Long-Term Neuropsychiatric Developmental Defects after Neonatal Organophosphate Exposure: Mitigation by Synthetic Neurosteroids.

Children are much more susceptible to the neurotoxic effects of organophosphate (OP) pesticides and nerve agents than adults. OP poisoning in children leads to acute seizures and neuropsychiatric sequela, including the development of long-term disabilities and cognitive impairments. Despite these risks, there are few chronic rodent models that use pediatric OP exposure for studying neurodevelopmental consequences and interventions. Here, we investigated the protective effect of the neurosteroid ganaxolone (GX) on the long-term developmental impact of neonatal exposure to the OP compound, diisopropyl-fluorophosphate (DFP). Pediatric postnatal day-28 rats were acutely exposed to DFP, and at 3 and 10 months after exposure, they were evaluated using a series of cognitive and behavioral tests with or without the postexposure treatment of GX. Analysis of the neuropathology was performed after 10 months. DFP-exposed animals displayed significant long-term deficits in mood, anxiety, depression, and aggressive traits. In spatial and nonspatial cognitive tests, they displayed striking impairments in learning and memory. Analysis of brain sections showed significant loss of neuronal nuclei antigen(+) principal neurons, parvalbumin(+) inhibitory interneurons, and neurogenesis, along with increased astrogliosis, microglial neuroinflammation, and mossy fiber sprouting. These detrimental neuropathological changes are consistent with behavioral dysfunctions. In the neurosteroid GX-treated cohort, behavioral and cognitive deficits were significantly reduced and were associated with strong protection against long-term neuroinflammation and neurodegeneration. In conclusion, this pediatric model replicates the salient features of children exposed to OPs, and the protective outcomes from neurosteroid intervention support the viability of developing this strategy for mitigating the long-term effects of acute OP exposure in children. SIGNIFICANCE STATEMENT: An estimated 3 million organophosphate exposures occur annually worldwide, with children comprising over 30% of all victims. Our understanding of the neurodevelopmental consequences in children exposed to organophosphates is limited. Here, we investigated the long-term impact of neonatal exposure to diisopropyl-fluorophosphate in pediatric rats. Neurosteroid treatment protected against major deficits in behavior and memory and was well correlated with neuropathological changes. Overall, this pediatric model is helpful to screen novel therapies to mitigate long-term developmental deficits of organophosphate exposure.

Experimental Models of Gulf War Illness, a Chronic Neuropsychiatric Disorder in Veterans.

Gulf War illness (GWI) is a chronic multifaceted condition with debilitating pain and fatigue, as well as sleep, behavioral, and cognitive impairments in war veterans. Currently, there is no effective treatment or cure for GWI; therefore, there is a critical need to develop experimental models to help better understand its mechanisms and interventions related to GWI-associated neuropsychiatric disorders. Chemical neurotoxicity appears to be one cause of GWI, and its symptoms manifest as disruptions in neuronal function. However, the mechanisms underlying such incapacitating neurologic and psychiatric symptoms are poorly understood. The etiology of GWI is complex, and many factors including chemical exposure, psychological trauma, and environmental stressors have been associated with its development. Attempts have been made to create GWI-like symptomatic models, including through chronic induction in mice and rats. Here, we present a brief protocol of GWI in rats and mice, which exhibit robust neuropsychiatric signs and neuropathologic changes reminiscent of GWI. This article provides a guide to working protocols, application of therapeutic drugs, outcomes, troubleshooting, and data analysis. Our broad profiling of GWI-like symptoms in rodents reveals features of progressive morphologic and long-lasting neuropsychiatric features. Together, the GWI model in rodents shows striking consistency in recapitulating major hallmark features of GWI in veterans. These models help identify mechanisms and interventions for GWI. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Experimental induction of Gulf War illness in rats Support Protocol 1: Monitoring of Gulf War illness signs and neuroimaging analysis in rats Basic Protocol 2: Experimental induction of Gulf War illness in mice Support Protocol 2: Monitoring of Gulf War illness signs and neuropathology analysis in mice.

SUPPRESSOR OF PHYTOCHROME B-4 #3 reduces the expression of PIF-activated genes and increases expression of growth repressors to regulate hypocotyl elongation in short days.

SUPPRESSOR OF PHYTOCHROME B-4 #3 (SOB3) is a member of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family of transcription factors that are involved in light-mediated growth in Arabidopsis thaliana, affecting processes such as hypocotyl elongation. The majority of the research on the AHLs has been conducted in continuous light. However, there are unique molecular events that promote growth in short days (SD) compared to constant light conditions. Therefore, we investigated how AHLs affect hypocotyl elongation in SD. Firstly, we observed that AHLs inhibit hypocotyl growth in SD, similar to their effect in constant light. Next, we identified AHL-regulated genes in SD-grown seedlings by performing RNA-seq in two sob3 mutants at different time points. Our transcriptomic data indicate that PHYTOCHROME INTERACTING FACTORS (PIFs) 4, 5, 7, and 8 along with PIF-target genes are repressed by SOB3 and/or other AHLs. We also identified PIF target genes that are repressed and have not been previously described as AHL-regulated, including PRE1, PIL1, HFR1, CDF5, and XTR7. Interestingly, our RNA-seq data also suggest that AHLs activate the expression of growth repressors to control hypocotyl elongation, such as HY5 and IAA17. Notably, many growth-regulating and other genes identified from the RNA-seq experiment were differentially regulated between these two sob3 mutants at the time points tested. Surprisingly, our ChIP-seq data suggest that SOB3 mostly binds to similar genes throughout the day. Collectively, these data suggest that AHLs affect gene expression in a time point-specific manner irrespective of changes in binding to DNA throughout SD.

The NAC transcription factor ATAF2 promotes ethylene biosynthesis and response in Arabidopsis thaliana seedlings.

Arabidopsis thaliana activating factor 2 (ATAF2) plays extensive regulatory roles in pathogenesis, seedling development, and stress responses. Here, we performed transcriptome analysis on ATAF2 loss- and gain-of-function mutants to identify differentially expressed genes (DEGs). Gene ontology analyses on DEGs reveal that ATAF2 enhances seedling responses to multiple hormone and stress signals. In particular, our transcriptome analysis suggests that ATAF2 promotes ethylene biosynthesis and responses via activating relevant genes. This novel role of ATAF2 was further demonstrated by using multiple ATAF2-null and overexpression lines for reverse transcription quantitative PCR verification, ethylene production measurements, and assays of seedlings growth responses to the ethylene immediate biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC). ACC suppresses ATAF2 expression to form a negative feedback regulation loop.

Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species.

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.

Two ATAF transcription factors ANAC102 and ATAF1 contribute to the suppression of cytochrome P450-mediated brassinosteroid catabolism in Arabidopsis.

PHYB ACTIVATION TAGGED SUPPRESSOR 1 (BAS1) and SUPPRESSOR OF PHYB-4 7 (SOB7) are two cytochrome P450 enzymes that inactivate brassinosteroids (BRs) in Arabidopsis. The NAC transcription factor (TF) ATAF2 (ANAC081) and the core circadian clock regulator CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) both suppress the expression of BAS1 and SOB7 via direct promoter binding. Additionally, BRs cause feedback suppression on ATAF2 expression. Here, we report that two ATAF-subgroup TFs, ANAC102 and ATAF1 (ANAC002), also contribute to the transcriptional suppression of BAS1 and SOB7. ANAC102 and ATAF1 gene-knockout mutants exhibit elevated expression of both BAS1 and SOB7, expanded tissue-level accumulation of their protein products and reduced hypocotyl growth in response to exogenous BR treatments. Similar to ATAF2, both ANAC102 and ATAF1 are transcriptionally suppressed by BRs and white light. Neither BAS1 nor SOB7 expression is further elevated in ATAF double or triple mutants, suggesting that the suppression effect of these three ATAFs is not additive. In addition, ATAF single, double, and triple mutants have similar levels of BR responsiveness with regard to hypocotyl elongation. ATAF2, ANAC102, ATAF1, and CCA1 physically interact with itself and each other, suggesting that they may coordinately suppress BAS1 and SOB7 expression via protein-protein interactions. Despite the absence of CCA1-binding elements in their promoters, ANAC102 and ATAF1 have similar transcript circadian oscillation patterns as that of CCA1, suggesting that these two ATAF genes may be indirectly regulated by the circadian clock.

Overexpression of AtAHL20 causes delayed flowering in Arabidopsis via repression of FT expression.

BACKGROUND: The 29-member Arabidopsis AHL gene family is classified into three main classes based on nucleotide and protein sequence evolutionary differences. These differences include the presence or absence of introns, type and/or number of conserved AT-hook and PPC domains. AHL gene family members are divided into two phylogenetic clades, Clade-A and Clade-B. A majority of the 29 members remain functionally uncharacterized. Furthermore, the biological significance of the DNA and peptide sequence diversity, observed in the conserved motifs and domains found in the different AHL types, is a subject area that remains largely unexplored. RESULTS: Transgenic plants overexpressing AtAHL20 flowered later than the wild type under both short and long days. Transcript accumulation analyses showed that 35S:AtAHL20 plants contained reduced FT, TSF, AGL8 and SPL3 mRNA levels. Similarly, overexpression of AtAHL20's orthologue in Camelina sativa, Arabidopsis' closely related Brassicaceae family member species, conferred a late-flowering phenotype via suppression of CsFT expression. However, overexpression of an aberrant AtAHL20 gene harboring a missense mutation in the AT-hook domain's highly conserved R-G-R core motif abolished the late-flowering phenotype. Data from targeted yeast-two-hybrid assays showed that AtAHL20 interacted with itself and several other Clade-A Type-I AHLs which have been previously implicated in flowering-time regulation: AtAHL19, AtAHL22 and AtAHL29. CONCLUSION: We showed via gain-of-function analysis that AtAHL20 is a negative regulator of FT expression, as well as other downstream flowering time regulating genes. A similar outcome in Camelina sativa transgenic plants overexpressing CsAHL20 suggest that this is a conserved function. Our results demonstrate that AtAHL20 acts as a photoperiod-independent negative regulator of transition to flowering.

The Turnera Style S-Locus Gene TsBAHD Possesses Brassinosteroid-Inactivating Activity When Expressed in Arabidopsis thaliana.

Heterostyly distinct hermaphroditic floral morphs enforce outbreeding. Morphs differ structurally, promote cross-pollination, and physiologically block self-fertilization. In Turnera the self-incompatibility (S)-locus controlling heterostyly possesses three genes specific to short-styled morph genomes. Only one gene, TsBAHD, is expressed in pistils and this has been hypothesized to possess brassinosteroid (BR)-inactivating activity. We tested this hypothesis using heterologous expression in Arabidopsis thaliana as a bioassay, thereby assessing growth phenotype, and the impacts on the expression of endogenous genes involved in BR homeostasis and seedling photomorphogenesis. Transgenic A. thaliana expressing TsBAHD displayed phenotypes typical of BR-deficient mutants, with phenotype severity dependent on TsBAHD expression level. BAS1, which encodes an enzyme involved in BR inactivation, was downregulated in TsBAHD-expressing lines. CPD and DWF, which encode enzymes involved in BR biosynthesis, were upregulated. Hypocotyl growth of TsBAHD dwarfs responded to application of brassinolide in light and dark in a manner typical of plants over-expressing genes encoding BR-inactivating activity. These results provide empirical support for the hypothesis that TsBAHD possesses BR-inactivating activity. Further this suggests that style length in Turnera is controlled by the same mechanism (BR inactivation) as that reported for Primula, but using a different class of enzyme. This reveals interesting convergent evolution in a biochemical mechanism to regulate floral form in heterostyly.

Self-transcriptional repression of the Arabidopsis NAC transcription factor ATAF2 and its genetic interaction with phytochrome A in modulating seedling photomorphogenesis.

MAIN CONCLUSION: The NAC transcription factor ATAF2 suppresses its own transcription via self-promoter binding. ATAF2 genetically interacts with the circadian regulator CCA1 and phytochrome A to modulate seedling photomorphogenesis in Arabidopsis thaliana. ATAF2 (ANAC081) is a NAC (NAM, ATAF and CUC) transcription factor (TF) that participates in the regulation of disease resistance, stress tolerance and hormone metabolism in Arabidopsis thaliana. We previously reported that ATAF2 promotes Arabidopsis hypocotyl growth in a light-dependent manner via transcriptionally suppressing the brassinosteroid (BR)-inactivating cytochrome P450 genes BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1). Assays using low light intensities suggest that the photoreceptor phytochrome A (PHYA) may play a more critical role in ATAF2-regulated photomorphogenesis than phytochrome B (PHYB) and cryptochrome 1 (CRY1). In addition, ATAF2 is also regulated by the circadian clock. The core circadian TF CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) physically interacts with ATAF2 at the DNA-protein and protein-protein levels, and both differentially suppress BAS1- and SOB7-mediated BR catabolism. In this research, we show that ATAF2 can bind its own promoter as a transcriptional self-repressor. This self-feedback-suppression loop is a typical feature of multiple circadian-regulated genes. Additionally, ATAF2 and CCA1 synergistically suppress seedling photomorphogenesis as reflected by the light-dependent hypocotyl growth analysis of their single and double gene knock-out mutants. Similar fluence-rate response assays using ATAF2 and photoreceptor (PHYB, CRY1 and PHYA) knock-out mutants demonstrate that PHYA is required for ATAF2-regulated photomorphogenesis in a wide range of light intensities. Furthermore, disruption of PHYA can suppress the BR-insensitive hypocotyl-growth phenotype of ATAF2 loss-of-function seedlings in the light, but not in darkness. Collectively, our results provide a genetic interaction synopsis of the circadian-clock-photomorphogenesis-BR integration node involving ATAF2, CCA1 and PHYA.

Improving seed size, seed weight and seedling emergence in Camelina sativa by overexpressing the Atsob3-6 gene variant.

Seedling stand establishment is a critical factor affecting crop yield in low-precipitation agricultural regions. This is especially true for small seeded crops, such as Camelina (Camelina sativa) and canola (Brassica napus), that need to be planted shallow. Deeper planting would be desirable so that seeds can access soil moisture and bigger seeds could improve emergence and stand establishment by providing the energy necessary for seedling elongation. AHL (AT-Hook Containing, Nuclear Localized) genes play an important role in seedling growth and development. AHL proteins contain two structural units, the DNA-binding AT-hook motif and the Plant and Prokaryote Conserved (PPC) domain, required for protein-protein interactions. Our previous studies demonstrate that AtAHL29/SOB3 (Suppressor of phytochrome B-4 #3) regulates seedling development in Arabidopsis (Arabidopsis thaliana). Activation-tagged overexpression of AtSOB3 (Atsob3-D) represses the long-hypocotyl phenotype of an Arabidopsis phytochrome B mutant. In contrast, overexpression of the Atsob3-6 variant (Atsob3-6-OX), with a non-functional AT-hook, confers a long-hypocotyl phenotype. In this study, we demonstrate the role of Atsob3-D and Atsob3-6-OX in modulating seed size and hypocotyl length in the brassicas Arabidopsis and Camelina. In Arabidopsis, Atsob3-D reduces seed weight whereas Atsob3-6-OX increases seed weight and size when compared to the wild type. Similarly, Atsob3-6-OX transgenic Camelina seedlings are taller than the wild type, and produce larger and heavier seeds. These larger Atsob3-6-OX Camelina seeds also confer better emergence in deep-soil planting when compared to the wild type. Taken together, Atsob3-6-OX increases seed size, seed weight, seedling hypocotyl length and stand establishment in the oilseed crop Camelina.

Emerging Molecular Links Between Plant Photomorphogenesis and Virus Resistance.

Photomorphogenesis refers to photoreceptor-mediated morphological changes in plant development that are triggered by light. Multiple photoreceptors and transcription factors (TFs) are involved in the molecular regulation of photomorphogenesis. Likewise, light can also modulate the outcome of plant-virus interactions since both photosynthesis and many viral infection events occur in the chloroplast. Despite the apparent association between photosynthesis and virus infection, little is known about whether there are also interplays between photomorphogenesis and plant virus resistance. Recent research suggests that plant-virus interactions are potentially regulated by several photoreceptors and photomorphogenesis regulators, including phytochromes A and B (PHYA and PHYB), cryptochromes 2 (CRY2), phototropin 2 (PHOT2), the photomorphogenesis repressor constitutive photomorphogenesis 1 (COP1), the NAM, ATAF, and CUC (NAC)-family TF ATAF2, the Aux/IAA protein phytochrome-associated protein 1 (PAP1), the homeodomain-leucine zipper (HD-Zip) TF HAT1, and the core circadian clock component circadian clock associated 1 (CCA1). Particularly, the plant growth promoting brassinosteroid (BR) hormones play critical roles in integrating the regulatory pathways of plant photomorphogenesis and viral defense. Here, we summarize the current understanding of molecular mechanisms linking plant photomorphogenesis and defense against viruses, which represents an emerging interdisciplinary research topic in both molecular plant biology and virology.

Production location of the gelling agent Phytagel has a significant impact on Arabidopsis thaliana seedling phenotypic analysis.

BACKGROUND: Recently, it was found that 1% Phytagel plates used to conduct Arabidopsis thaliana seedling phenotypic analysis no longer reproduced previously published results. This Phytagel, which is produced in China (Phytagel C), has replace American-made Phytagel (Phytagel), which is no longer commercially available. In this study, we present the impact of Phytagel produced in the United States vs. China on seedling phenotypic analysis. As a part of this study, an alternative gelling agent has been identified that is capable of reproducing previously published seedling morphometrics. RESULTS: Phytagel and Phytagel C were investigated based on their ability to reproduce the subtle phenotype of the sob3-4 esc-8 double mutant. Fluence-rate-response analysis of seedlings grown on 1% Phytagel C plates failed to replicate the sob3-4 esc-8 subtle phenotype seen on 1% Phytagel. Furthermore, root penetrance analysis showed a significant difference between sob3-4 esc-8 seedlings grown on 1% Phytagel and 1% Phytagel C. It was also found that 1% Phytagel C was significantly harder than 1% Phytagel. As a replacement for Phytagel C, Gellan was tested. 1% Gellan was able to reproduce the subtle phenotype of sob3-4 esc-8. Furthermore, there was no significant difference in root penetration of the wild type or sob3-4 esc-8 seedlings between 1% Phytagel and 1% Gellan. This may be due to the significant reduction in hardness in 1% Gellan plates compared to 1% Phytagel plates. Finally, we tested additional concentrations of Gellan and found that seedlings on 0.6% Gellan looked more uniform while also being able to reproduce previously published results. CONCLUSIONS: Phytagel has been the standard gelling agent for several studies involving the characterization of subtle seedling phenotypes. After production was moved to China, Phytagel C was no longer capable of reproducing these previously published results. An alternative gelling agent, Gellan, was able to reproduce previously published seedling phenotypes at both 1% and 0.6% concentrations. The information provided in this manuscript is beneficial to the scientific community as whole, specifically phenomics labs, as it details key problematic differences between gelling agents that should be performing identically (Phytagel and Phytagel C).

Complete Genome Sequence of the Citrobacter freundii Type Strain.

Citrobacter freundii is a species of facultative anaerobic Gram-negative bacteria of the family Enterobacteriaceae The complete genome is composed of a single chromosomal circle of 4,957,773 bp with a G+C content of 52%.

AT-Hook Transcription Factors Restrict Petiole Growth by Antagonizing PIFs.

Upon detecting abiotic or biotic stress, plants generally reduce their growth, enabling resources to be conserved and diverted to stress response mechanisms. In Arabidopsis thaliana, the AT-hook motif nuclear-localized (AHL) transcription factor family has been implicated in restricting rosette growth in response to stress. However, the mechanism by which AHLs repress growth in rosettes is unknown. In this study, we establish that SUPPRESSOR OF PHYTOCHROME B4-#3 (SOB3) and other AHLs restrict petiole elongation by antagonizing the growth-promoting PHYTOCHROME-INTERACTING FACTORs (PIFs). Our data show that high levels of SOB3 expression lead to a short-petiole phenotype similar to that conferred by removal of PIF4. Conversely, the dominant-negative sob3-6 mutant has long petioles, a phenotype which is PIF-dependent. We further show that AHLs repress the expression of many PIF-activated genes, several of which are involved in hormone-mediated promotion of growth. Additionally, a subset of PIF-activated, AHL-repressed genes are directly bound by both SOB3 and PIFs. Finally, SOB3 reduces binding of PIF4 to shared target loci. Collectively, our results demonstrate that AHLs repress petiole growth by antagonizing PIF-mediated transcriptional activation of genes associated with growth and hormone pathways. By elucidating a mechanism via which the stress-responsive AHL transcription factor family influences growth in petioles, this study identifies a key step in the gene regulatory network controlling leaf growth in response to the environment.

CIRCADIAN CLOCK ASSOCIATED 1 and ATAF2 differentially suppress cytochrome P450-mediated brassinosteroid inactivation.

Brassinosteroids (BRs) are a group of steroid hormones regulating plant growth and development. Since BRs do not undergo transport among plant tissues, their metabolism is tightly regulated by transcription factors (TFs) and feedback loops. BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1) are two BR-inactivating cytochrome P450s identified in Arabidopsis thaliana. We previously found that a TF ATAF2 (ANAC081) suppresses BAS1 and SOB7 expression by binding to the Evening Element (EE) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-binding site (CBS) on their promoters. Both the EE and CBS are known binding targets of the circadian regulatory protein CCA1. Here, we confirm that CCA1 binds the EE and CBS motifs on BAS1 and SOB7 promoters, respectively. Elevated accumulations of BAS1 and SOB7 transcripts in the CCA1 null mutant cca1-1 indicate that CCA1 is a repressor of their expression. When compared with either cca1-1 or the ATAF2 null mutant ataf2-2, the cca1-1 ataf2-2 double mutant shows higher SOB7 transcript accumulations and a stronger BR-insensitive phenotype of hypocotyl elongation in white light. CCA1 interacts with ATAF2 at both DNA-protein and protein-protein levels. ATAF2, BAS1, and SOB7 are all circadian regulated with distinct expression patterns. These results demonstrate that CCA1 and ATAF2 differentially suppress BAS1- and SOB7-mediated BR inactivation.

Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development.

Whitefly-transmitted begomoviruses cause serious damage to many economically important food, feed, and fiber crops. Numerous vegetable crops are severely affected and chilli leaf curl virus (ChiLCV) is the most dominant and widely distributed begomovirus in chilli (Capsicum annuum) throughout the Indian subcontinent. Recently, CRISPR-Cas9 technology was used as a means to reduce geminivirus replication in infected plants. However, this approach was shown to have certain limitations such as the evolution of escape mutants. In this study, we used a novel, multiplexed guide RNA (gRNA) based CRISPR-Cas9 approach that targets the viral genome at two or more sites simultaneously. This tactic was effective in eliminating the ChiLCV genome without recurrence of functional escape mutants. Six individual gRNA spacer sequences were designed from the ChiLCV genome and in vitro assays confirmed the cleavage behaviour of these spacer sequences. Multiplexed gRNA expression clones, based on combinations of the above-mentioned spacer sequences, were developed. A total of nine-duplex and two-triplex CRISPR-Cas9 constructs were made. The efficacy of these constructs was tested for inhibition of ChiLCV infection in Nicotiana benthamiana. Results indicated that all the constructs caused a significant reduction in viral DNA accumulation. In particular, three constructs (gRNA5+4, gRNA5+2 and gRNA1+2) were most effective in reducing the viral titer and symptoms. T7E1 assay and sequencing of the targeted viral genome did not detect any escape mutants. The multiplexed genome-editing technique could be an effective way to trigger a high level of resistance against begemoviruses. To our knowledge, this is the first report of demonstrating the effectiveness of a multiplexed gRNA-based plant virus genome editing to minimize and eliminate escape mutant formation.

Putative Auxin and Light Responsive Promoter Elements From the Tomato spotted wilt tospovirus Genome, When Expressed as cDNA, Are Functional in Arabidopsis.

Members of the virus order Bunyavirales cause serious diseases in animals, humans and plants. Family Tospoviridae in this order contains only one genus Orthotospovirus, and members in this genus exclusively infect plants. Tomato spotted wilt tospovirus (TSWV) is considered one of the most economically important plants viruses. Little is known about the regulatory elements in the TSWV genome. Here we show that, when in the cDNA form, the 5'-upstream region of the TSWV-coded GN/GC gene (pGN/GC) possesses putative cis-regulatory elements, including an auxin responsive element (AuxRE) for binding of auxin response factors (ARFs), as well as a circadian clock-associated 1 (CCA1) protein binding site (CBS). Due to the lack of a reverse genetics system, we verified the functionality of these elements in Arabidopsis. pGN/GC showed light-suppressive promoter activity in transgenic Arabidopsis, and mutation in the CBS was sufficient to switch the activity to light inducible. Additionally, exogenous auxin treatments repressed the promoter activity of both wild type and CBS-mutated pGN/GC. Mutation in AuxRE in both promoters abolished their sensitivity to auxin. As transcriptional repressors, both CCA1 and ARF2 were able to bind to pGN/GC directly. To our knowledge, this is the first report that a 5'-terminal sequence of an RNA virus has light-and hormone-responsive promoter activities when expressed as cDNA in host plant's nuclear background. Our findings suggest new clues on the possible origin, evolution and function of the TSWV genomic sequence and its non-coding regions.

Neural Processing and Production of Gesture in Children and Adolescents With Autism Spectrum Disorder.

Individuals with autism spectrum disorder (ASD) demonstrate impairments in non-verbal communication, including gesturing and imitation deficits. Reduced sensitivity to biological motion (BM) in ASD may impair processing of dynamic social cues like gestures, which in turn may impede encoding and subsequent performance of these actions. Using both an fMRI task involving observation of action gestures and a charade style paradigm assessing gesture performance, this study examined the brain-behavior relationships between neural activity during gesture processing, gesturing abilities and social symptomology in a group of children and adolescents with and without ASD. Compared to typically developing (TD) controls, participants with ASD showed atypical sensitivity to movement in right posterior superior temporal sulcus (pSTS), a region implicated in action processing, and had poorer overall gesture performance with specific deficits in hand posture. The TD group showed associations between neural activity, gesture performance and social skills, that were weak or non-significant in the ASD group. These findings suggest that those with ASD demonstrate abnormalities in both processing and production of gestures and may reflect dysfunction in the mechanism underlying perception-action coupling resulting in atypical development of social and communicative skills.

Synopsis of the SOFL Plant-Specific Gene Family.

SUPPRESSOR OF PHYB-4#5DOMINANT (sob5-D) was previously identified as a suppressor of the phyB-4 long-hypocotyl phenotype in Arabidopsis thaliana Overexpression of SOB5 conferred dwarf phenotypes similar to those observed in plants containing elevated levels of cytokinin (CK) nucleotides and nucleosides. Two SOB-FIVE- LIKE (SOFL) proteins, AtSOFL1 and AtSOFL2, which are more similar at the protein level to each other than they are to SOB5, conferred similar phenotypes to the sob5-D mutant when overexpressed. We used protein sequences of founding SOFL gene family members to perform database searches and identified a total of 289 SOFL homologs in genomes of 89 angiosperm species. Phylogenetic analysis results implied that the SOFL gene family emerged during the expansion of angiosperms and later evolved into four distinct clades. Among the newly identified gene family members are four previously unreported Arabidopsis SOFLs Multiple sequence alignment of the 289 SOFL protein sequences revealed two highly conserved domains; SOFL-A and SOFL-B. We used overexpression and site-directed mutagenesis studies to demonstrate that SOFL domains are necessary for SOB5 and AtSOFL1's overexpression phenotypes. Examination of the subcellular localization patterns of founding Arabidopsis thaliana SOFLs suggested they may be localized in the cytoplasm and/or the nucleus. Overall, we report that SOFLs are a plant-specific gene family characterized by two conserved domains that are important for function.