An update on evolutionary, structural, and functional studies of receptor-like kinases in plants.

All living organisms must develop mechanisms to cope with and adapt to new environments. The transition of plants from aquatic to terrestrial environment provided new opportunities for them to exploit additional resources but made them vulnerable to harsh and ever-changing conditions. As such, the transmembrane receptor-like kinases (RLKs) have been extensively duplicated and expanded in land plants, increasing the number of RLKs in the advanced angiosperms, thus becoming one of the largest protein families in eukaryotes. The basic structure of the RLKs consists of a variable extracellular domain (ECD), a transmembrane domain (TM), and a conserved kinase domain (KD). Their variable ECDs can perceive various kinds of ligands that activate the conserved KD through a series of auto- and trans-phosphorylation events, allowing the KDs to keep the conserved kinase activities as a molecular switch that stabilizes their intracellular signaling cascades, possibly maintaining cellular homeostasis as their advantages in different environmental conditions. The RLK signaling mechanisms may require a coreceptor and other interactors, which ultimately leads to the control of various functions of growth and development, fertilization, and immunity. Therefore, the identification of new signaling mechanisms might offer a unique insight into the regulatory mechanism of RLKs in plant development and adaptations. Here, we give an overview update of recent advances in RLKs and their signaling mechanisms.

The receptor-like kinase EMS1 and BRI1 coordinately regulate stamen elongation via the transcription factors BES1/BZR1 in Arabidopsis.

Plants possess a large family of receptor kinase proteins to mediate cell-to-cell and cell-to-environment communication, and these regulations are essential for plant growth and development as well as resistance to biotic or abiotic stresses. EMS1 is a receptor kinase which involved in tapetum cell fate determination during anther development, while brassinosteroid (BR) receptor, BRI1, controls most aspects of plant growth and development. Although EMS1 and BRI1 are known to regulate independent biological processes, they interact with identical components of the downstream signaling pathways. However, the biological processes other than the tapetum development controlled by the EMS1 signal are not clear. Here, we report that EMS1 signaling-related mutants exhibited an insufficient stamen elongation phenotype, similar to BR signaling mutants. Transgenic expression of BRI1 restored the short filament phenotype of ems1. Conversely, co-expression of EMS1 and TPD1 also restored the short filaments of BRI1 mutants, bri1. Genetic experiments confirmed that EMS1 and BRI1 regulate filament elongation through their downstream transcription factors BES1/BZR1. Molecular analysis suggested that the decrease in BR signaling output in filaments of the ems1 mutant caused deficient filament development. Moreover, in vitro and in vivo experiments proved BES1 interacts with filament-specific transcription factor MYB21. Together, we found that the two receptor-like kinases (RLKs) EMS1 and BRI1 are cooperatively involved in the regulation of filament elongation via the transcription factors BES1/BZR1. These results indicated that the biological processes regulated by EMS1 and BRI1 in plants are both independent and interactive, which provides us with insights into multidimensional molecular control of the RLK pathway.

Kinase regulators evolved into two families by gain and loss of ability to bind plant steroid receptors.

All biological functions evolve by fixing beneficial mutations and removing deleterious ones. Therefore, continuously fixing and removing the same essential function to separately diverge monophyletic gene families sounds improbable. Yet, here we report that brassinosteroid insensitive1 kinase inhibitor1 (BKI1)/membrane-associated kinase regulators (MAKRs) regulating a diverse function evolved into BKI1 and MAKR families from a common ancestor by respectively enhancing and losing ability to bind brassinosteroid receptor brassinosteroid insensitive1 (BRI1). The BKI1 family includes BKI1, MAKR1/BKI1-like (BKL) 1, and BKL2, while the MAKR family contains MAKR2-6. Seedless plants contain only BKL2. In seed plants, MAKR1/BKL1 and MAKR3, duplicates of BKL2, gained and lost the ability to bind BRI1, respectively. In angiosperms, BKL2 lost the ability to bind BRI1 to generate MAKR2, while BKI1 and MAKR6 were duplicates of MAKR1/BKL1 and MAKR3, respectively. In dicots, MAKR4 and MAKR5 were duplicates of MAKR3 and MAKR2, respectively. Importantly, BKI1 localized in the plasma membrane, but BKL2 localized to the nuclei while MAKR1/BKL1 localized throughout the whole cell. Importantly, BKI1 strongly and MAKR1/BKL1 weakly inhibited plant growth, but BKL2 and the MAKR family did not inhibit plant growth. Functional study of the chimeras of their N- and C-termini showed that only the BKI1 family was partially reconstructable, supporting stepwise evolution by a seesaw mechanism between their C- and N-termini to alternately gain an ability to bind and inhibit BRI1, respectively. Nevertheless, the C-terminal BRI1-interacting motif best defines the divergence of BKI1/MAKRs. Therefore, BKI1 and MAKR families evolved by gradually gaining and losing the same function, respectively, extremizing divergent evolution and adding insights into gene (BKI1/MAKR) duplication and divergence.

Two Conserved Amino Acids Characterized in the Island Domain Are Essential for the Biological Functions of Brassinolide Receptors.

Brassinosteroids (BRs) play important roles in plant growth and development, and BR perception is the pivotal process required to trigger BR signaling. In angiosperms, BR insensitive 1 (BRI1) is the essential BR receptor, because its mutants exhibit an extremely dwarf phenotype in Arabidopsis. Two other BR receptors, BRI1-like 1 (BRL1) and BRI1-like 3 (BRL3), are shown to be not indispensable. All BR receptors require an island domain (ID) responsible for BR perception. However, the biological functional significance of residues in the ID remains unknown. Based on the crystal structure and sequence alignments analysis of BR receptors, we identified two residues 597 and 599 of AtBRI1 that were highly conserved within a BR receptor but diversified among different BR receptors. Both of these residues are tyrosine in BRI1, while BRL1/BRL3 fixes two phenylalanines. The experimental findings revealed that, except BRI1Y597F and BRI1Y599F, substitutions of residues 597 and 599 with the remaining 18 amino acids differently impaired BR signaling and, surprisingly, BRI1Y599F showed a weaker phenotype than BRI1Y599 did, implying that these residues were the key sites to differentiate BR receptors from a non-BR receptor, and the essential BR receptor BRI1 from BRL1/3, which possibly results from positive selection via gain of function during evolution.

Evolutionary Analysis and Functional Identification of Ancient Brassinosteroid Receptors in Ceratopteris richardii.

Phytohormones play an important role in the adaptive evolution of terrestrial plants. Brassinosteroids (BRs) are essential hormones that regulate multiple aspects of plant growth and development in angiosperms, but the presence of BR signaling in non-seed plants such as ferns remains unknown. Here, we found that BR promotes the growth of Ceratopteris richardii, while the synthetic inhibitor PCZ inhibits the growth. Using full-length transcriptome sequencing, we identified four BRI1-like receptors. By constructing chimeric receptors, we found that the kinase domains of these four receptors could trigger BR downstream signaling. Further, the extracellular domains of two receptors were functionally interchangeable with that of BRI1. In addition, we identified a co-receptor, CtSERK1, that could phosphorylate with CtBRL2s in vitro. Together, these proved the presence of a receptor complex in Ceratopteris richardii that might perceive BR and activate downstream hormone signaling. Our results shed light on the biological and molecular mechanisms of BR signaling in ferns and the role of BR hormone signaling in the adaptive evolution of terrestrial plants.

A Non-redundant Function of MNS5: A Class I α-1, 2 Mannosidase, in the Regulation of Endoplasmic Reticulum-Associated Degradation of Misfolded Glycoproteins.

Endoplasmic Reticulum-Associated Degradation (ERAD) is one of the major processes in maintaining protein homeostasis. Class I α-mannosidases MNS4 and MNS5 are involved in the degradation of misfolded variants of the heavily glycosylated proteins, playing an important role for glycan-dependent ERAD in planta. MNS4 and MNS5 reportedly have functional redundancy, meaning that only the loss of both MNS4 and MNS5 shows phenotypes. However, MNS4 is a membrane-associated protein while MNS5 is a soluble protein, and both can localize to the endoplasmic reticulum (ER). Furthermore, MNS4 and MNS5 differentially demannosylate the glycoprotein substrates. Importantly, we found that their gene expression patterns are complemented rather than overlapped. This raises the question of whether they indeed work redundantly, warranting a further investigation. Here, we conducted an exhaustive genetic screen for a suppressor of the bri1-5, a brassinosteroid (BR) receptor mutant with its receptor downregulated by ERAD, and isolated sbi3, a suppressor of bri1-5 mutant named after sbi1 (suppressor of bri1). After genetic mapping together with whole-genome re-sequencing, we identified a point mutation G343E in AT1G27520 (MNS5) in sbi3. Genetic complementation experiments confirmed that sbi3 was a loss-of-function allele of MNS5. In addition, sbi3 suppressed the dwarf phenotype of bri1-235 in the proteasome-independent ERAD pathway and bri1-9 in the proteasome-dependent ERAD pathway. Importantly, sbi3 could only affect BRI1/bri1 with kinase activities such that it restored BR-sensitivities of bri1-5, bri1-9, and bri1-235 but not null bri1. Furthermore, sbi3 was less tolerant to tunicamycin and salt than the wild-type plants. Thus, our study uncovers a non-redundant function of MNS5 in the regulation of ERAD as well as plant growth and ER stress response, highlighting a need of the traditional forward genetic approach to complement the T-DNA or CRISPR-Cas9 systems on gene functional study.

Pan-brassinosteroid signaling revealed by functional analysis of NILR1 in land plants.

Brassinosteroid (BR) signaling has been identified from the ligand BRs sensed by the receptor Brassinosteroid Insensitive 1 (BRI1) to the final activation of Brassinozole Resistant 1/bri1 EMS-Suppressor 1 through a series of transduction events. Extensive studies have been conducted to characterize the role of BR signaling in various biological processes. Our previous study has shown that Excess Microsporocytes 1 (EMS1) and BRI1 control different aspects of plant growth and development via conserved intracellular signaling. Here, we reveal that another receptor, NILR1, can complement the bri1 mutant in the absence of BRs, indicating a pathway that resembles BR signaling activated by NILR1. Genetic analysis confirms the intracellular domains of NILR1, BRI1 and EMS1 have a common signal output. Furthermore, we demonstrate that NILR1 and BRI1 share the coreceptor BRI1 Associated Kinase 1 and substrate BSKs. Notably, the NILR1-mediated downstream pathway is conserved across land plants. In summary, we provide evidence for the signaling cascade of NILR1, suggesting pan-brassinosteroid signaling initiated by a group of distant receptor-ligand pairs in land plants.

The Role of SBI2/ALG12/EBS4 in the Regulation of Endoplasmic Reticulum-Associated Degradation (ERAD) Studied by a Null Allele.

Redundancy and lethality is a long-standing problem in genetics but generating minimal and lethal phenotypes in the knockouts of the same gene by different approaches drives this problem to a new high. In Asn (N)-linked glycosylation, a complex and ubiquitous cotranslational and post-translational protein modification required for the transfer of correctly folded proteins and endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, ALG12 (EBS4) is an α 1, 6-mannosyltransferase catalyzing a mannose into Glc3Man9GlcNAc2. In Arabidopsis, T-DNA knockout alg12-T is lethal while likely ebs4 null mutants isolated by forward genetics are most healthy as weak alleles, perplexing researchers and demanding further investigations. Here, we isolated a true null allele, sbi2, with the W258Stop mutation in ALG12/EBS4. sbi2 restored the sensitivity of brassinosteroid receptor mutants bri1-5, bri1-9, and bri1-235 with ER-trapped BRI1 to brassinosteroids. Furthermore, sbi2 maturated earlier than the wild-type. Moreover, concomitant with impaired and misfolded proteins accumulated in the ER, sbi2 had higher sensitivity to tunicamycin and salt than the wild-type. Our findings thus clarify the role of SBI2/ALG12/EBS4 in the regulation of the ERAD of misfolded glycoproteins, and plant growth and stress response. Further, our study advocates the necessity and importance of using multiple approaches to validate genetics study.

Glass FRP-Reinforced Geopolymer Based Columns Comprising Hybrid Fibres: Testing and FEA Modelling.

This study seeks to evaluate the effectiveness of glass-FRP-reinforced geopolymer concrete columns integrating hybrid fibres (GFGC columns) and steel bar-reinforced geopolymer concrete columns incorporating hybrid fibres (SFGC columns) under eccentric and concentric loadings. Steel fibre (SF) and polypropylene fibres (PF) are two types of fibres that are mixed into hybrid fibre-reinforced geopolymer concrete (HFRGC). Eighteen circular concrete columns with a cross-section of 300 mm × 1200 mm were cast and examined under axial loading up to failure. Nine columns were cast with glass-FRP rebars, whereas the other nine were cast with steel rebars. Using ABAQUS, a nonlinear finite element model was established for the GFGC and SFGC columns. The HFRGC material was modelled using a simplified concrete damage plasticity model, whereas the glass-FRP material was simulated as a linear elastic material. It was observed that GFGC columns had up to 20% lower axial strength (AST) and up to 24% higher ductility indices than SFGC columns. The failure modes of both GFGC and SFGC columns were analogous. Both GFGC and SFGC columns revealed the same effect of eccentricity in the form of a decline in AST. A novel statistical model was suggested for predicting the AST of GFGC columns. The outcomes of the experiments, finite element simulations, and theoretical results show that the models can accurately determine the AST of GFGC columns.

Kinase Function of Brassinosteroid Receptor Specified by Two Allosterically Regulated Subdomains.

Plants acquire the ability to adapt to the environment using transmembrane receptor-like kinases (RLKs) to sense the challenges from their surroundings and respond appropriately. RLKs perceive a variety of ligands through their variable extracellular domains (ECDs) that activate the highly conserved intracellular kinase domains (KDs) to control distinct biological functions through a well-developed downstream signaling cascade. A new study has emerged that brassinosteroid-insensitive 1 (BRI1) family and excess microsporocytes 1 (EMS1) but not GASSHO1 (GSO1) and other RLKs control distinct biological functions through the same signaling pathway, raising a question how the signaling pathway represented by BRI1 is specified. Here, we confirm that BRI1-KD is not functionally replaceable by GSO1-KD since the chimeric BRI1-GSO1 cannot rescue bri1 mutants. We then identify two subdomains S1 and S2. BRI1 with its S1 and S2 substituted by that of GSO1 cannot rescue bri1 mutants. Conversely, chimeric BRI1-GSO1 with its S1 and S2 substituted by that of BRI1 can rescue bri1 mutants, suggesting that S1 and S2 are the sufficient requirements to specify the signaling function of BRI1. Consequently, all the other subdomains in the KD of BRI1 are functionally replaceable by that of GSO1 although the in vitro kinase activities vary after replacements, suggesting their functional robustness and mutational plasticity with diverse kinase activity. Interestingly, S1 contains αC-ß4 loop as an allosteric hotspot and S2 includes kinase activation loop, proposedly regulating kinase activities. Further analysis reveals that this specific function requires ß4 and ß5 in addition to αC-ß4 loop in S1. We, therefore, suggest that BRI1 specifies its kinase function through an allosteric regulation of these two subdomains to control its distinct biological functions, providing a new insight into the kinase evolution.

Less Conserved LRRs Is Important for BRI1 Folding.

Brassinosteroid insensitive 1 (BRI1) is a multidomain plant leucine-rich repeat receptor-like kinase (LRR-RLK), belongs to the LRR X subfamily. BRI1 perceives plant hormone brassinosteroids (BRs) through its extracellular domain that constitutes of LRRs interrupted by a 70 amino acid residue island domain (ID), which activates the kinase domain (KD) in its intracellular domain to trigger BR response. Thus, the KD and the ID of BRI1 are highly conserved and greatly contribute to BR functions. In fact, most bri1 mutants are clustered in or surrounded around the ID and the KD. However, the role of the less conserved LRR domains, particularly the first few LRRs after the signal peptide, is elusive. Here, we report the identification of a loss-of-function mutant bri1-235 that carries a mutation in the less conserved fourth LRR of BRI1 extracellular domain in Arabidopsis. This mutant had a base alteration from C to T, resulting in an amino acid substitution from serine to phenylalanine at the 156th position of BRI1. Compared with the wild-type plants, bri1-235 exhibited round leaves, prolonged life span, shorter stature, and approximately normal fertility under light conditions. The bri1-235 mutant was less sensitive to exogenous brassinolide under normal conditions. Importantly, both wild-type BRI1 expression and a sbi1 mutant that activates BRI1 rescued bri1-235 and resembled the wild type. Furthermore, bri1-235 protein was localized in endoplasmic reticulum rather than plasma membrane, suggestive of a cause for reducing BR sensitive in bri1-235. Taken together, our findings provide an insight into the role of the less conserved LRRs of BRI1, shedding light on the role of LRRs in a variety of LRR-RLKs that control numerous processes of plant growth, development, and stress response.

Polymorphism of the IL28B gene (rs8099917, rs12979860) and virological response of Pakistani hepatitis C virus genotype 3 patients to pegylated interferon therapy.

BACKGROUND: The gold standard treatment for chronic hepatitis C virus (HCV) infection is pegylated interferon (PEG-IFN) in combination with ribavirin. Most patients treated with PEG-IFN achieve a sustained virological response (SVR). However host genetic factors play a vital role in the spontaneous and treatment-induced clearance of HCV infection from these infected patients. In the current study, polymorphisms of IL28B (rs8099917 and rs12979860) were analyzed and their association with the virological response to PEG-IFN alpha treatment was determined. METHODS: One hundred and fifty HCV genotype 3 patients were assessed to study the correlation of IL28B with a therapeutic regimen of PEG-IFN alpha plus ribavirin. Twenty patients were excluded due to a refusal to participate in the study and 25 patients failed to meet the inclusion criteria. Of the 105 patients recruited, 49 (46.7%) were male and 56 (53.3%) were female. In order to determine single nucleotide polymorphisms of rs8099917 and rs12979860, the sample was amplified by PCR and then IL28B typing was carried out by restriction fragment length polymorphism (RFLP) followed by standard sequencing. RESULTS: We found three types of genotype in rs8099917 of IL28B: wild-type TT in 60.0% of patients, heterozygous GT minor genotype in 36.2%, and GG in 3.8%. The frequency of the CC genotype of rs12979860 was 54.3%, CT was 37.1%, and TT was 8.6%. Overall, SVR was achieved in 68.6% of patients. A higher SVR was achieved for patients with the favorable genotype CC of rs12979860, with 84.2% as compared to 56.4% and 22.2% for minor genotype CT and TT, respectively (p=0.0001). We did not find a significant association for SVR to antiviral treatment in patients with genotype TT (rs8099917) (71.9%, p=0.36). The rapid virological response (RVR) rate was significantly higher in patients with major genotype TT (88.9%, p=0.04). These results show that IL28B polymorphism is highly associated with SVR to therapy in the Pakistani population infected with HCV genotype 3. CONCLUSIONS: HCV-infected patients carrying homozygous C/C have a higher chance of SVR. In addition, patients who carry T/T (rs8099917) have a higher chance of RVR.

Intramedullary lesion expansion on magnetic resonance imaging in patients with motor complete cervical spinal cord injury.

OBJECT: The authors performed a study to determine if lesion expansion occurs in humans during the early hours after spinal cord injury (SCI), as has been established in rodent models of SCI, and to identify factors that might predict lesion expansion. METHODS: The authors studied 42 patients with acute cervical SCI and admission American Spinal Injury Association Impairment Scale Grades A (35 patients) and B (7 patients) in whom 2 consecutive MRI scans were obtained 3-134 hours after trauma. They recorded demographic data, clinical information, Injury Severity Score (ISS), admission MRI-documented spinal canal and cord characteristics, and management strategies. RESULTS: The characteristics of the cohort were as follows: male/female ratio 37:5; mean age, 34.6 years; and cause of injury, motor vehicle collision, falls, and sport injuries in 40 of 42 cases. The first MRI study was performed 6.8 ±2.7 hours (mean ± SD) after injury, and the second was performed 54.5 ± 32.3 hours after injury. The rostrocaudal intramedullary length of the lesion on the first MRI scan was 59.2 ± 16.1 mm, whereas its length on the second was 88.5 ± 31.9 mm. The principal factors associated with lesion length on the first MRI study were the time between injury and imaging (p = 0.05) and the time to decompression (p = 0.03). The lesion's rate of rostrocaudal intramedullary expansion in the interval between the first and second MRI was 0.9 ± 0.8 mm/hour. The principal factors associated with the rate of expansion were the maximum spinal cord compression (p = 0.03) and the mechanism of injury (p = 0.05). CONCLUSIONS: Spinal cord injury in humans is characterized by lesion expansion during the hours following trauma. Lesion expansion has a positive relationship with spinal cord compression and may be mitigated by early surgical decompression. Lesion expansion may be a novel surrogate measure by which to assess therapeutic effects in surgical or drug trials.