Protein Of Unknown Function Research Articles

Differential domains and endoproteolytic processing in dominant surface proteins of unknown function from Mycoplasma hyopneumoniae and Mycoplasma flocculare

Mycoplasma hyopneumoniae causes porcine enzootic pneumonia (PEP), a chronic respiratory disease that leads to severe economic losses in the pig industry. Swine infection and PEP development depend on the adhesion of the pathogen to the swine respiratory tract and the host immune response, but these and other disease determinants are not fully understood. For instance, M. hyopneumoniae has a large repertoire of proteins of unknown function (PUFs) and some of them are abundant in the cell surface, where they likely mediate so far unknown pathogen-host interactions. Moreover, these surface PUFs may undergo endoproteolytic processing to generate larger repertoires of proteoforms to further complicate this scenario. Here, we investigated the five PUFs more represented on the surface of M. hyopneumoniae pathogenic strain 7448 in comparison with their orthologs from the nonpathogenic M. hyopneumoniae J strain and the closely related commensal species Mycoplasma flocculare. Comparative in silico analyses of deduced amino acid sequences and proteomic data identified differential domains, disordered regions and repeated motifs. We also provide evidence of differential endoproteolytic processing and antigenicity. Phylogenetic analyses were also performed with ortholog sequences, showing higher conservation of three of the assessed PUFs among Mycoplasma species related to respiratory diseases. Overall, our data point out to M. hyopneumoniae surface-dominant PUFs likely associated with pathogenicity.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Non-structural genes of novel lemur adenoviruses reveal codivergence of virus and host.

Adenoviruses (AdVs) are important human and animal pathogens and are frequently used as vectors for gene therapy and vaccine delivery. Surprisingly, there are only scant data regarding primate AdV origin and evolution, especially in the most basal primate hosts. We detect and sequence AdVs from faeces of two Madagascan lemur species. Complete genome sequence analyses define a new AdV species with a particularly large gene encoding a protein of unknown function in the early gene region 3. Unexpectedly, the new AdV species is not most similar to human or other simian AdVs but to bat adenovirus C. Genome characterisation shows signals of virus-host codivergence in non-structural genes, which show lower diversity than structural genes. Outside a lemur species mixing zone, recombination less frequently separates structural genes, as in human adenovirus C. The evolutionary history of lemur AdVs likely involves both a host switch and codivergence with the lemur hosts.

Listeria monocytogenes GlmR Is an Accessory Uridyltransferase Essential for Cytosolic Survival and Virulence.

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions against these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell-wall stress, cytosolic survival, inflammasome avoidance, and, ultimately, virulence in vivo. In this study, a genetic suppressor screen revealed that blocking utilization of UDP N-acetylglucosamine (UDP-GlcNAc) by a nonessential wall teichoic acid decoration pathway restored resistance to lysozyme and partially restored virulence of ΔglmR mutants. In parallel, metabolomic analysis revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggests that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell-wall stress responses and virulence in vivo. Taken together, these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes. IMPORTANCE Bacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK), to survive in the host cytosol. GlmR is important for resistance to some cell-wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell-wall metabolite, UDP-GlcNAc, and suppressors that increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc, and this enzymatic activity is conserved in both Bacillus subtilis and Staphylococcus aureus. These results highlight the importance of accessory cell wall metabolism enzymes in responding to cell-wall stress in a variety of Gram-positive bacteria.

New Genes Identified as Modulating Salt Tolerance in Maize Seedlings Using the Combination of Transcriptome Analysis and BSA

(1) Background: Salt stress is an abiotic factor that limits maize yield and quality. A highly salt-tolerance inbred AS5 and a salt-sensitive inbred NX420 collected from Ningxia Province, China, were used to identify new genes for modulating salt resistance in maize. (2) Methods: To understand the different molecular bases of salt tolerance in AS5 and NX420, we performed BSA-seq using an F2 population for two extreme bulks derived from the cross between AS5 and NX420. Transcriptomic analysis was also conducted for AS5 and NX420 at the seedling stage after treatment with 150 mM of NaCl for 14 days. (3) Results: AS5 had a higher biomass and lower Na+ content than NX420 in the seedling stage after treatment with 150 mM NaCl for 14 days. One hundred and six candidate regions for salt tolerance were mapped on all of the chromosomes through BSA-seq using F2 in an extreme population. Based on the polymorphisms identified between both parents, we detected 77 genes. A large number of differentially expressed genes (DEGs) at the seedling stage under salt stress between these two inbred lines were detected using transcriptome sequencing. GO analysis indicated that 925 and 686 genes were significantly enriched in the integral component of the membrane of AS5 and NX420, respectively. Among these results, two and four DEGs were identified as overlapping in these two inbred lines using BSA-seq and transcriptomic analysis, respectively. Two genes (Zm00001d053925 and Zm00001d037181) were detected in both AS5 and NX420; the transcription level of Zm00001d053925 was induced to be significantly higher in AS5 than in NX420 (41.99 times versus 6.06 times after 150 mM of NaCl treatment for 48 h), while the expression of Zm00001d037181 showed no significant difference upon salt treatment in both lines. The functional annotation of the new candidate genes showed that it was an unknown function protein. (4) Conclusions: Zm00001d053925 is a new functional gene responding to salt stress in the seedling stage, which provides an important genetic resource for salt-tolerant maize breeding.

C-Type Natriuretic Peptide Acts as a Microorganism-Activated Regulator of the Skin Commensals Staphylococcus epidermidis and Cutibacterium acnes in Dual-Species Biofilms.

The effect of C-type natriuretic peptide in a concentration closer to the normal level in human blood plasma was studied on the mono-species and dual-species biofilms of the skin commensal bacteria Cutibacterium acnes HL043PA2 and Staphylococcus epidermidis ATCC14990. Despite the marginal effect of the hormone on cutibacteria in mono-species biofilms, the presence of staphylococci in the community resulted in a global shift of the CNP effect, which appeared to increase the competitive properties of C. acnes, its proliferation and the metabolic activity of the community. S. epidermidis was mostly inhibited in the presence of CNP. Both bacteria had a significant impact on the gene expression levels revealed by RNA-seq. CNP did not affect the gene expression levels in mono-species cutibacterial biofilms; however, in the presence of staphylococci, five genes were differentially expressed in the presence of the hormone, including two ribosomal proteins and metal ABC transporter permease. In staphylococci, the Na-translocating system protein MpsB NADH-quinone oxidoreductase subunit L was downregulated in the dual-species biofilms in the presence of CNP, while in mono-species biofilms, two proteins of unknown function were downregulated. Hypothetically, at least one of the CNP mechanisms of action is via the competition for zinc, at least on cutibacteria.

Diffusion Alignment Coefficient (DAC): A Novel Similarity Metric for Protein-Protein Interaction Network.

Interaction networks can be used to predict the functions of unknown proteins using known interactions and proteins with known functions. Many graph theory or diffusion-based methods have been proposed, using the assumption that the topological properties of a protein in a network are related to its biological function. Here we seek to improve function prediction by finding more similar neighbors with a new diffusion-based alignment technique to overcome the topological information loss of the node. In this study, we introduce the Diffusion Alignment Coefficient (DAC) algorithm, which combines diffusion, longest common subsequence, and longest common substring techniques to measure the similarity of two nodes in protein interaction networks. As a proof of concept, our experiments, conducted on a real PPI networks S.cerevisiae and Homo Sapiens, demonstrated that our method obtained better results than competitors for MIPS and MSigDB Collections hallmark gene set functional categories. This is the first study to develop a measure of node function similarity using alignment to consider the positions of nodes in protein-protein interaction networks. According to the experimental results, the use of spatial information belonging to the nodes in the network has a positive effect on the detection of more functionally similar neighboring nodes.

CsgI (YccT) Is a Novel Inhibitor of Curli Fimbriae Formation in Escherichia coli Preventing CsgA Polymerization and Curli Gene Expression.

Curli fimbriae are amyloids-found in bacteria (Escherichia coli)-that are involved in solid-surface adhesion and bacterial aggregation during biofilm formation. The curli protein CsgA is coded by a csgBAC operon gene, and the transcription factor CsgD is essential to induce its curli protein expression. However, the complete mechanism underlying curli fimbriae formation requires elucidation. Herein, we noted that curli fimbriae formation was inhibited by yccT-i.e., a gene that encodes a periplasmic protein of unknown function regulated by CsgD. Furthermore, curli fimbriae formation was strongly repressed by CsgD overexpression caused by a multicopy plasmid in BW25113-the non-cellulose-producing strain. YccT deficiency prevented these CsgD effects. YccT overexpression led to intracellular YccT accumulation and reduced CsgA expression. These effects were addressed by deleting the N-terminal signal peptide of YccT. Localization, gene expression, and phenotypic analyses revealed that YccT-dependent inhibition of curli fimbriae formation and curli protein expression was mediated by the two-component regulatory system EnvZ/OmpR. Purified YccT inhibited CsgA polymerization; however, no intracytoplasmic interaction between YccT and CsgA was detected. Thus, YccT-renamed CsgI (curli synthesis inhibitor)-is a novel inhibitor of curli fimbriae formation and has a dual role as an OmpR phosphorylation modulator and CsgA polymerization inhibitor.

CYSRT1: An Antimicrobial Epidermal Protein that Can Interact with Late Cornified Envelope Proteins

Late cornified envelope (LCE) proteins are small cationic epidermal proteins with antimicrobial properties, and the combined deletion of LCE3B and LCE3C genes is a risk factor for psoriasis that affects skin microbiome composition. In a yeast two-hybrid screen, we identified CYSRT1 as an interacting partner of members of all LCE groups except LCE6. These interactions were confirmed in a mammalian cell system by coimmunoprecipitation. CYSRT1 is a protein of unknown function that is specifically expressed in cutaneous and oral epithelia and spatially colocalizes with LCE proteins in the upper layers of the suprabasal epidermis. Constitutive CYSRT1 expression is present in fully differentiated epidermis and can be further induced invivo by disruption of the skin barrier upon stratum corneum removal. Transcriptional regulation correlates to keratinocyte terminal differentiation but not to skin bacteria exposure. Similar to LCEs, CYSRT1 was found to have antibacterial activity against Pseudomonas aeruginosa. Comparative gene sequence analysis and protein amino acid alignment indicate that CYSRT1 is highly conserved among vertebrates and has putative antimicrobial activity. To summarize, we identified CYSRT1 in the outer skin layer, where it colocalizes with LCE proteins and contributes to the constitutive epidermal antimicrobial host defense repertoire.

Identifying esterase and lipase activity in proteins of unknown function.

Over 3800 structures in the Protein Data Bank (PDB) have unknown function. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) curriculum uses authentic inquiry to teach students to use structural bioinformatics tools to compare these structures to known enzymes and predict a function. We have predicted esterase or lipase function for several structures after performing both global and local structure alignments to identify similar structures in the PDB as well as specific amino acids that may be active site motifs. This function was supported by sequence alignment data from BLAST and PFam. SwissDock was then used for ligand docking to determine ester and lipid substrates to use for testing activity. Students in the biochemistry lab next used standard wet-lab biochemistry techniques to express and purify the target enzymes and performed kinetic assays using p-nitrophenyl acetate to test for esterase activity. After completing the BASIL curriculum, students continued with independent research projects to develop assays to test for lipase function. We have focused our efforts on the protein structures with PDB IDs 2O14 and 4Q7Q, as they showed the most promise through initial enzymatic testing. Both proteins show significant esterase activity in hydrolyzing both p-nitrophenyl acetate and p-nitrophenyl butyrate. Additionally, both proteins are able to hydrolyze p-nitrophenyl dodecanoate when sodium deoxycholate is present in the buffer solution for solubility of the substrate. We have also incorporated the p-nitrophenyl dodecanoate in DMPC vesicles, and testing of substrate hydrolysis with these vesicles is underway. We have also tested pancreatic lipase under all these conditions as a positive control for the experiments. Students will be creating a Proteopedia page for each protein. This project has been supported in part by NSF IUSE 1709355 and 2141908.

The BASIL cure: Using structure to predict function in protein biochemistry.

We have developed an undergraduate biochemistry lab curriculum based on authentic inquiry. This course-based undergraduate research experience (CURE) allows a large number of students to gain skills often learned only in the traditional one-on-one mentor-student research model by bringing a research project into the undergraduate teaching lab. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) uses a combined computational and wet lab approach to study proteins of known structure but unknown function. Over 3800 structures in the Protein Data Bank (PDB) have unknown function. Students following the BASIL curriculum use a combination of sequence and structure alignment tools to study these structures with the goal of identifying possible enzymes. They then use molecular docking to predict what model substrates fit near a proposed active site. Students can produce the target enzymes in the lab using standard wet-lab biochemistry techniques for expression and purification, and they then perform kinetic assays with model substrates selected from their docking studies. We have successfully used this curriculum in biochemistry lab courses for majors and non-majors. The curriculum is modular and can be used as a whole or individual parts may be incorporated into an existing course - either lecture or lab. Curriculum materials are available free-of-charge at basilbiochem.org. We are currently investigating how this curriculum can be used in a variety of academic settings, and we welcome new collaborators. We can offer synchronous support via virtual meetings and asynchronous support via Slack. This project is supported in part by NSF IUSE 2141908.

A Revised View of the LSU Gene Family: New Functions in Plant Stress Responses and Phytohormone Signaling.

LSUs (RESPONSE TO LOW SULFUR) are plant-specific proteins of unknown function that were initially identified during transcriptomic studies of the sulfur deficiency response in Arabidopsis. Recent functional studies have shown that LSUs are important hubs of protein interaction networks with potential roles in plant stress responses. In particular, LSU proteins have been reported to interact with members of the brassinosteroid, jasmonate signaling, and ethylene biosynthetic pathways, suggesting that LSUs may be involved in response to plant stress through modulation of phytohormones. Furthermore, in silico analysis of the promoter regions of LSU genes in Arabidopsis has revealed the presence of cis-regulatory elements that are potentially responsive to phytohormones such as ABA, auxin, and jasmonic acid, suggesting crosstalk between LSU proteins and phytohormones. In this review, we summarize current knowledge about the LSU gene family in plants and its potential role in phytohormone responses.

Discovering and prioritizing candidate resistance genes against soybean pests by integrating GWAS and gene coexpression networks

Soybean is one of the most important legume crops worldwide. Soybean pests have a considerable impact on crop yield. Here, we integrated publicly available genome-wide association studies and transcriptomic data to prioritize candidate resistance genes against the insects Aphis glycines and Spodoptera litura, and the nematode Heterodera glycines. We identified 171, 7, and 228 high-confidence candidate resistance genes against A. glycines, S. litura, and H. glycines, respectively. We found some overlap of candidate genes between insect species, but not between insects and H. glycines. Although 15% of the prioritized candidate genes encode proteins of unknown function, the vast majority of the candidates are related to plant immunity processes, such as transcriptional regulation, signaling, oxidative stress, recognition, and physical defense. Based on the number of resistance alleles, we selected the ten most promising accessions against each pest species in the soybean USDA germplasm. The most resistant accessions do not reach the maximum theoretical resistance potential, indicating that they might be further improved to increase resistance in breeding programs or through genetic engineering. Finally, the coexpression networks we inferred in this work are available in a user-friendly web application (https://soypestgcn.venanciogroup.uenf.br/) and an R/Shiny package (https://github.com/almeidasilvaf/SoyPestGCN) that serve as a public resource to explore soybean-pest interactions at the transcriptional level.

The Oxidative Stress-Induced Hypothetical Protein PG_0686 in Porphyromonas gingivalis W83 Is a Novel Diguanylate Cyclase.

The survival/adaptation of Porphyromonas gingivalis to the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. Several functional classes of genes, depending on the severity and duration of the exposure, were induced in P. gingivalis under H2O2-induced oxidative stress. The PG_0686 gene was highly upregulated under prolonged oxidative stress. PG_0686, annotated as a hypothetical protein of unknown function, is a 60 kDa protein that carries several domains including hemerythrin, PAS10, and domain of unknown function (DUF)-1858. Although PG_0686 showed some relatedness to several diguanylate cyclases (DGCs), it is missing the classical conserved, active site sequence motif (GGD[/E]EF), commonly observed in other bacteria. PG_0686-related proteins are observed in other anaerobic bacterial species. The isogenic mutant P. gingivalis FLL361 (ΔPG_0686::ermF) showed increased sensitivity to H2O2, and decreased gingipain activity compared to the parental strain. Transcriptome analysis of P. gingivalis FLL361 showed the dysregulation of several gene clusters/operons, known oxidative stress resistance genes, and transcriptional regulators, including PG_2212, CdhR and PG_1181 that were upregulated under normal anaerobic conditions. The intracellular level of c-di-GMP in P. gingivalis FLL361 was significantly decreased compared to the parental strain. The purified recombinant PG_0686 (rPG_0686) protein catalyzed the formation of c-di-GMP from GTP. Collectively, our data suggest a global regulatory property for PG_0686 that may be part of an unconventional second messenger signaling system in P. gingivalis. Moreover, it may coordinately regulate a pathway(s) vital for protection against environmental stress, and is significant in the pathogenicity of P. gingivalis and other anaerobes. IMPORTANCE Porphyromonas gingivalis is an important etiological agent in periodontitis and other systemic diseases. There is still a gap in our understanding of the mechanisms that P. gingivalis uses to survive the inflammatory microenvironment of the periodontal pocket. The hypothetical PG_0686 gene was highly upregulated under prolonged oxidative stress. Although the tertiary structure of PG_0686 showed little relatedness to previously characterized diguanylate cyclases (DGCs), and does not contain the conserved GGD(/E)EF catalytic domain motif sequence, an ability to catalyze the formation of c-di-GMP from GTP is demonstrated. The second messenger pathway for c-di-GMP was previously predicted to be absent in P. gingivalis. PG_0686 paralogs are identified in other anaerobic bacteria. Thus, PG_0686 may represent a novel class of DGCs, which is yet to be characterized. In conclusion, we have shown, for the first time, evidence for the presence of c-di-GMP signaling with environmental stress protective function in P. gingivalis.

Lack of a protective effect of the Tmem106b “protective SNP” in the Grn knockout mouse model for frontotemporal lobar degeneration

Genetic variants in TMEM106B are a common risk factor for frontotemporal lobar degeneration and the most important modifier of disease risk in patients with progranulin (GRN) mutations (FTLD-GRN). TMEM106B is encoding a lysosomal transmembrane protein of unknown molecular function. How it mediates its disease-modifying function remains enigmatic. Several TMEM106B single nucleotide polymorphisms (SNPs) are significantly associated with disease risk in FTLD-GRN carriers, of which all except one are within intronic sequences of TMEM106B. Of note, the non-coding SNPs are in high linkage disequilibrium with the coding SNP rs3173615 located in exon six of TMEM106B, resulting in a threonine to serine change at amino acid 185 in the minor allele, which is protective in FTLD-GRN carriers. To investigate the functional consequences of this variant in vivo, we generated and characterized a knockin mouse model harboring the Tmem106bT186S variant. We analyzed the effect of this protective variant on FTLD pathology by crossing Tmem106bT186S mice with Grn−/− knockout mice, a model for GRN-mediated FTLD. We did not observe the amelioration of any of the investigated Grn−/− knockout phenotypes, including transcriptomic changes, lipid alterations, or microgliosis in Tmem106bT186S/T186S × Grn−/− mice, indicating that the Tmem106bT186S variant is not protective in the Grn−/− knockout mouse model. These data suggest that effects of the associated SNPs not directly linked to the amino acid exchange in TMEM106B are critical for the modifying effect.

Complete genome sequence of a novel fusarivirus from Rhizoctonia solani AG-3 PT strain 3P-2-2.

Here, we describe a novel mycovirus, tentatively designated as "Rhizoctonia solani fusarivirus 6" (RsFV6), which was discovered in Rhizoctonia solani AG-3 PT strain 3P-2-2. The virus has a single-stranded positive-sense RNA (+ssRNA) genome of 6141 nucleotides containing two open reading frames (ORFs) and a poly(A) tail. ORF1 encodes a large polypeptide of 1,862 amino acids (aa) with conserved RNA-dependent RNA polymerase (RdRp) and helicase (Hel) domains. ORF2 encodes a putative 167-aa protein of unknown function. BLASTp searches revealed that the ORF1-encoded polypeptide showed the highest sequence similarity (70.67% identity) to that of Rhizoctonia solani fusarivirus 3 (RsFV3), which was isolated from Rhizoctonia solani AG-2-2LP. Multiple sequence alignments and phylogenetic analysis based on RdRp and Hel sequences indicated that RsFV6 could be a novel member of the genus Alphafusarivirus family Fusariviridae.

Identification and functional study of fhx-L1, a major silk component in Bombyx mori

The silkworm cocoon was composed of fibroins, sericins, protease inhibitors, and proteins of unknown function. In this study, we focused on fhx-L1 (fibrohexamerin-like1), which was the homolog of fibroin fhx (fibrohexamerin). We identified 154 fhx family genes in 44 Lepidoptera insects, and seven fhx-Ls were found in Bombyx mori. Fhx-L1 was the most abundant of these proteins in silk and was specifically expressed in the silk gland. Immunofluorescence analysis showed that fhx-L1 was secreted into the whole sericin layers, similar to sericin1 (ser1). Western blotting revealed that the fhx-L1 protein contains N-linked oligosaccharide chains. CRISPR/Cas9-mediated gene editing was used to generate a homozygous mutant of fhx-L1 (fhx-L1KO). The cocoon of fhx-L1KO was larger and fluffier than that of the wild-type (WT), which was attributed to the lower adhesion between silk fibers. We also found that the content of β-sheet in the mutant silk was lower than in the WT silk, which resulted in further deterioration of the mechanical properties of the fhx-L1KO silk. Our study revealed the properties and function of fhx-L1 as a major structural component in silk. Then, our study provided a potential insight for in-depth study of silk protein function.

Integrated GWAS and transcriptomic analysis reveal the candidate salt-responding genes regulating Na+/K+ balance in barley (Hordeum vulgare L.).

Salt stress is one of the main abiotic stresses affecting crop yield and quality. Barley has strong salt tolerance, however, the underlying genetic basis is not fully clear, especially in the seedling stage. This study examined the ionic changes in barley core germplasms under the control and salt conditions. Genome-wide association study (GWAS) analysis revealed 54 significant SNPs from a pool of 25,342 SNPs distributed in 7 chromosomes (Chr) of the Illumina Barley 50K SNP array. These SNPs are associated with ion homeostasis traits, sodium (Na+) and potassium (K+) content, and Na+/K+ ratio representing five genomic regions on Chr 2, 4, 5, 6, and 7 in the leaves of worldwide barley accessions. And there are 3 SNP peaks located on the Chr 4, 6, and 7, which could be the "hot spots" regions for mining and identifying candidate genes for salt tolerance. Furthermore, 616 unique candidate genes were screened surrounding the significant SNPs, which are associated with transport proteins, protein kinases, binding proteins, and other proteins of unknown function. Meanwhile, transcriptomic analysis (RNA-Seq) was carried out to compare the salt-tolerant (CM72) and salt-sensitive (Gairdner) genotypes subjected to salt stress. And there was a greater accumulation of differentially expressed genes(DEGs) in Gairdner compared to CM72, mainly enriched in metabolic pathway, biosynthesis of secondary metabolites, photosynthesis, signal transduction,emphasizing the different transcriptional response in both genotypes following salt exposure. Combined GWAS and RNA-Seq analysis revealed 5 promising salt-responding genes (PGK2, BASS3, SINAT2, AQP, and SYT3) from the hot spot regions, which were verified between the salt-tolerant and salt-sensitive varieties by qRT-PCR. In all, these results provide candidate SNPs and genes responsible for salinity responding in barley, and a new idea for studying such genetic basis in similar crops.

TMEM161B regulates cerebral cortical gyration, Sonic Hedgehog signaling, and ciliary structure in the developing central nervous system

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation invitro and disrupted cortical histogenesis invivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.

TMEM161B modulates radial glial scaffolding in neocortical development

TMEM161B encodes an evolutionarily conserved widely expressed novel 8-pass transmembrane protein of unknown function in human. Here we identify TMEM161B homozygous hypomorphic missense variants in our recessive polymicrogyria(PMG) cohort. Patients carrying TMEM161B mutations exhibit striking neocortical PMG and intellectual disability. Tmem161b knockout mice fail to develop midline hemispheric cleavage, whereas knock-in of patient mutations and patient-derived brain organoids show defects in apical cell polarity and radial glial scaffolding. We found that TMEM161B modulates actin filopodia, functioning upstream of the Rho-GTPase CDC42. Our data link TMEM161B with human PMG, likely regulating radial glia apical polarity during neocortical development.