Reactive oxygen species signalling is involved in alkamide-induced altered root development.

Alkamides are alpha unsaturated N-acylamides structurally related to N-acyl ethanolamides (NAEs) and N-acyl-L-homoserine lactones (AHLs). Studies have shown that alkamides induce prominent changes in root architecture, a significant metabolic readjustment, and transcriptional reprogramming. Some alkamide responses have been associated with redox signalling; however, this involvement and ROS sources have not been fully described. We utilized a genetic approach to address ROS signalling in alkamide-induced processes and found that in Arabidopsis, treatment with the alkamide affinin (50μM) increased the in-situ accumulation of H2O2 in lateral root emergence sites and reduced H2O2 accumulation in primary root meristems implying that altered root growth was dependent on endogenous H2O2. Results show that ROS sourced from PRX34, RBOHC and RBOHD were involved in promotion of lateral root emergence by alkamides. RBOHC was required for affinin-induced enhanced root hair expansion. Furthermore, affinin-induced changes in lateral root emergence, but not root hair length, were dependent on a change in extracellular pH. Finally, reverse genetic experiments suggest heterotrimeric G-proteins were involved in plant response to alkamides; nevertheless, further studies with additional higher order G-protein mutants will be required to resolve this question. These results support that alkamides recruit specific ROS signaling programs to mediate alterations in root architecture.

Reverse Genetic Screen for Deleterious Recessive Variants in the Local Simmental Cattle Population of Switzerland

We herein report the result of a large-scale reverse genetic screen in the Swiss Simmental population, a local dual-purpose cattle breed. We aimed to detect possible recessively inherited variants affecting protein-coding genes, as such deleterious variants can impair fertility and rearing success significantly. We used 115,000 phased SNP data of almost 10 thousand cattle with pedigree data. This revealed evidence for 11 genomic regions of 1.17 Mb on average, with haplotypes (SH1 to SH11) showing a significant depletion in homozygosity and an allele frequency between 3.2 and 10.6%. For the proposed haplotypes, it was unfortunately not possible to evaluate associations with fertility traits as no corresponding data were available. For each haplotype region, possible candidate genes were listed based on their known function in development and disease. Subsequent mining of single-nucleotide variants and short indels in the genomes of 23 sequenced haplotype carriers allowed us to identify three perfectly linked candidate causative protein-changing variants: a SH5-related DIS3:p.Ile678fs loss-of-function variant, a SH8-related CYP2B6:p.Ile313Asn missense variant, and a SH9-related NUBPL:p.Ser143Tyr missense variant. None of these variants occurred in homozygous state in any of more than 5200 sequenced cattle of various breeds. Selection against these alleles in order to reduce reproductive failure and animal loss is recommended.

Manipulation of Cannabinoid Biosynthesis via Transient RNAi Expression

Cannabis sativa L. produces unique phytocannabinoids, which are used for their pharmaceutical benefits. To date, there are no reports of in vivo engineering targeting the cannabinoid biosynthesis genes to greater elucidate the role each of these genes play in synthesis of these medically important compounds. Reported here is the first modulation of cannabinoid biosynthesis genes using RNAi via agroinfiltration. Vacuum infiltrated leaf segments of the Cannbio-2 C. sativa strain, transfected with different RNAi constructs corresponding to THCAS, CBDAS, and CBCAS gene sequences, showed significant downregulation of all cannabinoid biosynthesis genes using real-time quantitative PCR. Using RNAi, significant off-targeting occurs resulting in the downregulation of highly homologous transcripts. Significant (p < 0.05) downregulation was observed for THCAS (92%), CBDAS (97%), and CBCAS (70%) using pRNAi-GG-CBDAS-UNIVERSAL. Significant (p < 0.05) upregulation of CBCAS (76%) and non-significant upregulation of THCAS (13%) were observed when transfected with pRNAi-GG-CBCAS, suggesting the related gene’s ability to synthesize multiple cannabinoids. Using this approach, increased understanding of the relationship between cannabinoid biosynthesis genes can be further elucidated. This RNAi approach enables functional genomics screens for further reverse genetic studies as well as the development of designer cannabis strains with over-expression and/or downregulation of targeted cannabinoid biosynthesis genes. Functional genomics screens, such as these, will further provide insights into gene regulation of cannabinoid biosynthesis in Cannabis.

Forward and reverse genetic dissection of morphogenesis identifies filament-competent Candida auris strains

Candida auris is an emerging healthcare-associated pathogen of global concern. Recent reports have identified C. auris isolates that grow in cellular aggregates or filaments, often without a clear genetic explanation. To investigate the regulation of C. auris morphogenesis, we applied an Agrobacterium-mediated transformation system to all four C. auris clades. We identified aggregating mutants associated with disruption of chitin regulation, while disruption of ELM1 produced a polarized, filamentous growth morphology. We developed a transiently expressed Cas9 and sgRNA system for C. auris that significantly increased targeted transformation efficiency across the four C. auris clades. Using this system, we confirmed the roles of C. auris morphogenesis regulators. Morphogenic mutants showed dysregulated chitinase expression, attenuated virulence, and altered antifungal susceptibility. Our findings provide insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic tools described here will allow for efficient manipulation of the C. auris genome.

Characterization and reverse genetic establishment of cattle derived Akabane virus in China

Background Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. Results An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. Conclusion We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Distribution and Functions of Monodehydroascorbate Reductases in Plants: Comprehensive Reverse Genetic Analysis of Arabidopsis thaliana Enzymes

Monodehydroascorbate reductase (MDAR) is an enzyme involved in ascorbate recycling. Arabidopsis thaliana has five MDAR genes that encode two cytosolic, one cytosolic/peroxisomal, one peroxisomal membrane-attached, and one chloroplastic/mitochondrial isoform. In contrast, tomato plants possess only three enzymes, lacking the cytosol-specific enzymes. Thus, the number and distribution of MDAR isoforms differ according to plant species. Moreover, the physiological significance of MDARs remains poorly understood. In this study, we classify plant MDARs into three classes: class I, chloroplastic/mitochondrial enzymes; class II, peroxisomal membrane-attached enzymes; and class III, cytosolic/peroxisomal enzymes. The cytosol-specific isoforms form a subclass of class III and are conserved only in Brassicaceae plants. With some exceptions, all land plants and a charophyte algae, Klebsormidium flaccidum, contain all three classes. Using reverse genetic analysis of Arabidopsis thaliana mutants lacking one or more isoforms, we provide new insight into the roles of MDARs; for example, (1) the lack of two isoforms in a specific combination results in lethality, and (2) the role of MDARs in ascorbate redox regulation in leaves can be largely compensated by other systems. Based on these findings, we discuss the distribution and function of MDAR isoforms in land plants and their cooperation with other recycling systems.

Discovering the DNA-Binding Consensus of the Thermus thermophilus HB8 Transcriptional Regulator TTHA1359

Transcription regulatory proteins, also known as transcription factors, function as molecular switches modulating the first step in gene expression, transcription initiation. Cyclic-AMP receptor proteins (CRPs) and fumarate and nitrate reduction regulators (FNRs) compose the CRP/FNR superfamily of transcription factors, regulating gene expression in response to a spectrum of stimuli. In the present work, a reverse-genetic methodology was applied to the study of TTHA1359, one of four CRP/FNR superfamily transcription factors in the model organism Thermus thermophilus HB8. Restriction Endonuclease Protection, Selection, and Amplification (REPSA) followed by next-generation sequencing techniques and bioinformatic motif discovery allowed identification of a DNA-binding consensus for TTHA1359, 5′–AWTGTRA(N)6TYACAWT–3′, which TTHA1359 binds to with high affinity. By bioinformatically mapping the consensus to the T. thermophilus HB8 genome, several potential regulatory TTHA1359-binding sites were identified and validated in vitro. The findings contribute to the knowledge of TTHA1359 regulatory activity within T. thermophilus HB8 and demonstrate the effectiveness of a reverse-genetic methodology in the study of putative transcription factors.

Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum

Clostridium acetobutylicum is a promising biocatalyst for the production of n-butanol at high yield from renewable resources. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it was previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remained unknown until this study, were necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purified, identified and characterized the proteins responsible for both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase activities and demonstrated the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrated the yield of butanol formation was limited by the level of expression of CAC_0764, the ferredoxin-NADP+ reductase encoding gene. The integration of these enzymes into metabolic engineering strategies introduces new opportunities for developing a homobutanologenic C. acetobutylicum strain.