An Efficient Root Transformation System for Recalcitrant Vicia sativa

Common vetch (Vicia sativa) is a multi-purpose legume widely used in pasture and crop rotation systems. Vetch seeds have desirable nutritional characteristics and are often used to feed ruminant animals. Although transcriptomes are available for vetch, problems with genetic transformation and plant regeneration hinder functional gene studies in this legume species. Therefore, the aim of this study was to develop a simple, efficient and rapid hairy root transformation system for common vetch to facilitate functional gene analysis. At first, we infected the hypocotyls of 5-day-old in vitro or in vivo, soil-grown seedlings with Rhizobium rhizogenes K599 using a stabbing method and produced transgenic hairy roots after 24 days at 19 and 50% efficiency, respectively. We later improved the hairy root transformation in vitro by infecting different explants (seedling, hypocotyl-epicotyl, and shoot) with R. rhizogenes. We observed hairy root formation at the highest efficiency in shoot and hypocotyl-epicotyl explants with 100 and 93% efficiency, respectively. In both cases, an average of four hairy roots per explant were obtained, and about 73 and 91% of hairy roots from shoot and hypocotyl-epicotyl, respectively, showed stable expression of a co-transformed marker β-glucuronidase (GUS). In summary, we developed a rapid, highly efficient, hairy root transformation method by using R. rhizogenes on vetch explants, which could facilitate functional gene analysis in common vetch.

Targeted gene knock-in reduces variation between transformants in the mushroom-forming fungus Schizophyllum commune

Gene integration in mushroom-forming fungi currently occurs by the ectopic integration of a plasmid. The locus of integration is unpredictable and, problematically, this generally results in a high variability in gene expression and phenotypes between the transformants. Here, we developed an approach for targeted gene integration (knock-in) in the basidiomycete Schizophyllum commune by replacing a 75-bp non-coding region of the genome with a selection marker and an arbitrary gene of interest using CRISPR-Cas9 ribonucleoproteins. To assess the suitability of our method, we compared targeted integration and ectopic integration of the gene encoding the red fluorescent protein dTomato. Targeted integration resulted in a higher average fluorescence intensity and less variability between the transformants. This method may be applied to any gene construct and may therefore greatly increase the efficiency of functional gene analysis in S. commune.

A Methodological Advance of Tobacco Rattle Virus-Induced Gene Silencing for Functional Genomics in Plants

As a promising high-throughput reverse genetic tool in plants, virus-induced gene silencing (VIGS) has already begun to fulfill some of this promise in diverse aspects. However, review of the technological advancements about widely used VIGS system, tobacco rattle virus (TRV)-mediated gene silencing, needs timely updates. Hence, this article mainly reviews viral vector construction, inoculation method advances, important influential factors, and summarizes the recent applications in diverse plant species, thus providing a better understanding and advice for functional gene analysis related to crop improvements.

Metagenomic Approaches to Analyze Antimicrobial Resistance: An Overview

Antimicrobial resistance is a major global public health problem, which develops when pathogens acquire antimicrobial resistance genes (ARGs), primarily through genetic recombination between commensal and pathogenic microbes. The resistome is a collection of all ARGs. In microorganisms, the primary method of ARG acquisition is horizontal gene transfer (HGT). Thus, understanding and identifying HGTs, can provide insight into the mechanisms of antimicrobial resistance transmission and dissemination. The use of high-throughput sequencing technologies has made the analysis of ARG sequences feasible and accessible. In particular, the metagenomic approach has facilitated the identification of community-based antimicrobial resistance. This approach is useful, as it allows access to the genomic data in an environmental sample without the need to isolate and culture microorganisms prior to analysis. Here, we aimed to reflect on the challenges of analyzing metagenomic data in the three main approaches for studying antimicrobial resistance: (i) analysis of microbial diversity, (ii) functional gene analysis, and (iii) searching the most complete and pertinent resistome databases.

Clonal sector analysis and cell ablation confirm a function for DORNROESCHEN-LIKE in founder cells and the vasculature in Arabidopsis

Main conclusion Inducible lineage analysis and cell ablation via conditional toxin expression in cells expressing the DORNRÖSCHEN-LIKE transcription factor represent an effective and complementary adjunct to conventional methods of functional gene analysis. Abstract Classical methods of functional gene analysis via mutational and expression studies possess inherent limitations, and therefore, the function of a large proportion of transcription factors remains unknown. We have employed two complementary, indirect methods to obtain functional information for the AP2/ERF transcription factor DORNRÖSCHEN-LIKE (DRNL), which is dynamically expressed in flowers and marks lateral organ founder cells. An inducible, two-component Cre–Lox system was used to express beta-glucuronidase GUS in cells expressing DRNL, to perform a sector analysis that reveals lineages of cells that transiently expressed DRNL throughout plant development. In a complementary approach, an inducible system was used to ablate cells expressing DRNL using diphtheria toxin A chain, to visualise the phenotypic consequences. These complementary analyses demonstrate that DRNL functionally marks founder cells of leaves and floral organs. Clonal sectors also included the vasculature of the leaves and petals, implicating a previously unidentified role for DRNL in provasculature development, which was confirmed in cotyledons by closer analysis of drnl mutants. Our findings demonstrate that inducible gene-specific lineage analysis and cell ablation via conditional toxin expression represent an effective and informative adjunct to conventional methods of functional gene analysis.

Efficient CRISPR-mediated base editing inAgrobacteriumspp.

Agrobacteriumspp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. AlthoughAgrobacteriumspp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis ofAgrobacteriumhas been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of bothAgrobacterium tumefaciensandAgrobacterium rhizogenes. As an example, we generated EHA105 strains with loss-of-function mutations inrecA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development byA. rhizogenesK599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of “engineering the engineer,” leading to improvedAgrobacteriumstrains for more efficient plant transformation and gene editing.

p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation

Actinosynnema species produce diverse natural products with important biological activities, which represent an important resource of antibiotic discovery. Advances in genome sequencing and bioinformatics tools have accelerated the exploration of the biosynthetic gene clusters (BGCs) encoding natural products. Herein, the completed BGCs of dnacin B1 were first discovered in two Actinosynnema pretiosum subsp. auranticum strains DSM 44131T (hereafter abbreviated as strain DSM 44131T) and X47 by comparative genome mining strategy. The BGC for dnacin B1 contains 41 ORFs and spans a 66.9 kb DNA region in strain DSM 44131T. Its involvement in dnacin B1 biosynthesis was identified through the deletion of a 9.7 kb region. Based on the functional gene analysis, we proposed the biosynthetic pathway for dnacin B1. Moreover, p-amino-phenylalanine (PAPA) unit was found to be the dnacin B1 precursor for the quinone moiety formation, and this was confirmed by heterologous expression of dinV, dinE and dinF in Escherichia coli. Furthermore, nine potential PAPA aminotransferases (APAT) from the genome of strain DSM 44131T were explored and expressed. Biochemical evaluation of their amino group transformation ability was carried out with p-amino-phenylpyruvic acid (PAPP) or PAPA as the substrate for the final product formation. Two of those, APAT4 and APAT9, displayed intriguing aminotransferase ability for the formation of PAPA. The proposed dnacin B1 biosynthetic machinery and PAPA biosynthetic investigations not only enriched the knowledge of tetrahydroisoquinoline (THIQ) biosynthesis, but also provided PAPA building blocks to generate their structurally unique homologues.

Transcriptome Assembly of Asian Buffalo Leech Hirudinaria Manillensis and An Accurate Identification of Genes Involved in Anticoagulant Biosynthesis

Background: The Asian buffalo leech Hirudinaria manillensis is a valuable animal widely used in traditional Chinese medicine to cure blood-clotting. However, although the content of the anticoagulant genes in H. manillensis were identified, their actual expression profile remains undetermined. Herein, in this study we conducted transcriptome analysis on this species to address this subject.Results: A high qualified accurate gene expression profile of H. manillensis transcripts was obtained. By identifying genes upregulated during feeding, anticoagulant-related genes and signaling pathways were identified. The assembled Unigenes were compared in various mainstream databases, and a total of 16,682 Unigenes received annotation information. In addition, the Unigenes were evaluated in terms of length distribution, GC content, and expression level. The data showed good sequencing quality and high reliability. A total of 155 anticoagulant-related genes were found in this study, including those involved in different types of degradation of the extracellular matrix and inhibition of platelet aggregation.Conclusions：Substantial transcriptome information was obtained by transcriptome sequencing of H. manillensis. This information should help to provide a further molecular theoretical basis for functional gene analysis, genomics, genetic diversity analysis, and molecular marker development of H. manillensis and for the anticoagulation-related genes of this species and its medicinal value as an anticoagulant.

Efficient Cas9 multiplex editing using unspaced gRNA arrays engineering in a Potato virus X vector

SummarySystems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as the Cas nucleases or the guide RNA (gRNA). While the Cas nucleases are constant elements in editing approaches, gRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of gRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing (VIGE). In this work, we engineered Potato virus X (PVX) to build a vector able to easily express one or more gRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expressed Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced gRNAs achieving highly efficient multiplex editing in a few days in adult plant tissues. We also demonstrate that genome modifications are inherited in plants regenerated from infected tissues. In sum, the new PVX VIGE vector allows easy, fast and efficient expression of gRNAs arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in important crops of the family Solanaceae.