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Retinoblastoma binding protein 4 (Rbbp4) is a component of transcription regulatory complexes that control cell cycle gene expression. Previous work indicated that Rbbp4 cooperates with the Rb tumor suppressor to block cell cycle entry. Here, we use genetic analysis to examine the interactions of Rbbp4, Rb, and Tp53 in zebrafish neural progenitor cell cycle regulation and survival.Rbbp4 is upregulated across the spectrum of human embryonal and glial brain cancers. Transgenic rescue of rbbp4 mutant embryos shows Rbbp4 is essential for zebrafish neurogenesis. Rbbp4 loss leads to apoptosis and γ-H2AX in the developing brain that is suppressed by tp53 knockdown or maternal zygotic deletion. Mutant retinal neural precursors accumulate in M phase and fail to initiate G0 gene expression. rbbp4; rb1 mutants show an additive effect on the number of M phase cells. In rbbp4 mutants, Tp53 acetylation is detected; however, Rbbp4 overexpression did not rescue DNA damage-induced apoptosis.Rbbp4 is necessary for neural progenitor cell cycle progression and initiation of G0 independent of Rb. Tp53-dependent apoptosis in the absence of Rbpb4 correlates with Tp53 acetylation. Together these results suggest that Rbbp4 is required for cell cycle exit and contributes to neural progenitor survival through the regulation of Tp53 acetylation.

The ability to regulate gene activity spatially and temporally is essential to investigate cell-type-specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here, we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4off and rb1off integration alleles resulted in strong mRFP expression,>99% reduction of endogenous gene expression, and recapitulated known indel loss-of-function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4on and rb1on integration alleles did not cause phenotypes in combination with a loss-of-function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox-responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics.

The ability to regulate gene activity spatially and temporally is essential to investigate cell-type-specific gene function during development and in postembryonic processes and disease models. The Cre/

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/loxsystem has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/loxregulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron inhdac1, rbbp4, andrb1. Active, gene off orientationhdac1-UFlip-Offandrb1-UFlip-Offintegration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientationrbbp4-UFlip-Onandrb1-UFlip-Onintegration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs ofloxPandlox2272sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstraterbbp4-UFlip-Onandrb1-UFlip-Ongene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP inrbbp4-UFlip-Off, or 2A-KalTA4 inrb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/loxresponsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

Nonhost resistance (NHR), which protects all members of a plant species from non-adapted or nonhost plant pathogens, is the most common form of plant immunity. NHR provides the most durable and robust form of broad-spectrum immunity against non-adaptive pathogens pathogenic to other crop species. In a mutant screen for loss of Arabidopsis (Arabidopsis thaliana) NHR against the soybean pathogen, Phytophthora sojae, the Phytophthora sojae susceptible 30 (pss30) mutant was identified. The pss30 mutant is also susceptible to the soybean pathogen Fusarium virguliforme. PSS30 encodes a folate transporter, AtFOLT1, which was previously localized to chloroplasts and implicated in the transport of folate from the cytosol to plastids. We have shown that two Arabidopsis folate biosynthetic mutants with reduced folate levels exhibited a loss of nonhost immunity against P. sojae. As compared to the wild-type Col-0 ecotype, the steady state folate levels are reduced in the pss1, atfolt1 and two folate biosynthetic mutants suggesting that folate is required for expression of nonhost immunity. Overexpression of AtFOLT1 enhances immunity of transgenic soybean (Glycine max, (L.) Merr.) lines against two serious soybean pathogens, the fungal pathogen F. virguliforme and soybean cyst nematode (SCN) Heterodera glycines. Transgenic lines showing enhanced SCN resistance also showed a trend of increased levels of folate accumulation. This study thus suggests that folate contributes to nonhost plant immunity and overexpression of a nonhost resistance gene could be a suitable strategy for generating broad-spectrum disease resistance in crop plants.

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Acinetobacter baumannii is a successful human pathogen which has emerged as one of the most problematic and highly antibiotic-resistant Gram-negative bacteria worldwide. Multidrug efflux is a major mechanism that A. baumannii uses to counteract the action of multiple classes of antibiotics, such as β-lactams, tetracyclines, fluoroquinolones, and aminoglycosides. Here, we report a cryo-electron microscopy (cryo-EM) structure of the prevalent A. baumannii AdeB multidrug efflux pump, which indicates a plausible pathway for multidrug extrusion. Overall, our data suggest a mechanism for energy coupling that powers up this membrane protein to export antibiotics from bacterial cells. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens., Resistance-nodulation-cell division multidrug efflux pumps are membrane proteins that catalyze the export of drugs and toxic compounds out of bacterial cells. Within the hydrophobe-amphiphile subfamily, these multidrug-resistant proteins form trimeric efflux pumps. The drug efflux process is energized by the influx of protons. Here, we use single-particle cryo-electron microscopy to elucidate the structure of the Acinetobacter baumannii AdeB multidrug efflux pump embedded in lipidic nanodiscs to a resolution of 2.98 Å. We found that each AdeB molecule within the trimer preferentially takes the resting conformational state in the absence of substrates. We propose that proton influx and drug efflux are synchronized and coordinated within the transport cycle.

The immutans ( im ) variegation mutant of Arabidopsis defines the gene for PTOX (plastid terminal oxidase), a versatile plastoquinol oxidase in chloroplast membranes. In this report we used im to gain insight into the function of PTOX in etioplasts of dark-grown seedlings. We discovered that PTOX helps control the redox state of the plastoquinone (PQ) pool in these organelles, and that it plays an essential role in etioplast metabolism by participating in the desaturation reactions of carotenogenesis and in one or more redox pathways mediated by PGR5 (PROTON GRADIENT REGULATION 5) and NDH (NAD(P)H dehydrogenase), both of which are central players in cyclic electron transport. We propose that these elements couple PTOX with electron flow from NAD(P)H to oxygen, and by analogy to chlororespiration (in chloroplasts) and chromorespiration (in chromoplasts), we suggest that they define a respiratory process in etioplasts that we have termed "etiorespiration". We further show that the redox state of the PQ pool in etioplasts might control chlorophyll biosynthesis, perhaps by participating in mechanisms of retrograde (plastid-to-nucleus) signaling that coordinate biosynthetic and photoprotective activities required to poise the etioplast for light development. We conclude that PTOX is an important component of metabolism and redox sensing in etioplasts.

This paper reports a highly economical and accessible approach to generate different discrete relative humidity conditions in spatially separated wells of a modified multi-well plate for humidity assay of plant-pathogen interactions with good throughput. We demonstrated that a discrete humidity gradient could be formed within a few minutes and maintained over a period of a few days inside the device. The device consisted of a freeway channel in the top layer, multiple compartmented wells in the bottom layer, a water source, and a drying agent source. The combinational effects of evaporation, diffusion, and convection were synergized to establish the stable discrete humidity gradient. The device was employed to study visible and molecular disease phenotypes of soybean in responses to infection by Phytophthora sojae, an oomycete pathogen, under a set of humidity conditions, with two near-isogenic soybean lines, Williams and Williams 82, that differ for a Phytophthora resistance gene (Rps1-k). Our result showed that at 63% relative humidity, the transcript level of the defense gene GmPR1 was at minimum in the susceptible soybean line Williams and at maximal level in the resistant line Williams 82 following P. sojae CC5C infection. In addition, we investigated the effects of environmental temperature, dimensional and geometrical parameters, and other configurational factors on the ability of the device to generate miniature humidity environments. This work represents an exploratory effort to economically and efficiently manipulate humidity environments in a space-limited device and shows a great potential to facilitate humidity assay of plant seed germination and development, pathogen growth, and plant-pathogen interactions. Since the proposed device can be easily made, modified, and operated, it is believed that this present humidity manipulation technology will benefit many laboratories in the area of seed science, plant pathology, and plant-microbe biology, where humidity is an important factor that influences plant disease infection, establishment, and development.

Fusarium virguliforme causes the serious disease sudden death syndrome (SDS) in soybean. Host resistance to this pathogen is partial and is encoded by a large number of quantitative trait loci, each conditioning small effects. Breeding SDS resistance is therefore challenging and identification of single-gene encoded novel resistance mechanisms is becoming a priority to fight this devastating this fungal pathogen. In this transcriptomic study we identified a few putative soybean defense genes, expression of which is suppressed during F. virguliforme infection. The F. virguliforme infection-suppressed genes were broadly classified into four major classes. The steady state transcript levels of many of these genes were suppressed to undetectable levels immediately following F. virguliforme infection. One of these classes contains two novel genes encoding ankyrin repeat-containing proteins. Expression of one of these genes, GmARP1, during F. virguliforme infection enhances SDS resistance among the transgenic soybean plants. Our data suggest that GmARP1 is a novel defense gene and the pathogen presumably suppress its expression to establish compatible interaction.

The immutans (im) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. IM codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white im sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen Pseudomonas syringae. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions.

Introduction: Antiphospholipid syndrome (APS) is an autoimmune disorder caused by "antiphospholipid" antibodies (aPL) directed against β2-glycoprotein I (β2GPI). In the presence of β2GPI, aPL activate endothelial cells through TLR4-mediated and other pathways. Previous studies suggest that a region encompassing amino acids 39-43 of β2GPI domain 1 comprises an important binding site for pathogenic aPL. However, due to difficulty in expressing full-length recombinant β2GPI, antibody specificity has only been tested using individual, recombinant β2GPI domains. Further definition of antibody specificity using intact recombinant β2GPI provides a new approach for diagnostic and mechanistic studies. In the present study we describe the expression of full-length, wild-type recombinant β2GPI and site-directed mutants encompassing the putative β2GPI binding region in domain 1. Binding of APS patient-derived anti-β2GPI antibodies to these proteins and their ability to support anti-β2GPI-mediated endothelial cell activation was determined. Methods: Full length APOH cDNA with a modified signal peptide (spm) was cloned into the lentiviral vector pLenti CMV Puro DEST. We generated two mutants spanning the 39 to 43 amino acid region: SEGVG (R39S; G40E; M42V; R43G) and AAGMA (R39A; G40A; R43A). The final recombinant plasmids were termed as pDEST-spmAPOH-WT; pDEST-spmAPOH-SEGVG and pDEST-spmAPOH-AAGMA. Lentivirus was produced using the Lentiviral Gateway Expression kit using GP2-293 (HEK) cells. Stable cell lines were generated by transducing HEK cells with APOH-lentivirus and selecting against puromycin. Stable cell lines were transferred to serum-free media and grown in suspension. Cell culture supernatants containing secreted β2GPI were filtered and concentrated, and recombinant β2GPI was purified using a hitrap-heparin column. Anti-β2GPI antibody binding to plasma-derived and rβ2GPI was determined using a standard β2GPI ELISA and SPR (Surface Plasmon Resonance) on a Biacore 3000. Biosensor analysis was performed by crosslinking β2GPI to carboxymethyl-dextran coated sensor chips using amine coupling. Anti-β2GPI antibodies at concentrations ranging from 1-15,000 nM, were flowed through channels until equilibrium binding was achieved, at which point dissociation was assessed over a 10 minute interval. The BIAevaluation program was used to calculate association and dissociation rates. Endothelial cell activation was assessed by measuring cell surface E-selectin expression after incubating cells with control IgG or anti-β2GPI antibodies in the presence or absence of wild-type or mutant rβ2GPI. Molecular modeling was performed using PyMol. Results: After transduction with lentivirus encoding β2GPI with a modified signal peptide, HEK-293 cells were able to express and efficiently secrete rβ2GPI and site-directed mutants. Using a standard β2GPI-ELISA and SPR we found no significant differences in binding affinity of aPL towards plasma-derived and wild-type rβ2GPI. However, in the anti-β2GPI ELISA, binding of patient-derived aPL (APS21) to both mutants was reduced by ~70% compared to wild-type rβ2GPI. Consistent with binding specificity for a region encompassing aa 39-43, the Kd for binding of APS21 anti-β2GPI antibodies to wild-type and rβ2GPI-SEGVG determined by SPR was 20 nM and 5000 nM, respectively. Moreover, the ability of patient-derived APS21 anti-β2GPI antibodies to activate endothelial cells was reduced by >60% in the presence of rβ2GPI-SEGVG compared to wild-type rβ2GPI. Molecular modeling of β2GPI demonstrated that a mutation in the aa 39-43 region is predicted to cause a change from a net positive to a net negative charge without any structural change (Figure 1). Conclusion: These studies are the first to assess binding of human anti-β2GPI antibodies to rβ2GPI and site-directed mutants expressed in mammalian cells. No significant differences between binding of aPL to plasma derived and wild-type rβ2GPI were observed. APS21 aPL has specificity towards epitope 39-43 of β2GPI domain 1 and its binding affinity to rβ2GPI-SEGVG was significantly reduced. Functional studies demonstrate the importance of β2GPI aa 39 to 43 in supporting endothelial cell activation by anti-β2GPI antibodies. These recombinant proteins should facilitate further studies concerning the role of aPL-β2GPI interactions in the diagnosis and pathogenesis of APS. Disclosures No relevant conflicts of interest to declare.

The immutans (im) variegation mutant of Arabidopsis has green and white-sectored leaves due to the absence of fully functional PTOX (plastid terminal oxidase), a plastoquinol oxidase in thylakoid membranes. PTOX appears to be at the nexus of a growing number of biochemical pathways in the plastid, including carotenoid biosynthesis, PSI cyclic electron flow, and chlororespiration. During the early steps of chloroplast biogenesis, PTOX serves as an alternate electron sink and is a prime determinant of the redox poise of the developing photosynthetic apparatus. Whereas a lack of PTOX causes the formation of photooxidized plastids in the white sectors of im, compensating mechanisms allow the green sectors to escape the effects of the mutation. This manuscript provides an update on PTOX, the mechanism of immutans variegation, and findings about im compensatory mechanisms.

The immutans (im) variegation mutant of Arabidopsis thaliana is caused by an absence of PTOX, a plastid terminal oxidase bearing similarity to mitochondrial alternative oxidase (AOX). In an activation tagging screen for suppressors of im, we identified one suppression line caused by overexpression of AOX2. AOX2 rescued the im defect by replacing the activity of PTOX in the desaturation steps of carotenogenesis. Similar results were obtained when AOX1a was reengineered to target the plastid. Chloroplast-localized AOX2 formed monomers and dimers, reminiscent of AOX regulation in mitochondria. Both AOX2 and AOX1a were present in higher molecular weight complexes in plastid membranes. The presence of these proteins did not generally affect steady state photosynthesis, aside from causing enhanced nonphotochemical quenching in both lines. Because AOX2 was imported into chloroplasts using its own transpeptide, we propose that AOX2 is able to function in chloroplasts to supplement PTOX activity during early events in chloroplast biogenesis. We conclude that the ability of AOX1a and AOX2 to substitute for PTOX in the correct physiological and developmental contexts is a striking example of the capacity of a mitochondrial protein to replace the function of a chloroplast protein and illustrates the plasticity of the photosynthetic apparatus.