R2 Repeat Research Articles

Genome-wide analysis of MYB transcription factors and their responses to salt stress in Casuarina equisetifolia

BackgroundMYB transcription factors are a kind of DNA binding protein that can specifically interact with the promoter region. Members of MYB TFs are widely involved in plant growth and development, secondary metabolism, stress response, and hormone signal transduction. However, there is no report of comprehensive bioinformatics analysis on the MYB family of Casuarina equisetifolia.ResultsIn this study, bioinformatics methods were used to screen out 182 MYB transcription factors from the Casuarina equisetifolia genome database, including 69 1R-MYB, 107 R2R3-MYB, 4 R1R2R3-MYB, and 2 4R-MYB. The C. equisetifolia R2R3-MYB genes were divided into 29 groups based on the phylogenetic topology and the classification of the MYB superfamily in Arabidopsis thaliana, while the remaining MYB genes (1R-MYB, R1R2R3-MYB, and 4R-MYB) was divided into 19 groups. Moreover, the conserved motif and gene structure analysis shown that the members of the CeqMYBs were divided into the same subgroups with mostly similar gene structures. In addition, many conserved amino acids in the R2 and R3 domains of CeqMYBs by WebLogo analysis, especially tryptophan residues (W), with 3 conserved W in R2 repeat and 2 conserved W in R3 repeat. Combining promoter and GO annotation analysis, speculated on the various biological functions of CeqMYBs, thus 32 MYB genes were selected to further explore its response to salt stress by using qPCR analysis technique. Most CeqMYB genes were differentially regulated following multiple salt treatments.ConclusionsSeven genes (CeqMYB164, CeqMYB4, CeqMYB53, CeqMYB32, CeqMYB114, CeqMYB71 and CeqMYB177) were assigned to the “response to salt stress” by GO annotation. Among them, the expression level of CeqMYB4 was up-regulated under various salt treatments, indicating CeqMYB4 might participated in the response to salt stress. Our results provide important information for the biological function of C. equisetifolia, as well as offer candidate genes for further study of salt stress mechanism.

Inclusion of the C-Terminal Domain in the β-Sheet Core of Heparin-Fibrillized Three-Repeat Tau Protein Revealed by Solid-State Nuclear Magnetic Resonance Spectroscopy.

Many neurodegenerative diseases such as Alzheimer's disease are characterized by pathological β-sheet filaments of the tau protein, which spread in a prion-like manner in patient brains. To date, high-resolution structures of tau filaments obtained from patient brains show that the β-sheet core only includes portions of the microtubule-binding repeat domains and excludes the C-terminal residues, indicating that the C-terminus is dynamically disordered. Here, we use solid-state NMR spectroscopy to identify the β-sheet core of full-length 0N3R tau fibrillized using heparin. Assignment of 13C and 15N chemical shifts of the rigid core of the protein revealed a single predominant β-sheet conformation, which spans not only the R3, R4, R' repeats but also the entire C-terminal domain (CT) of the protein. This massive β-sheet core qualitatively differs from all other tau fibril structures known to date. Using long-range correlation NMR experiments, we found that the R3 and R4 repeats form a β-arch, similar to that seen in some of the brain-derived tau fibrils, but the R1 and R3 domains additionally stack against the CT, reminiscent of previously reported transient interactions of the CT with the microtubule-binding repeats. This expanded β-sheet core structure suggests that the CT may have a protective effect against the formation of pathological tau fibrils by shielding the amyloidogenic R3 and R4 domains, preventing side-on nucleation. Truncation and post-translational modification of the CT in vivo may thus play an important role in the progression of tauopathies.

Direct Observation of the Self-Aggregation of R3R4 Bi-repeat of Tau Protein.

Different cryo-EM derived atomic models of in vivo tau filaments from patients with tauopathies consisted of R3 and R4 repeats of the microtubule-binding domain. In comparison, only the R3 repeat forms the core of the heparin-induced fibrils of the three repeat tau isoforms. For developing therapeutics, it is desirable to have an in vitro tau aggregation system producing fibrils corresponding to the disease morphology. Here we report the self-aggregation of truncated tau segment R3R4 peptide without requiring heparin for aggregation induction. We used NMR spectroscopy and other biophysical methods to monitor the self-aggregation of R3R4. We identified the hexapeptide region in R3 and β-turn region in R4 as the aggregation initiating region of the protein. The solid-state NMR of self-aggregated R3R4 fibrils demonstrated that in addition to R3 residues, residues of R4 were also part of the fibril filaments. The presence of both R3 and R4 residues in the aggregation process and the core of fibril filaments suggest that the aggregation of R3R4 might resemble the in vivo aggregation process.

Curli-Mediated Self-Assembly of a Fibrous Protein Scaffold for Hydroxyapatite Mineralization.

Nanostructures formed by self-assembled peptides have been increasingly exploited as functional materials for a wide variety of applications, from biotechnology to energy. However, it is sometimes challenging to assemble free short peptides into functional supramolecular structures, since not all peptides have the ability to self-assemble. Here, we report a self-assembly mechanism for short functional peptides that we derived from a class of fiber-forming amyloid proteins called curli. CsgA, the major subunit of curli fibers, is a self-assembling β-helical subunit composed of five pseudorepeats (R1-R5). We first deleted the internal repeats (R2, R3, R4), known to be less essential for the aggregation of CsgA monomers into fibers, forming a truncated CsgA variant (R1/R5). As a proof-of-concept to introduce functionality in the fibers, we then genetically substituted the internal repeats by a hydroxyapatite (HAP)-binding peptide, resulting in a R1/HAP/R5 construct. Our method thus utilizes the R1/R5-driven self-assembly mechanism to assemble the HAP-binding peptide and form hydrogel-like materials in macroscopic quantities suitable for biomineralization. We confirmed the expression and fibrillar morphology of the truncated and HAP-containing curli-like amyloid fibers. X-ray diffraction and TEM showed the functionality of the HAP-binding peptide for mineralization and formation of nanocrystalline HAP. Overall, we show that fusion to the R1 and R5 repeats of CsgA enables the self-assembly of functional peptides into micron long fibers. Further, the mineral-templating ability that the R1/HAP/R5 fibers possesses opens up broader applications for curli proteins in the tissue engineering and biomaterials fields.

Alternatively Spliced BnaPAP2.A7 Isoforms Play Opposing Roles in Anthocyanin Biosynthesis of Brassica napus L.

Brassica napus L. (rapeseed, oilseed rape, and canola) and varieties of its two diploid parents, B. oleracea and B. rapa, display a large amount of variation in anthocyanin pigmentation of the leaf, stem, and fruit. Here, we demonstrate that BnaPAP2.A7, an ortholog of the B. oleracea anthocyanin activator BoMYB2 that confers purple traits, positively regulates anthocyanin biosynthesis in leaves of B. napus. Sequencing of BnaPAP2.A7 and transgenic analysis suggests that activation of this gene in purple rapeseed may result from a single nucleotide and/or 2bp insertion in its promoter region. BnaPAP2.A7 gives rise to three splice variants, designated BnaPAP2.A7-744, BnaPAP2.A7-910, and BnaPAP2.A7-395 according to the length of the transcripts. While BnaPAP2.A7-744 encodes a full-length R2R3-MYB, both BnaPAP2.A7-910 and BnaPAP2.A7-395 encode truncated proteins that lack both a partial R3 repeat and the complete C terminal domain, and so in vitro are unable to interact with the Arabidopsis bHLH protein AtTT8. Although expression of either BnaPAP2.A7-910 or BnaPAP2.A7-395 in green rapeseed does not result in purple leaves, both genes do modify genome-wide gene expression, with a strong repression of anthocyanin-related genes. We have demonstrated that BnaPAP.A7 regulates anthocyanin accumulation in leaves of B. napus and propose a potential mechanism for modulation of anthocyanin biosynthesis by alternative splicing.

Isolation and functional validation of stress tolerant EaMYB18 gene and its comparative physio-biochemical analysis with transgenic tobacco plants overexpressing SoMYB18 and SsMYB18.

MYB transcription factors are one of the most important mediators for the survival of plants under multiple stress responses. In the present study, EaMYB18, encoding a single R3 repeat MYB DNA binding domain was isolated from stress-tolerant wild relative species of sugarcane Erianthus arundinaceus. In silico analysis of 948bp coding mRNA sequence of EaMYB18 exhibited the presence of four exons and three introns. Further, the EaMYB18 gene was transformed in tobacco and its stable inheritance was confirmed through antibiotic resistance screening, PCR amplification and Southern hybridization blotting. Results of the estimation of MDA, proline, total chlorophyll and antioxidant activities of EaMYB18 transgenic tobacco lines exhibited least oxidative damage under drought and cold stress over the untransformed ones, the over-expression of EaMYB18 has improved drought and cold stress tolerance ability in tobacco. The comparative physiological and biochemical analysis of transgenic tobacco plants overexpressing SoMYB18, SsMYB18 and EaMYB18, revealed that the EaMYB18 and SsMYB18 transgenic plants demonstrated effective tolerance to drought and cold stresses, while SoMYB18 showed improved tolerance to salt stress alone. Amongst these three genes, EaMYB18 displayed the highest potential for drought and cold stress tolerances as compared to SoMYB18 and SsMYB18 genes.

Structure-function Relationships in Biofilms Characterizing the Staphylococcal Autolysin R2 Repeat Domain

Staphylococcal autolysin is an enzyme involved in the hydrolysis of peptidoglycan contained in cell walls. Certain structures of bacterial cell walls serve as receptors for autolysin repeats, such as the R2ab repeat, and these repeat domains possess unique ligand binding properties. The autolysin repeat domains have also been implicated in bacterial attachment to surfaces during the early stages of biofilm formation. The R2ab repeat consists of two SH3b domains that were found to interact with serum proteins vitronectin and fibronectin. Knowledge of the solution structure and binding energetics of R2ab is therefore necessary to understand its role in interacting with serum proteins and inorganic surfaces. We measured the thermodynamic stability of R2ab, and the Tm and ∆H were found to be 51.2 °C and 24.4 kcal mol−1, respectively. No evidence for multi-state unfolding was observed. We also determined the solution structure of R2ab and studied its backbone dynamics. Analysis of 15N NMR relaxation experiments reveals that the two domains maintain a rigid conformation, and no inter-domain motions are observed. Order parameters were in agreement with the relaxation data, revealing a dynamic C-terminus. Finally, we investigated R2ab behavior in the presence of 30 nm silica nanoparticles (SiNPs), an approximate model for a glass surface. Our binding capacity data suggests that R2ab denatures on the SiNP surface. In future studies, the R2ab binding site with fibronectin will be identified using chemical shift perturbation. Together, these experiments may result in a better understanding of how R2ab interacts with surfaces and proteins, potentially leading to new approaches for slowing biofilm formation.

Structural insights into target DNA recognition by R2R3-MYB transcription factors.

As the largest group of MYB family transcription factors, R2R3-MYB proteins play essential roles during plant growth and development. However, the structural basis underlying how R2R3-MYBs recognize the target DNA remains elusive. Here, we report the crystal structure of Arabidopsis WEREWOLF (WER), an R2R3-MYB protein, in complex with its target DNA. Structural analysis showed that the third α-helices in both the R2 and R3 repeats of WER fit in the major groove of the DNA, specifically recognizing the DNA motif 5′-AACNGC-3′. In combination with mutagenesis, in vitro binding and in vivo luciferase assays, we showed that K55, N106, K109 and N110 are critical for the function of WER. Although L59 of WER is not involved in DNA binding in the structure, ITC analysis suggested that L59 plays an important role in sensing DNA methylation at the fifth position of cytosine (5mC). Like 5mC, methylation at the sixth position of adenine (6mA) in the AAC element also inhibits the interaction between WER and its target DNA. Our study not only unravels the molecular basis of how WER recognizes its target DNA, but also suggests that 5mC and 6mA modifications may block the interaction between R2R3-MYB transcription factors and their target genes.

Plant Polyphenols Inhibit Functional Amyloid and Biofilm Formation in Pseudomonas Strains by Directing Monomers to Off-Pathway Oligomers.

Self-assembly of proteins to β-sheet rich amyloid fibrils is commonly observed in various neurodegenerative diseases. However, amyloid also occurs in the extracellular matrix of bacterial biofilm, which protects bacteria from environmental stress and antibiotics. Many Pseudomonas strains produce functional amyloid where the main component is the highly fibrillation-prone protein FapC. FapC fibrillation may be inhibited by small molecules such as plant polyphenols, which are already known to inhibit formation of pathogenic amyloid, but the mechanism and biological impact of inhibition is unclear. Here, we elucidate how polyphenols modify the self-assembly of functional amyloid, with particular focus on epigallocatechin gallate (EGCG), penta-O-galloyl-β-d-glucose (PGG), baicalein, oleuropein, and procyanidin B2. We find EGCG and PGG to be the best inhibitors. These compounds inhibit amyloid formation by redirecting the aggregation of FapC monomers into oligomeric species, which according to small-angle X-ray scattering (SAXS) measurements organize into core-shell complexes of short axis diameters 25–26 nm consisting of ~7 monomers. Using peptide arrays, we identify EGCG-binding sites in FapC’s linker regions, C and N-terminal parts, and high amyloidogenic sequences located in the R2 and R3 repeats. We correlate our biophysical observations to biological impact by demonstrating that the extent of amyloid inhibition by the different inhibitors correlated with their ability to reduce biofilm, highlighting the potential of anti-amyloid polyphenols as therapeutic agents against biofilm infections.

Specificity of MYB interactions relies on motifs in ordered and disordered contexts.

Physical interactions between members of the MYB and bHLH transcription factor (TF) families regulate many important biological processes in plants. Not all reported MYB–bHLH interactions can be explained by the known binding sites in the R3 repeat of the MYB DNA-binding domain. Noteworthy, most of the sequence diversity of MYB TFs lies in their non-MYB regions, which contain orphan small subgroup-defining motifs not yet linked to molecular functions. Here, we identified the motif mediating interaction between MYB TFs from subgroup 12 and their bHLH partners. Unlike other known MYB–bHLH interactions, the motif locates to the centre of the predicted disordered non-MYB region. We characterised the core motif, which enabled accurate prediction of previously unknown bHLH-interacting MYB TFs in Arabidopsis thaliana, and we confirmed its functional importance in planta. Our results indicate a correlation between the MYB–bHLH interaction affinity and the phenotypic output controlled by the TF complex. The identification of an interaction motif outside R3 indicates that MYB–bHLH interactions must have arisen multiple times, independently and suggests many more motifs of functional relevance to be harvested from subgroup-specific studies.

In vitro 0N4R tau fibrils contain a monomorphic β-sheet core enclosed by dynamically heterogeneous fuzzy coat segments

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single β-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit β-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

Protection Efficacy of the rLTB-R1 Chimera against Experimental Swine Mycoplasmal Pneumonia

Background: Mycoplasma hyopneumoniaeis the etiological agent of the Swine Mycoplasmal Pneumonia (SMP), one of the most economically significant diseases in the swine industry worldwide. Commonly used vaccines for SMP control consist of inactivated whole cells (bacterins). These vaccines are efficacious against M. hyopneumoniaechallenge, but do not prevent colonization by the pathogen or completely eliminate pneumonia. P97 adhesin is conserved in the M. pneumoniae virulent strains, therefore it is an attractive target to be used in recombinant vaccines against M. hyopneumoniae. The aim of the present study was to evaluate protection afforded by rLTB-R1, a recombinant chimera composed by LTB fused with the R1 repeat region of P97 adhesin of M. hyopneumoniae, in specific-pathogen-free (SPF) piglets vaccinated by intranasal or intramuscular route and challenged with a pathogenic strain of M. hyopneumoniae.Materials, Methods & Results:PCR products of the LTB and R1 coding sequences were fused, then cloned into pETDEST42™ expression vector. The rLTB-R1 was expressed in Escherichia coli BL21 (DE3) Salt induction (SI).They were allocated into three groups: four piglets were intranasally vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IN rLTB-R1 group); four piglets were intramuscularly vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IM rLTB-R1 group);three piglets were intranasally and intramuscularly inoculated with 1 mL of PBS (control group).Two weeks after the last immunization (28 day), piglets were intratracheally challenged with 10 mL of a suspension containing 109color-changing unit (CCU) of pathogenic M. hyopneumoniae 7448 strain on three consecutive days. Until the challenge (28 days), intranasal and intramuscular vaccination with rLTB-R1 induced seroconversions of anti-R1 systemic antibodies of 1.6 and 4.6 ×, respectively. The IN rLTB-R1 group had no pulmonary lesion, rLTB-R1 conferred protection against experimental SMP. On the other hand, IM rLTB-R1 and control groups had on average 7.24% and 8.46% of pulmonary lesion, respectively, showing that intramuscular vaccination with rLTB-R1 did not confer protection.Discussion:The rLTB-R1, when intranasally administrated to mice, elicited production of anti-R1 IgA in trachea and bronchi as well as specific Th1 response, suggesting an adequate stimulation of the mucosal immune system. We believe that rLTB-R1 induced a similar immune response in piglets intranasally vaccinated, conferring protection against experimental SMP. The present study, the rLTB-R1 alone, without any chemical adjuvant, stimulated a significant seroconversion of anti-R1 systemic antibodies in pigs intramuscularly vaccinated, showing the potential of LTB as a parenteral adjuvant in swine vaccination.Previous work has shown that the intramuscular administration route was evaluated in pigs because mice intramuscularly vaccinated with rLTB-R1 presented significant levels of anti-R1 IgA in trachea and bronchi, suggesting that rLTB can stimulate some degree of mucosal immunity even if not delivered by a mucosal route.However, in the present study, piglets intramuscularly vaccinated with rLTB-R1 presented high levels of anti-R1 systemic antibodies, they were not protected.On the other hand, intranasal vaccination of piglets with rLTB-R1 elicited low levels of anti-R1 systemic antibodies (1.6 × at 28 days), but it conferred full protection against experimental SMP. The present study demonstrated that intranasal vaccination of piglets with rLTB-R1 conferred protection against experimental SMP. A more detailed analysis of the protective immune response induced by rLTB-R1 in pigs is currently being performed.

Genome-wide characterization and expression analyses of the MYB superfamily genes during developmental stages in Chinese jujube.

The MYB transcription factor (TF) superfamily, one of the largest gene superfamilies, regulates a variety of physiological processes in plants. Although many MYB superfamily genes have been identified in plants, the MYB TFs in Chinese jujube (Ziziphus jujuba Mill.) have not been fully identified and characterized. Additionally, the functions of these genes remain unclear. In total, we identified 171 MYB superfamily genes in jujube and divided them into five subfamilies containing 99 genes of the R2R3-MYB subfamily, 58 genes of the MYB-related subfamily, four genes of the R1R2R3-MYB subfamily, one gene of the 4R-MYB subfamily, and nine genes of the atypical MYB subfamily. The 99 R2R3-MYB genes of jujube were divided into 35 groups, C1–C35, and the 58 MYB-related genes were divided into the following groups: the R-R-type, CCA1-like, I-box-binding-like, TBP-like, CPC-like, and Chinese jujube-specific groups. ZjMYB genes in jujube were well supported by additional highly conserved motifs and exon/intron structures. Most R1 repeats of MYB-related proteins comprised the R2 repeat and had highly conserved EED and EEE residue groups in jujube. Three tandem duplicated gene pairs were found on 12 chromosomes in jujube. According to an expression analysis of 126 ZjMYB genes, MYB-related genes played important roles in jujube development and fruit-related biological processes. The total flavonoid content of jujube fruit decreased as ripening progressed. A total of 93 expressed genes were identified in the RNA-sequencing data from jujube fruit, and 56 ZjMYB members presented significant correlations with total flavonoid contents by correlation analysis. Five pairs of paralogous MYB genes within jujube were composed of nine jujube MYB genes. A total of 14 ZjMYB genes had the same homology to the MYB genes of Arabidopsis and peach, indicating that these 14 MYB genes and their orthologs probably existed before the ancestral divergence of the MYB superfamily. We used a synteny analysis of MYB genes between jujube and Arabidopsis to predict that the functions of the ZjMYBs involve flavonoid/phenylpropanoid metabolism, the light signaling pathway, auxin signal transduction, and responses to various abiotic stresses (cold, drought, and salt stresses). Additionally, we speculate that ZjMYB108 is an important TF involved in the flavonoid metabolic pathway. This comprehensive analysis of MYB superfamily genes in jujube lay a solid foundation for future comprehensive analyses of ZjMYB gene functions.

Cloning and Optimizing the Culture Parameters for Expression of R1 and R2 Repeat Regions of P97 Adhesin from Mycoplasma hyopneumoniae in Escherichia coli

Cloning and Optimizing the Culture Parameters for Expression of R1 and R2 Repeat Regions of P97 Adhesin from Mycoplasma hyopneumoniae in Escherichia coli

Two amino acid changes in the R3 repeat cause functional divergence of two clustered MYB10 genes in peach.

R2R3-MYB genes play a pivotal role in regulating anthocyanin accumulation. Here, we report two tandemly duplicated R2R3-MYB genes in peach, PpMYB10.1 and PpMYB10.2, with the latter showing lower ability to induce anthocyanin accumulation than the former. Site-directed mutation assay revealed two amino acid changes in the R3 repeat, Arg/Lys66 and Gly/Arg93, responsible for functional divergence between these two PpMYB10 genes. Anthocyanin-promoting activity of PpMYB10.2 was significantly increased by a single amino acid replacement of Arg93 with Gly93. However, either the Gly93 → Arg93 or Arg66 → Lys66 substitutions alone showed little impact on anthocyanin-promoting activity of PpMYB10.1, but simultaneous substitutions caused a significant decrease. Reciprocal substitution of Arg/Gly93 could significantly alter binding affinity to PpbHLH3, while the Arg66 → Lys66 substitution is predicted to affect the folding of the MYB DNA-binding domain, instead of PpbHLH3-binding affinity. Overall, the change of anthocyanin-promoting activity was accompanied with that of bHLH-binding affinity, suggesting that DNA-binding affinity of R2R3-MYBs depends on their bHLH partners. Our study is helpful for understanding of functional evolution of R2R3-MYBs and their interaction with DNA targets.

The R2R3MYB Gene Family in Phyllostachys edulis: Genome-Wide Analysis and Identification of Stress or Development-Related R2R3MYBs.

The MYB transcription factor (TF) is one of the largest gene families in plants and involved to multiple biological processes. However, little is known about the MYB family and its functional role in the genome of moso bamboo. In the present study, a total of 114 R2R3MYB genes were first identified from moso bamboo genome and full-length non-chimeric (FLNC) reads. Phylogenetic analysis coupled with gene structure analysis and motif determination resulted in the division of these PheR2R3MYBs into 17 subgroups. The position of eight proteins along an external branch in the phylogenetic tree suggested their relatively ancient origin. The genes in this group were all substituted by (Met, M)/(Arg, R) at conservative W residues in both R2 and R3 repeats, and half were found to possess no transcriptional activation activity. The analysis of evolutionary patterns and divergence suggests that the expansion of PheMYBs was mainly attributable to whole genome duplication (WGD) under different selection pressures. Expressional analysis based on microarray and qRT-PCR data performed diverse expression patterns of R2R3MYBs in response to both various abiotic stimuli and flower development. Furthermore, the co-expression analysis of R2R3MYBs suggested an intricate interplay of growth- and stress-related responses. Finally, we found a hub gene, PheMYB4, was involved in a complex proteins interaction network. Further functional analysis indicated that ectopic overexpression of its homologous gene, PheMYB4-1, could increase tolerance to cold treatment and sensitivity to drought and salt treatment of transgenic Arabidopsis seedlings. These findings provide comprehensive insights into the MYB family members in moso bamboo and offer candidate MYB genes for further studies on their roles in stress resistance.

Genome-wide identification and classification of MYB superfamily genes in peach.

The MYB transcription factor superfamily is one of the largest superfamilies modulating various biological processes in plants. Over the past few decades, many MYB superfamily genes have been identified and characterized in some plant species. However, genes belonging to the MYB superfamily in peach (Prunus persica) have not been comprehensively identified and characterized although the genome sequences of peach were released several years ago. In total, this study yielded a set of 256 MYB superfamily genes that was divided into five subfamilies: the R2R3-MYB (2R-MYB), R1R2R3-MYB (3R-MYB), MYB-related (1R-MYB), 4R-MYB, and Atypical-MYB subfamilies. These subfamilies contained 128, 4, 109, 1, and 14 members, respectively. The 128 R2R3-MYB subfamily genes in peach were further clustered into 35 groups, and the 109 MYB-related subfamily genes were further clustered into 6 groups: the CCA1-like, CPC-like, TBP-like, I-box-binding-like, R-R-type, and Peach-specific groups. The motif compositions and exon/intron structures within each group within the R2R3-MYB or MYB-related subfamily in peach were highly conserved. The logo sequences of the R2 and R3 repeats of R2R3-MYB subfamily members were highly conserved with those in these repeats of several other plant species. Except for 48 novel peach-specific MYB genes, the remaining 208 out of 256 MYB genes in peach were conserved with the corresponding 198 MYB genes in A. thaliana. Additionally, the 256 MYB genes unevenly distributed on chromosomes 1 to 8 of the peach genome. Eighty-one orthologous pairs of peach/A. thaliana MYB genes were identified among 256 MYB genes in peach and 198 MYB genes in A. thaliana in this study. In addition, 146 pairs of paralogous MYB genes were identified on the eight chromosomes of peach. The expression levels of some of the 51 MYB genes selected for qRT-PCR analysis decreased or increased with red-fleshed fruit development, while the expression patterns of some genes followed no clear rules over the five developmental stages of fruits. This study laid the foundation for further functional analysis of MYB superfamily genes in peach and enriched the knowledge of MYB superfamily genes in plant species.

A common antigenic motif recognized by naturally occurring human VH5\u201351/VL4\u20131 anti-tau antibodies with distinct functionalities

Misfolding and aggregation of tau protein are closely associated with the onset and progression of Alzheimer’s Disease (AD). By interrogating IgG+ memory B cells from asymptomatic donors with tau peptides, we have identified two somatically mutated VH5–51/VL4–1 antibodies. One of these, CBTAU-27.1, binds to the aggregation motif in the R3 repeat domain and blocks the aggregation of tau into paired helical filaments (PHFs) by sequestering monomeric tau. The other, CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of tau seeds and mediates the uptake of tau aggregates into microglia by binding PHFs. Crystal structures revealed that the combination of VH5–51 and VL4–1 recognizes a common Pro-Xn-Lys motif driven by germline-encoded hotspot interactions while the specificity and thereby functionality of the antibodies are defined by the CDR3 regions. Affinity improvement led to improvement in functionality, identifying their epitopes as new targets for therapy and prevention of AD.

Isothermal Titration Calorimetry and Vesicle Leakage Assays Highlight the Differential Behaviors of Tau Repeat Segments Upon Interaction with Anionic Lipid Membranes

Alzheimer's Disease (AD) is the sixth-leading cause of death in the United States and is due to protein misfolding. One of the proteins implicated in this misfolding is Tau, which is a protein that stabilizes the cytoskeletal structure in neurons. Tau contains four repeat segments (R1-R4) that are crucial to its function, but are also the site of the misfolding that forms neurofibrillary tangles (NFTs). These NFTs inhibit signals between neurons and invokes processes which cause degradation of the neuronal cell. A number of previous studies reveal that anionic membranes interact with tau and induce misfolding of the protein. Here, we have investigated how each repeat segment of tau interacts with anionic and neutral membranes using isothermal titration calorimeter (ITC) and leakage assays. We have determined the equilibrium binding constants for peptide: membrane interactions, along with the thermodynamic parameters ΔH, ΔS, and ΔCp. We found that R2 and R3 repeat segments interact more favorably with anionic membranes, and none of the segments interacted strongly with neutral membranes. The differential behaviors of these sequences was also evident in their driving forces to membrane binding - both classical and nonclassical hydrophobic effects were observed among these sequences. Leakage assays showed that all sequences display lytic activity to anionic membranes but not neutral membranes. Taken together, these results align with models of NFT formation that invoke misfolding events at the membrane surface. Further exploration will include experiments using NMR to see how the phosphates on the side chains of the amino acids play a role in the structural composition of the repeat segments.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin.

BACKGROUNDThe B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant.OBJECTIVESWe aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead.METHODSBALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response.FINDINGSWestern blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide.MAIN CONCLUSIONSThe results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.