The SC10-RNase promoter displays changes in DNA methylation patterns through pistil development in self-incompatible Nicotiana alata.

In Solanaceae, self-incompatibility is a genetic mechanism that prevents endogamy in plant populations. Expression of the S-determinants, S-RNase, and SLF, is tightly regulated during pistil and pollen development. However, the molecular mechanism of gene expression regulation in S-RNase-based self-incompatibility systems must be better understood. Here, we identified a 1.3 Kbp sequence upstream to the coding region of the functional SC10-RNase allele from the self-incompatible Nicotiana alata, which directs SC10-RNase expression in mature pistils. This SC10-RNase promoter includes a 300 bp region with minimal elements that sustain the SC10-RNase expression. Likewise, a fragment of a transposable element from the Gypsy family of retrotransposons is also present at the -320 bp position. Nevertheless, its presence does not affect the expression of the SC10-RNase in mature pistils. Additionally, we determined that the SC10-RNase promoter undergoes different DNA methylation states during pistil development, being the mCHH methylation context the most frequent close to the transcription start site at pistil maturity. We hypothesized that the Gypsy element at the SC10-RNase promoter might contribute to the DNA methylation remodeling on the three sequence contexts analyzed here. We propose that mCHH methylation enrichment and other regulatory elements in the S-RNase coding region regulate the specific and abundant SC10-RNase expression in mature pistils in N. alata.

PbrBZR1 interacts with PbrARI2.3 to mediate brassinosteroid-regulated pollen tube growth during self-incompatibility signaling in pear.

S-RNase-mediated self-incompatibility (SI) prevents self-fertilization and promotes outbreeding to ensure genetic diversity in many flowering plants, including pear (Pyrus sp.). Brassinosteroids (BRs) have well-documented functions in cell elongation, but their molecular mechanisms in pollen tube growth, especially in the SI response, remain elusive. Here, exogenously applied brassinolide (BL), an active BR, countered incompatible pollen tube growth inhibition during the SI response in pear. Antisense repression of BRASSINAZOLE-RESISTANT1 (PbrBZR1), a critical component of BR signaling, blocked the positive effect of BL on pollen tube elongation. Further analyses revealed that PbrBZR1 binds to the promoter of EXPANSIN-LIKE A3 (PbrEXLA3) to activate its expression. PbrEXLA3 encodes an expansin that promotes pollen tube elongation in pear. The stability of dephosphorylated PbrBZR1 was substantially reduced in incompatible pollen tubes, where it is targeted by ARIADNE2.3 (PbrARI2.3), an E3 ubiquitin ligase that is strongly expressed in pollen. Our results show that during the SI response, PbrARI2.3 accumulates and negatively regulates pollen tube growth by accelerating the degradation of PbrBZR1 via the 26S proteasome pathway. Together, our results show that an ubiquitin-mediated modification participates in BR signaling in pollen and reveal the molecular mechanism by which BRs regulate S-RNase-based SI.

The Snapdragon Genomes Reveal the Evolutionary Dynamics of the S-Locus Supergene.

The genus Antirrhinum has been used as a model to study self-incompatibility extensively. The multi-allelic S-locus, carrying a pistil S-RNase and dozens of S-locus F-box (SLF) genes, underlies the genetic control of self-incompatibility (SI) in Antirrhinum hispanicum. However, there have been limited studies on the genomic organization of the S-locus supergene due to a lack of high-quality genomic data. Here, we present the chromosome-level reference and haplotype-resolved genome assemblies of a self-incompatible A. hispanicum line, AhS7S8. For the first time, 2 complete A. hispanicum S-haplotypes spanning ∼1.2 Mb and containing a total of 32 SLFs were reconstructed, whereas most of the SLFs derived from retroelement-mediated proximal or tandem duplication ∼122 Mya. Back then, the S-RNase gene and incipient SLFs came into linkage to form the pro-type of type-1 S-locus in the common ancestor of eudicots. Furthermore, we detected a pleiotropic cis-transcription factor (TF) associated with regulating the expression of SLFs, and two miRNAs may control the expression of this TF. Interspecific S-locus and intraspecific S-haplotype comparisons revealed the dynamic nature and polymorphism of the S-locus supergene mediated by continuous gene duplication, segmental translocation or loss, and TE-mediated transposition events. Our data provide an excellent resource for future research on the evolutionary studies of the S-RNase-based self-incompatibility system.

BAG6-A from Fragaria viridis pollen modulates gametophyte development in diploid strawberry.

Male and female gametophyte development processes are essential steps in the life cycles of all land plants. Here, we characterized a gene, FviBAG6-A, screened from the Fragaria viridis (2 n = 2x=14) pollen cDNA library and physically interacted with S-RNase. Ubiquitinated of Sa-RNase might be determined by the interaction of FviBAG6-A in the ubiquitin-proteasome system during fertilization. We found that overexpression of FviBAG6-A in Arabidopsis caused shorter silique length, and decreased silique number. Moreover, overexpression of FviBAG6-A in Fragaria vesca (2 n = 2x=14) led to a greatly reduced seed number, with nearly 80% of the seeds aborted. Analyses of paraffin sections and reactive oxygen species (ROS) content revealed that the majority of severe pollen defects were likely due to the early degradation of the tapetum and middle layer as a result of ROS accumulation and abnormal development of the uninucleate megaspore mother. Moreover, the FviBAG6-A interact with the E3 ligase SIZ1 and contribute to the SUMOylation of FviBAG6-A , which may be induced by the high level of ROS content, further promoting gametophyte abortion in strawberry transgenic lines. This study characterized the FviBAG6-A and reveals its novel function in gametophyte development.

Self S-RNase inhibits ABF-LRX signaling to arrest pollen tube growth to achieve self-incompatibility in pear.

Gametophytic self-incompatibility (GSI) has been widely studied in flowering plants, but studies of the mechanisms underlying pollen tube growth arrest by self S-RNase in GSI species are limited. In the present study, two leucine-rich repeat extensin genes in pear (Pyrus bretschneideri), PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real-time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of the self-compatible cultivar 'Jinzhui' (harboring a spontaneous bud mutation) than in those of the self-incompatible cultivar 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 stimulated pollen tube growth and attenuated the inhibitory effects of self S-RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. These results indicate that abnormal expression of PbLRXA2.1 and PbLRXA2.2 is involved in the loss of self-incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by the ABRE-binding factor PbABF.D.2. Knockdown of PbABF.D.2 decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expression of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 was repressed by self S-RNase, suggesting that self S-RNase can arrest pollen tube growth by restricting the PbABF.D.2-PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide novel insight into pollen tube growth arrest by self S-RNase.

Genome-Wide Analysis of the RNase T2 Family and Identification of Interacting Proteins of Four ClS-RNase Genes in 'XiangShui' Lemon.

S-RNase plays vital roles in the process of self-incompatibility (SI) in Rutaceae plants. Data have shown that the rejection phenomenon during self-pollination is due to the degradation of pollen tube RNA by S-RNase. The cytoskeleton microfilaments of pollen tubes are destroyed, and other components cannot extend downwards from the stigma and, ultimately, cannot reach the ovary to complete fertilisation. In this study, four S-RNase gene sequences were identified from the 'XiangShui' lemon genome and ubiquitome. Sequence analysis revealed that the conserved RNase T2 domains within S-RNases in 'XiangShui' lemon are the same as those within other species. Expression pattern analysis revealed that S3-RNase and S4-RNase are specifically expressed in the pistils, and spatiotemporal expression analysis showed that the S3-RNase expression levels in the stigmas, styles and ovaries were significantly higher after self-pollination than after cross-pollination. Subcellular localisation analysis showed that the S1-RNase, S2-RNase, S3-RNase and S4-RNase were found to be expressed in the nucleus according to laser confocal microscopy. In addition, yeast two-hybrid (Y2H) assays showed that S3-RNase interacted with F-box, Bifunctional fucokinase/fucose pyrophosphorylase (FKGP), aspartic proteinase A1, RRP46, pectinesterase/pectinesterase inhibitor 51 (PME51), phospholipid:diacylglycerol acyltransferase 1 (PDAT1), gibberellin receptor GID1B, GDT1-like protein 4, putative invertase inhibitor, tRNA ligase, PAP15, PAE8, TIM14-2, PGIP1 and p24beta2. Moreover, S3-RNase interacted with TOPP4. Therefore, S3-RNase may play an important role in the SI of 'XiangShui' lemon.

QM/MM Well-Tempered Metadynamics Study of the Mechanism of XBP1 mRNA Cleavage by Inositol Requiring Enzyme 1α RNase.

A range of in silico methodologies were herein employed to study the unconventional XBP1 mRNA cleavage mechanism performed by the unfolded protein response (UPR) mediator Inositol Requiring Enzyme 1α (IRE1). Using Protein-RNA molecular docking along with a series of extensive restrained/unrestrained atomistic molecular dynamics (MD) simulations, the dynamical behavior of the system was evaluated and a reliable model of the IRE1/XBP1 mRNA complex was constructed. From a series of well-converged quantum mechanics molecular mechanics well-tempered metadynamics (QM/MM WT-MetaD) simulations using the Grimme dispersion interaction corrected semiempirical parametrization method 6 level of theory (PM6-D3) and the AMBER14SB-OL3 force field, the free energy profile of the cleavage mechanism was determined, along with intermediates and transition state structures. The results show two distinct reaction paths based on general acid-general base type mechanisms, with different activation energies that perfectly match observations from experimental mutagenesis data. The study brings unique atomistic insights into the cleavage mechanism of XBP1 mRNA by IRE1 and clarifies the roles of the catalytic residues H910 and Y892. Increased understanding of the details in UPR signaling can assist in the development of new therapeutic agents for its modulation.

Evaluation of the S-locus in Prunus domestica, characterization, phylogeny and 3D modelling.

Self-compatibility has become the primary objective of most prune (Prunus domestica) breeding programs in order to avoid the problems related to the gametophytic self-incompatibility (GSI) system present in this crop. GSI is typically under the control of a specific locus., known as the S-locus., which contains at least two genes. The first gene encodes glycoproteins with RNase activity in the pistils., and the second is an SFB gene expressed in the pollen. There is limited information on genetics of SI/SC in prune and in comparison., with other Prunus species, cloning., sequencing and discovery of different S-alleles is very scarce. Clear information about S-alleles can be used for molecular identification and characterization of the S-haplotypes. We determined the S-alleles of 36 cultivars and selections using primers that revealed 17 new alleles. In addition, our study describes for the first time the association and design of a molecular marker for self-compatibility in P. domestica. Our phylogenetic tree showed that the S-alleles are spread across the phylogeny, suggesting that like previous alleles detected in the Rosaceae., they were of trans-specific origin. We provide for the first time 3D models for the P. domestica SI RNase alleles as well as in other Prunus species, including P. salicina (Japanese plum), P. avium (cherry), P. armeniaca (apricot), P. cerasifera and P. spinosa.

Primary restriction of S-RNase cytotoxicity by a stepwise ubiquitination and degradation pathway in Petunia hybrida.

In self-incompatible Petunia species, the pistil S-RNase acts as cytotoxin to inhibit self-pollination but is polyubiquitinated by the pollen-specific nonself S-locus F-box (SLF) proteins and subsequently degraded by the ubiquitin-proteasome system (UPS), allowing cross-pollination. However, it remains unclear how S-RNase is restricted by the UPS. Using biochemical analyses, we first show that Petunia hybrida S3 -RNase is largely ubiquitinated by K48-linked polyubiquitin chains at three regions, R I, R II and R III. R I is ubiquitinated in unpollinated, self-pollinated and cross-pollinated pistils, indicating its occurrence before PhS3 -RNase uptake into pollen tubes, whereas R II and R III are exclusively ubiquitinated in cross-pollinated pistils. Transgenic analyses showed that removal of R II ubiquitination resulted in significantly reduced seed sets from cross-pollination and that of R I and R III to a lesser extent, indicating their increased cytotoxicity. Consistent with this, the mutated R II of PhS3 -RNase resulted in a marked reduction of its degradation, whereas that of R I and R III resulted in less reduction. Taken together, we demonstrate that PhS3 -RNase R II functions as a major ubiquitination region for its destruction and R I and R III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross-pollination in P. hybrida.

Ornithine decarboxylase genes contribute to S-RNase-independent pollen rejection.

Unilateral incompatibility (UI) manifests as pollen rejection in the pistil, typically when self-incompatible (SI) species are pollinated by self-compatible (SC) relatives. In the Solanaceae, UI occurs when pollen lack resistance to stylar S-RNases, but other, S-RNase-independent mechanisms exist. Pistils of the wild tomato Solanum pennellii LA0716 (SC) lack S-RNase yet reject cultivated tomato (Solanum lycopersicum, SC) pollen. In this cross, UI results from low pollen expression of a farnesyl pyrophosphate synthase gene (FPS2) in S. lycopersicum. Using pollen from fps2-/- loss-of-function mutants in S. pennellii, we identified a pistil factor locus, ui3.1, required for FPS2-based pollen rejection. We mapped ui3.1 to an interval containing 108 genes situated on the IL 3-3 introgression. This region includes a cluster of ornithine decarboxylase (ODC2) genes, with four copies in S. pennellii, versus one in S. lycopersicum. Expression of ODC2 transcript was 1,034-fold higher in S. pennellii than in S. lycopersicum styles. Pistils of odc2-/- knockout mutants in IL 3-3 or S. pennellii fail to reject fps2 pollen and abolish transmission ratio distortion (TRD) associated with FPS2. Pollen of S. lycopersicum express low levels of FPS2 and are compatible on IL 3-3 pistils, but incompatible on IL 12-3 × IL 3-3 hybrids, which express both ODC2 and ui12.1, a locus thought to encode the SI proteins HT-A and HT-B. TRD observed in F2 IL 12-3 × IL 3-3 points to additional ODC2-interacting pollen factors on both chromosomes. Thus, ODC2 genes contribute to S-RNase independent UI and interact genetically with ui12.1 to strengthen pollen rejection.

Structural insights in interactions between RNase from Bacillus Intermedius and rhamnogalacturonan I from potato.

Being biocompatible and biodegradable polymers, polysaccharides present a perspective material for drug delivery systems. This study aimed at unraveling the molecular details of interactions between rhamnogalacturonan I, brunched with galactan side chains, and RNase from Bacillus Intermedius, binase. FTIR- and NMR-spectroscopic analyses showed that binase interacts with side chains of the polysaccharide. In complexes with polysaccharide, the protein retains its native structure. The 2D-NMR techniques revealed eight protein residues responsive to polysaccharide binding. Further, computer simulations were carried out to provide the atomistic details of binase-polysaccharide complexes. Both blind and knowledge-based docking procedures elucidate the existence of epitopes on the binase surface with the preferential binding of galactan fragments. The refinement of these complexes by molecular dynamics simulations confirmed stable protein-polysaccharide interactions. The results of this study strengthen the knowledge on non-specific protein-carbohydrate interactions and outline the rhamnogalacturonan I as a possible matrix material for protein delivery systems.

Efficacy of alcohol-based hand sanitizers against human norovirus using RNase-RT-qPCR with validation by human intestinal enteroid replication.

Successful human norovirus (HuNoV) cultivation in stem cell-derived human intestinal enteroids (HIE) was recently reported. The purpose of this study was to evaluate the anti-HuNoV efficacy of two alcohol-based commercial hand sanitizers and 60% ethanol by suspension assay using RNase-RT-qPCR, with subsequent validation of efficacy by HuNoV cultivation using the HIE model. In suspension, when evaluated by RNase-RT-qPCR, 60% ethanol resulted in less than one log10 reduction in HuNoV genome equivalent copies (GEC) regardless of contact time (30 or 60s) or soil load. The two commercial products outperformed 60% ethanol regardless of contact time or soil load, providing 2·2-3·2 log10 HuNoV GEC reductions by suspension assay. Product B could not be validated in the HIE model due to cytotoxicity. Following a 60s exposure, viral replication in the HIE model increased 1·9 ± 0·2 log10 HuNoV GEC for the neutralization (positive) control and increased 0·9 ± 0·2 log10 HuNoV GEC in challenged HIE after treatment with 60% ethanol. No HuNoV replication in HIE was observed after a 60 s exposure to Product A.

NaTrxh is an essential protein for pollen rejection in Nicotiana by increasing S-RNase activity.

In self-incompatible Solanaceae, the pistil protein S-RNase contributes to S-specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S-RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S-RNase in vitro. Here, we show that expressing a redox-inactive mutant, NaTrxhSS , suppresses both S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys155 -Cys185 disulphide bond of SC10 -Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S-RNase activity by NaTrxh helps to explain why S-RNase alone could be insufficient for pollen rejection.

Self-assembly plasmonic metamaterials based on templated annealing for advanced biosensing.

In this paper, we introduce a novel method for the fabrication of self-assembly plasmonic metamaterials by exploiting fluid instabilities of optical thin films. Due to interplay between template reflow and spinodal dewetting, two metal nanoparticles of different sizes are generated on the top mesas of free-standing porous anodic aluminum oxide (AAO) template, which results in the apprearance of double resonant peaks in the extinction spectrum. These two resonant peaks possess refractive index resolution 3.27 × 10-4 and 2.53 × 10-4 RIU, respectively. This optical intensity modulation based plasmonic nanoplatform shows a dramatically surface sensing performance with outstanding detection capacity of biomolecules, because of the very small decay length of electric field at dual-modes. The detection ability for concanavalin A (Con A) demonstrats that the limit of detection of dual-modes reaches as small as 68 and 79 nM, respectively.

Efficient evaluation of a gene containment system for poplar through early flowering induction.

KEY MESSAGE: The early flowering system HSP::AtFT allowed a fast evaluation of a gene containment system based on the construct PsEND1::barnase-barstar for poplar. Transgenic lines showed disturbed pollen development and sterility. Vertical gene transfer through pollen flow from transgenic or non-native plant species into their crossable natural relatives is a major concern. Gene containment approaches have been proposed to reduce or even avoid gene flow among tree species. However, evaluation of genetic containment strategies for trees is very difficult due to the long-generation times. Early flowering induction would allow faster evaluation of genetic containment in this case. Although no reliable methods were available for the induction of fertile flowers in poplar, recently, a new early flowering approach was developed. In this study, early flowering poplar lines containing the gene construct PsEND1::barnase-barstar were obtained. The PsEND1 promoter was chosen due to its early expression pattern, its versality and efficiency for generation of male-sterile plants fused to the barnase gene. RT-PCRs confirmed barnase gene activity in flowers, and pollen development was disturbed, leading to sterile flowers. The system developed in this study represents a valuable tool for gene containment studies in forest tree species.

Opuntin B, the antiviral protein isolated from prickly pear (Opuntia ficus-indica (L.) Miller) cladode exhibits ribonuclease activity.

An antiviral protein, designated Opuntin B, was purified from Prickly Pear (Opuntia ficus-indica (L.) Miller) Cladode by heat treatment of the extract, protein precipitation by ammonium sulfate treatment followed by ion-exchange chromatography. Assessment of enzymatic activity of the purified protein showed that it degrades total plant genomic RNA, while causing electrophoretic mobility shifting of Cucumber mosaic virus (CMV) RNAs. However, heat-denatured viral RNA became sensitive to degradation upon treatment with antiviral protein. Opuntin B had no DNase activity on native and heat-denatured apricot genomic DNA, and on PCR-amplified coat protein gene of CMV. Using CMV as prey protein and Opuntin B as bait protein, no interaction was found between the antiviral protein and viral coat protein in far western dot blot analysis.

MiR-421 promotes the development of osteosarcoma by regulating MCPIP1 expression.

Despite improvements in surgical resection and adjuvant chemotherapy, the prognosis and outcomes of patients with osteosarcoma remains poor due to the occurrence of metastasis or relapse. Monocyte chemoattractant protein-1-induced protein-1 (MCPIP1), a zinc-finger RNA-binding protein, is known to regulate inflammatory responses and repress breast cancer growth. However, the regulation of MCPIP1 by microRNAs has not been clearly elucidated in osteosarcoma. In this study, we found that miR-421 expression was upregulated and MCPIP1 expression was downregulated in the osteosarcoma specimens from patients. Moreover, MCPIP1 expression was inversely correlated with miR-421 expression in the clinical samples. Furthermore, the upregulation of miR-421 and downregulation of MCPIP1 resulted in poor overall survival and severe disease progression, respectively, in the patients with osteosarcoma. Bioinformatics analysis and luciferase reporter gene assays confirmed that miR-421 specifically targets and binds to the 3'-UTR of MCPIP1. The overexpression of miR-421 induced cell proliferation, invasion, and migration, and the release of pro-inflammatory IL-6 in cultured human osteosarcoma cells. Additionally, the administration of miR-421 to tumor-bearing mice facilitated osteosarcoma growth by downregulating MCPIP1 expression. Taken together, these findings indicate that miR-421 is able to promote the development of osteosarcoma by regulating MCPIP1 expression, and can be a potential therapeutic target for osteosarcoma.

Removing DNA Contamination from RNA Samples by Treatment with RNase-Free DNase I.

RNA samples prepared using monophasic lysis reagents may contain small amounts of contaminating genomic DNA, which must be removed if the RNA will be used in subsequent analyses such as reverse transcriptase-polymerase chain reaction (RT-PCR) or quantitative real-time RT-PCR. In addition, the presence of contaminating DNA can render the quantitative determination of RNA in a sample inaccurate. The most common and effective method for removing trace to moderate amounts of DNA contamination from RNA samples is digestion with DNase I, as described here.

Proximity-dependent biotinylation mediated by TurboID to identify protein-protein interaction networks in yeast.

The use of proximity-dependent biotinylation assays coupled to mass spectrometry (PDB-MS) has changed the field of protein-protein interaction studies. However, despite the recurrent and successful use of BioID-based protein-protein interactions screening in mammalian cells, the implementation of PDB-MS in yeast has not been effective. Here, we report a simple and rapid approach in yeast to effectively screen for proximal and interacting proteins in their natural cellular environment by using TurboID, a recently described version of the BirA biotin ligase. Using the protein arginine methyltransferase Rmt3 and the RNA exosome subunits, Rrp6 and Dis3, the application of PDB-MS in yeast by using TurboID was able to recover protein-protein interactions previously identified using other biochemical approaches and provided new complementary information for a given protein bait. The development of a rapid and effective PDB assay that can systematically analyze protein-protein interactions in living yeast cells opens the way for large-scale proteomics studies in this powerful model organism.

ABHD5 blunts the sensitivity of colorectal cancer to fluorouracil via promoting autophagic uracil yield.

The efficacy of Fluorouracil (FU) in the treatment of colorectal cancer (CRC) is greatly limited by drug resistance. Autophagy has been implicated in chemoresistance, but the role of selective autophagic degradation in regulating chemoresistance remains unknown. In this study, we revealed a critical role of ABHD5 in charging CRC sensitivity to FU via regulating autophagic uracil yield. We demonstrated that ABHD5 localizes to lysosome and interacts with PDIA5 to prevent PDIA5 from interacting with RNASET2 and inactivating RNASET2. ABHD5 deficiency releases PDIA5 to directly interact with RNASET2 and leave RNASET2 in an inactivate state, which impairs RNASET2-mediated autophagic uracil yield and promotes CRC cells to uptake FU as an exogenous uracil, thus increasing their sensitivity to FU. Our findings for the first time reveal a novel role of ABHD5 in regulating lysosome function, highlighting the significance of ABHD5 as a compelling biomarker predicting the sensitivity of CRCs to FU-based chemotherapy.