Lotus japonicus cytokinin receptors work partially redundantly to mediate nodule formation.

Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.

Lotus japonicus SUNERGOS1 encodes a predicted subunit A of a DNA topoisomerase VI that is required for nodule differentiation and accommodation of rhizobial infection.

A symbiotic mutant of Lotus japonicus, called sunergos1-1 (suner1-1), originated from a har1-1 suppressor screen. suner1-1 supports epidermal infection by Mesorhizobium loti and initiates cell divisions for organogenesis of nodule primordia. However, these processes appear to be temporarily stalled early during symbiotic interaction, leading to a low nodule number phenotype. This defect is ephemeral and near wild-type nodule numbers are reached by suner1-1 at a later point after infection. Using an approach that combined map-based cloning and next-generation sequencing we have identified the causative mutation and show that the suner1-1 phenotype is determined by a weak recessive allele, with the corresponding wild-type SUNER1 locus encoding a predicted subunit A of a DNA topoisomerase VI. Our data suggest that at least one function of SUNER1 during symbiosis is to participate in endoreduplication, which is an essential step during normal differentiation of functional, nitrogen-fixing nodules.

BioBrick™ compatible vector system for protein expression in Rhodobacter sphaeroides.

We report here the creation of a modular, plasmid-based protein expression system utilizing elements of the native Rhodobacter puf promoter in a BioBrick(TM)-based vector system with DsRed encoding a red fluorescent reporter protein. A suite of truncations of the puf promoter were made to assess the influence of different portions of this promoter on expression of heterologous proteins. The 3' end of puf was found to be particularly important for increasing expression, with transformants accumulating significant quantities of DsRed under both aerobic and anaerobic growth conditions. Expression levels of this reporter protein in Rhodobacter sphaeroides were comparable to those achieved in Escherichia coli using the strong, constitutive P lac promoter, thus demonstrating the robustness of the engineered system. Furthermore, we demonstrate the ability to tune the designed expression system by modulating cellular DsRed levels based upon the promoter segment utilized and oxygenation conditions. Last, we show that the new expression system is able to drive expression of a membrane protein, proteorhodopsin, and that membrane purifications from R. sphaeroides yielded significant quantities of proteorhodopsin. This toolset lays the groundwork for the engineering of multi-step pathways, including recalcitrant membrane proteins, in R. sphaeroides.

Lotus japonicus symRK-14 uncouples the cortical and epidermal symbiotic program.

SYMRK is a leucine-rich-repeat (LRR)-receptor kinase that mediates intracellular symbioses of legumes with rhizobia and arbuscular mycorrhizal fungi. It participates in signalling events that lead to epidermal calcium spiking, an early cellular response that is typically considered as central for intracellular accommodation and nodule organogenesis. Here, we describe the Lotus japonicus symRK-14 mutation that alters a conserved GDPC amino-acid sequence in the SYMRK extracellular domain. Normal infection of the epidermis by fungal or bacterial symbionts was aborted in symRK-14. Likewise, epidermal responses of symRK-14 to bacterial signalling, including calcium spiking, NIN gene expression and infection thread formation, were significantly reduced. In contrast, no major negative effects on the formation of nodule primordia and cortical infection were detected. Cumulatively, our data show that the symRK-14 mutation uncouples the epidermal and cortical symbiotic program, while indicating that the SYMRK extracellular domain participates in transduction of non-equivalent signalling events. The GDPC sequence was found to be highly conserved in LRR-receptor kinases in legumes and non-legumes, including the evolutionarily distant bryophytes. Conservation of the GDPC sequence in nearly one-fourth of LRR-receptor-like kinases in the genome of Arabidopsis thaliana suggests, however, that this sequence might also play an important non-symbiotic function in this plant.

Seed development, seed germination and seedling growth in the R50 (sym16) pea mutant are not directly linked to altered cytokinin homeostasis.

R50 (sym16) is a pea nodulation mutant that accumulates cytokinin (CK) in its vegetative organs. Total CK content increases as the plant ages because of the low activity of the enzyme cytokinin oxidase/dehydrogenase (CKX) responsible for CK degradation. R50 exhibits a large seed with high relative water content, and its seedling establishes itself slowly. Whether these two traits are linked to abnormal CK levels was considered here. R50 was found to have a similar germination rate but a much slower epicotyl emergence than Sparkle, its wild-type (WT). At the onset of emergence, the starch grains in R50 cotyledons were larger than those of WT; furthermore, they did not degrade as fast as in WT because of low amylase activity. No differences between the pea lines were observed in the CK forms identified during seed embryogenesis. However, while CK content compared to that of WT was reduced early in R50 embryogenesis, it was elevated later on in its dry seeds where CKX activity was low, although CKX transcript abundance remained high. Transcripts of the two known PsCKX isoforms exhibited tissue- and development-specific profiles with no detectable PsCKX2 expression in cotyledons. There were more of both transcripts in R50 roots than in WT roots, but less of PsCKX2 than PsCKX1 in R50 shoots compared to WT shoots. Thus, although there is a definite CKX post-transcriptional defect in R50 dry seeds, an abnormal CK homeostasis is not the basis of the delay in R50 seedling establishment, which we linked to abnormal amylase activity early in development.

Telepsychology for Individuals With Spinal Cord Injury: Protocol for a Randomized Control Study of Video-Based Cognitive Behavioral Therapy.

Background: A substantial proportion of individuals with spinal cord injury (SCI) experience depression, which has been negatively associated with recovery and community participation after injury. Despite significant barriers to seeking and receiving in-person mental health care, little research has focused on the efficacy of telepsychology among individuals with SCI. Objectives: To describe the design and implementation of an ongoing single-center, randomized controlled, video-based cognitive behavioral therapy (CBT) intervention among individuals with SCI. Methods: Participants within 1 year of SCI will be randomized 1:1 to intervention or usual care in a 24-week study. Intervention participants will engage in 10 sessions of CBT over 12 weeks with a licensed clinical psychologist, using iPads via Apple FaceTime. Primary outcomes are depressive symptomatology, anxiety, and life satisfaction (as measured by the Patient Health Questionnaire-9, Generalized Anxiety Disorder-7 and Satisfaction with Life Scale, respectively) measured at three time points (baseline, 12 weeks, and 24 weeks). These and other measures are also assessed during monthly telephone surveys conducted between primary timepoints. Results: Recruitment is ongoing. Forty-six participants have been enrolled thus far. Conclusion: Telepsychology is a convenient, flexible, and effective alternative to traditional in-person services. We anticipate that intervention participants will experience improvements in depressive and anxiety symptoms and will have greater life satisfaction. Telepsychology interventions among individuals with SCI are tasked to maintain participant privacy, provide assistive technology and/or engage caregivers to minimize mobility limitations, and manage risk remotely. Challenges encountered include recruitment during the COVID-19 pandemic. Early intervention on symptoms of psychological morbidity using telepsychology may facilitate greater adaptation following SCI.

Genetic suppressors of the Lotus japonicus har1-1 hypernodulation phenotype.

Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.

Into the Root: How Cytokinin Controls Rhizobial Infection.

Leguminous plants selectively initiate primary responses to rhizobial nodulation factors (NF) that ultimately lead to symbiotic root nodule formation. Functioning downstream, cytokinin has emerged as the key endogenous plant signal for nodule differentiation, but its role in mediating rhizobial entry into the root remains obscure. Nonetheless, such a role is suggested by aberrant infection phenotypes of plant mutants with defects in cytokinin signaling. We postulate that cytokinin participates in orchestrating signaling events that promote rhizobial colonization of the root cortex and limit the extent of subsequent infection at the root epidermis, thus maintaining homeostasis of the symbiotic interaction. We further argue that cytokinin signaling must have been crucial during the evolution of plant cell predisposition for rhizobial colonization.

Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli.

Compartmentalization of designed metabolic pathways within protein based nanocompartments has the potential to increase reaction efficiency in multi-step biosynthetic reactions. We previously demonstrated proof-of-concept of this aim by targeting a functional enzyme to single cellular protein nanocompartments, which were formed upon recombinant expression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK in Escherichia coli. To optimize this system, increasing overall encapsulated enzyme reaction efficiency, factor(s) required for the production of more than one nanocompartment per cell must be identified. In this work we report that the cupin domain protein EutQ is required for assembly of more than one nanocompartment per cell. Overexpression of EutQ results in multiple nanocompartment assembly in our recombinant system. EutQ specifically interacts with the shell protein EutM in vitro via electrostatic interactions with the putative cytosolic face of EutM. These findings lead to the theory that EutQ could facilitate multiple nanocompartment biogenesis by serving as an assembly hub for shell proteins. This work offers insights into the biogenesis of Eut bacterial microcompartments, and also provides an improved platform for the production of protein based nanocompartments for targeted encapsulation of enzyme pathways.

Eut bacterial microcompartments: insights into their function, structure, and bioengineering applications.

Bacterial microcompartments (BMCs) are protein-based polyhedral organelles which serve to encapsulate and organize enzymes involved in key metabolic pathways. The sequestration of these pathways not only improves the overall reaction efficiency; it can also harbor toxic or volatile pathway intermediates, which would otherwise be detrimental to the cell. Genomic and phylogenetic analyses reveal the presence of these unique organelles in a diverse range of bacterial species, highlighting their evolutionary importance and the essential role that they play in bacterial cell survival. Functional and structural analyses of BMCs involved in ethanolamine utilization are developing our understanding of the self-assembly and encapsulation mechanisms employed by these protein supercomplexes. This knowledge will open up exciting new avenues of research with a range of potential engineering and biotechnological applications.

Common and not so common symbiotic entry.

Great advances have been made in our understanding of the host plant's common symbiosis functions, which in legumes mediate intracellular accommodation of both nitrogen-fixing bacteria and arbuscular mycorrhiza (AM) fungi. However, it has become apparent that additional plant genes are required specifically for bacterial entry inside the host root. In this opinion article, we consider Lotus japonicus nap1 and pir1 symbiotic mutants within the context of other deleterious mutations that impair an intracellular accommodation of bacteria but have no impact on the colonization of roots by AM fungi. We highlight a clear delineation of early signaling events during bacterial versus AM symbioses while suggesting a more intricate origin of the plant's ability for intracellular accommodation of bacteria.

Rearrangement of actin cytoskeleton mediates invasion of Lotus japonicus roots by Mesorhizobium loti.

Infection thread-dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.