor PSKR, a leucine-rich repeat receptor kinase (LRR-RK). The mechanisms underlying the recognition of PSK, the activation of PSKR and the identity of the components downstream of the initial binding remain elusive. Here we report the crystal structures of the extracellular LRR domain of PSKR in free, PSK- and co-receptor-bound forms. The structures reveal that PSK interacts mainly with a ß-strand from the island domain of PSKR, forming an anti-ß-sheet. The two sulfate moieties of PSK interact directly with PSKR, sensitizing PSKR recognition of PSK. Supported by biochemical, structural and genetic evidence, PSK binding enhances PSKR heterodimerization with the somatic embryogenesis receptor-like kinases (SERKs). However, PSK is not directly involved in PSKR-SERK interaction but stabilizes PSKR island domain for recruitment of a SERK. Our data reveal the structural basis for PSKR recognition of PSK and allosteric activation of PSKR by PSK, opening up new avenues for the design of PSKR-specific small molecules.