Small DNA elements can act as both insulators and silencers in plants.

Insulators are cis-regulatory elements that separate transcriptional units, whereas silencers are elements that repress transcription regardless of their position. In plants, these elements remain largely uncharacterized. Here, we use the massively parallel reporter assay Plant STARR-seq with short fragments of 8 large insulators to identify more than 100 fragments that block enhancer activity.

Small DNA elements that act as both insulators and silencers in plants.

Insulators are -regulatory elements that separate transcriptional units, whereas silencers are elements that repress transcription regardless of their position. In plants, these elements remain largely uncharacterized. Here, we use the massively parallel reporter assay Plant STARR-seq with short fragments of eight large insulators to identify more than 100 fragments that block enhancer activity.

A Novel, Precise and High-Throughput Technology for Viroid Detection in Cannabis (MFDetect).

Hop latent viroid (HLVd) is a severe disease of cannabis, causing substantial economic losses in plant yield and crop value for growers worldwide. The best way to control the disease is early detection to limit the spread of the viroid in grow facilities. This study describes MFDetect as a rapid, highly sensitive, and high-throughput tool for detecting HLVd in the early stages of plant development.

Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum.

Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive.

Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum.

For many important crops including sorghum, use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but also by the ability to regenerate a fertile plant from the engineered cell through tissue culture. Here, we report that Wuschel2 (Wus2)-enabled transformation increases not only the transformation efficiency, but also the CRISPR/Cas-targeted genome editing frequency in sorghum (Sorghum bicolor L.).

Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.

Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation.

An Efficient Gene Excision System in Maize.

Use of the morphogenic genes () and (), along with new ternary constructs, has increased the genotype range and the type of explants that can be used for maize transformation. Further optimizing the expression pattern for / has resulted in rapid maize transformation methods that are faster and applicable to a broader range of inbreds. However, expression of / can compromise the quality of regenerated plants, leading to sterility.

Efficient Gene Targeting in Maize Using Inducible CRISPR-Cas9 and Marker-free Donor Template.

CRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle before application of CRISPR-Cas9 technology is fully realized in plant breeding. Here, we report high-frequency, selectable marker-free intra-genomic gene targeting (GT) in maize.

Overexpression of VIRE2-INTERACTING PROTEIN2 in Arabidopsis regulates genes involved in Agrobacterium-mediated plant transformation and abiotic stresses.

Arabidopsis VIRE2-INTERACTING PROTEIN2 (VIP2) was previously described as a protein with a NOT domain, and Arabidopsis vip2 mutants are recalcitrant to Agrobacterium-mediated root transformation. Here we show that VIP2 is a transcription regulator and the C-terminal NOT2 domain of VIP2 interacts with VirE2. Interestingly, AtVIP2 overexpressor lines in Arabidopsis did not show an improvement in Agrobacterium-mediated stable root transformation, but the transcriptome analysis identified 1,634 differentially expressed genes compared to wild-type.

High efficiency Agrobacterium-mediated site-specific gene integration in maize utilizing the FLP-FRT recombination system.

An efficient Agrobacterium-mediated site-specific integration (SSI) technology using the flipase/flipase recognition target (FLP/FRT) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line (RTL) containing the corresponding heterologous FRT sites. A promoter-trap system consisting of a pre-integrated promoter followed by an FRT site enables efficient selection of events.

Novel Ternary Vectors for Efficient Sorghum Transformation.

Sorghum has been considered a recalcitrant crop for tissue culture and genetic transformation. A breakthrough in Agrobacterium-mediated sorghum transformation was achieved with the use of super-binary cointegrate vectors based on plasmid pSB1. However, even with pSB1, transformation capability was restricted to certain sorghum genotypes, excluding most of the important African sorghum varieties.

Maize Transformation Using the Morphogenic Genes Baby Boom and Wuschel2.

Despite the fact that maize transformation has been available for over 25 years, the technology has remained too specialized, labor-intensive, and inefficient to be useful for the majority of academic labs. Compounding this problem, future demands in maize genome engineering will likely require a step change beyond what researchers view as "traditional" maize transformation methods. Recently, we published on our use of constitutively expressed morphogenic transcription factors Baby Boom (Bbm) and Wuschel2 (Wus2) to improve maize transformation, which requires CRE-mediated excision before regeneration of healthy, fertile T0 plants.

Advancing Agrobacterium-Based Crop Transformation and Genome Modification Technology for Agricultural Biotechnology.

The last decade has seen significant strides in Agrobacterium-mediated plant transformation technology. This has not only expanded the number of crop species that can be transformed by Agrobacterium, but has also made it possible to routinely transform several recalcitrant crop species including cereals (e.g.

An improved ternary vector system for Agrobacterium-mediated rapid maize transformation.

A simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation. The super binary plasmid pSB1 is a mainstay of maize transformation.

Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application.

Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum.

Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens.

The MinION is a portable single-molecule DNA sequencing instrument that was released by Oxford Nanopore Technologies in 2014, producing long sequencing reads by measuring changes in ionic flow when single-stranded DNA molecules translocate through the pores. While MinION long reads have an error rate substantially higher than the ones produced by short-read sequencing technologies, they can generate de novo assemblies of microbial genomes, after an initial correction step that includes alignment of Illumina sequencing data or detection of overlaps between Oxford Nanopore reads to improve accuracy. In this study, MinION reads were generated from the multi-chromosome genome of Agrobacterium tumefaciens strain LBA4404.

Effect of Agrobacterium strain and plasmid copy number on transformation frequency, event quality and usable event quality in an elite maize cultivar.

Improving Agrobacterium -mediated transformation frequency and event quality by increasing binary plasmid copy number and appropriate strain selection is reported in an elite maize cultivar. Agrobacterium-mediated maize transformation is a well-established method for gene testing and for introducing useful traits in a commercial biotech product pipeline. To develop a highly efficient maize transformation system, we investigated the effect of two Agrobacterium tumefaciens strains and three different binary plasmid origins of replication (ORI) on transformation frequency, vector backbone insertion, single copy event frequency (percentage of events which are single copy for all transgenes), quality event frequency (percentage of single copy events with no vector backbone insertions among all events generated; QE) and usable event quality frequency (transformation frequency times QE frequency; UE) in an elite maize cultivar PHR03.

Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line.

An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds. This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media.

The role of RAR1 in Agrobacterium-mediated plant transformation.

RAR 1 is identified as a critical protein involved in plant innate immunity. We investigated the role of RAR 1 in Agrobacterium-mediated plant transformation based on the previous findings that accessory proteins associated with the E3 ligase complex such as SGT1, which tightly interacts with RAR 1, play a role in the transformation process. RAR1 gene silencing in Nicotiana benthamiana and Arabidopsis rar1 mutant analyses suggested that RAR1 is required for early stages of Agrobacterium-mediated plant transformation.

Several components of SKP1/Cullin/F-box E3 ubiquitin ligase complex and associated factors play a role in Agrobacterium-mediated plant transformation.

• Successful genetic transformation of plants by Agrobacterium tumefaciens requires the import of bacterial T-DNA and virulence proteins into the plant cell that eventually form a complex (T-complex). The essential components of the T-complex include the single stranded T-DNA, bacterial virulence proteins (VirD2, VirE2, VirE3 and VirF) and associated host proteins that facilitate the transfer and integration of T-DNA. The removal of the proteins from the T-complex is likely achieved by targeted proteolysis mediated by VirF and the plant ubiquitin proteasome complex.

Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens.

We investigated the effects of salicylic acid (SA) and systemic acquired resistance (SAR) on crown gall disease caused by Agrobacterium tumefaciens. Nicotiana benthamiana plants treated with SA showed decreased susceptibility to Agrobacterium infection. Exogenous application of SA to Agrobacterium cultures decreased its growth, virulence, and attachment to plant cells.

A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing.

Virus-induced gene silencing (VIGS) is a rapid and robust method for determining and studying the function of plant genes or expressed sequence tags (ESTs). However, only a few plant species are amenable to VIGS. There is a need for a systematic study to identify VIGS-efficient plant species and to determine the extent of homology required between the heterologous genes and their endogenous orthologs for silencing.

The phytotoxin coronatine contributes to pathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringae pv. tomato DC3000.

The roles of the phytotoxin coronatine (COR) and salicylic acid (SA)-mediated defenses in the interaction of Pseudomonas syringae pv. tomato DC3000 and tomato (Solanum lycopersicum) were investigated. Unlike findings reported for Arabidopsis thaliana, DC3000 mutants impaired for production of COR or one of its components, coronafacic acid (CFA) or coronamic acid (CMA), induced distinctly different disease lesion phenotypes in tomato.

Arabidopsis VIRE2 INTERACTING PROTEIN2 is required for Agrobacterium T-DNA integration in plants.

Agrobacterium tumefaciens-mediated genetic transformation is an efficient tool for genetic engineering of plants. VirE2 is a single-stranded DNA binding Agrobacterium protein that is transported into the plant cell and presumably protects the T-DNA from degradation. Using a yeast two-hybrid system, we identified Arabidopsis thaliana VIRE2-INTERACTING PROTEIN2 (VIP2) with a NOT domain that is conserved in both plants and animals.

Monitoring in planta bacterial infection at both cellular and whole-plant levels using the green fluorescent protein variant GFPuv.

* Green fluorescent protein (GFP) labeling of bacteria has been used to study their infection of and localization in plants, but strong autofluorescence from leaves and the relatively weak green fluorescence of GFP-labeled bacteria restrict its broader application to investigations of plant-bacterial interactions. * A stable and broad-host-range plasmid vector (pDSK-GFPuv) that strongly expresses GFPuv protein was constructed not only for in vivo monitoring of bacterial infection, localization, activity, and movement at the cellular level under fluorescence microscopy, but also for monitoring bacterial disease development at the whole-plant level under long-wavelength ultraviolet (UV) light. * The presence of pDSK-GFPuv did not have significant impact on the in vitro or in planta growth and virulence of phytobacteria.