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. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.

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. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom (Bbm) and Wuschel2 (Wus2) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL1 resulted in higher transformation frequency than strain LBA4404 THY- (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL1; 0.65% in LBA4404 THY-). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%-22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full-length site-specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development.

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., maize, sorghum, and wheat). However, the technology is limited by the random nature of DNA insertions, genotype dependency, low frequency of quality events, and variation in gene expression arising from genomic insertion sites. A majority of these deficiencies have now been addressed by improving the frequency of quality events, developing genotype-independent transformation capability in maize, developing an Agrobacterium-based site-specific integration technology for precise gene targeting, and adopting Agrobacterium-delivered CRISPR-Cas genes for gene editing. These improved transformation technologies are discussed in detail in this chapter.

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

KEY MESSAGE: 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. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86-103%) raw transformation frequency in an elite maize inbred.

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. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free 'quality events' ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84-7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9-mediated gene knockouts in Tx430.

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

KEY MESSAGE: 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 strain AGL0 gave a higher transformation frequency, but a reduced QE frequency than LBA4404 due to a higher number of vector backbone insertions. Higher binary plasmid copy number positively correlated with transformation frequency and usable event recovery. The above findings can be exploited to develop high-throughput transformation protocols, improve the quality of transgenic events in maize and other plants.

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

KEY MESSAGE: 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 use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.

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

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 MFDetectTM as a rapid, highly sensitive, and high-throughput tool for detecting HLVd in the early stages of plant development. Furthermore, in the largest research study conducted so far for HLVd detection in cannabis, we compared MFDetectTM with quantitative RT-PCR in a time course experiment using different plant tissues, leaves, petioles, and roots at different plant developmental stages to demonstrate both technologies are comparable. Our study found leaf tissue is a suitable plant material for HLVd detection, with the viroid titer increasing in the infected leaf tissue with the age of plants. The study showed that other tissue types, including petiole and roots, were equally sensitive to detection via MFDetectTM. The assay developed in this research allows the screening of thousands of plants in a week. The assay can be scaled easily to provide growers with a quick turnaround and a cost-effective diagnostic tool for screening many plants and tissue types at different stages of 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. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.

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. • We evaluated the involvement of the host SKP1/culin/F-box (SCF)-E3 ligase complex and its role in plant transformation. Gene silencing, mutant screening and gene expression studies suggested that the Arabidopsis homologs of yeast SKP1 (suppressor of kinetochore protein 1) protein, ASK1 and ASK2, are required for Agrobacterium-mediated plant transformation. • We identified the role for SGT1b (suppressor of the G2 allele of SKP1), an accessory protein that associates with SCF-complex, in plant transformation. We also report the differential expression of many genes that encode F-box motif containing SKP1-interacting proteins (SKIP) upon Agrobacterium infection. • We speculate that these SKIP genes could encode the plant specific F-box proteins that target the T-complex associated proteins for polyubiquitination and subsequent degradation by the 26S proteasome.

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.). Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased the regeneration capacity that resulted in higher transformation efficiency across different sorghum varieties. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrate up to 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled transformation, compared to without Wus2, across various targeted loci in different sorghum genotypes.

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. Heat shock-inducible Cas9 was used for generating targeted double-strand breaks and simultaneous mobilization of the donor template from pre-integrated T-DNA. The construct was designed such that release of the donor template and subsequent DNA repair activated expression of the selectable marker gene within the donor locus. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants, with up to 86% detected donor template release and 99% mutation rate being observed at the donor loci and the genomic target site, respectively. Unlike previous in planta or intra-genomic homologous recombination reports in which the original chimeric GT plants required extensive progeny screening in the next generation to identify non-chimeric GT individuals, our method provides non-chimeric heritable GT in one generation.

An Efficient Gene Excision System in Maize.

Use of the morphogenic genes Baby Boom (Bbm) and Wuschel2 (Wus2), 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 Bbm/Wus2 has resulted in rapid maize transformation methods that are faster and applicable to a broader range of inbreds. However, expression of Bbm/Wus2 can compromise the quality of regenerated plants, leading to sterility. We reasoned excising morphogenic genes after transformation but before regeneration would increase production of fertile T0 plants. We developed a method that uses an inducible site-specific recombinase (Cre) to excise morphogenic genes. The use of developmentally regulated promoters, such as Ole, Glb1, End2, and Ltp2, to drive Cre enabled excision of morphogenic genes in early embryo development and produced excised events at a rate of 25-100%. A different strategy utilizing an excision-activated selectable marker produced excised events at a rate of 53-68%; however, the transformation frequency was lower (13-50%). The use of inducible heat shock promoters (e.g. Hsp17.7, Hsp26) to express Cre, along with improvements in tissue culture conditions and construct design, resulted in high frequencies of T0 transformation (29-69%), excision (50-97%), usable quality events (4-15%), and few escapes (non-transgenic; 14-17%) in three elite maize inbreds. Transgenic events produced by this method are free of morphogenic and marker genes.

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. We recently developed a ternary vector system incorporating the pVIR accessory plasmid. The ternary vector system not only doubled the transformation frequency (TF) in Tx430, but also extended the transformation capability into an important African sorghum elite variety.

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. Moving beyond this first-generation system, we have developed a new expression system for Bbm and Wus2, using a non-constitutive maize phospholipid transferase protein promoter (Pltp pro) driving Bbm expression and a maize auxin-inducible promoter (Axig1 pro) for WUS2 expression. Using this combination of expression cassettes, abundant somatic embryos rapidly form on the scutella of Agrobacterium-transformed zygotic immature embryos. These somatic embryos are uniformly transformed and can be directly germinated into plants without a callus phase. Transformed plants are sent to the greenhouse in as little as 1 month, and these T0 plants match the seed-derived phenotype for the inbred and are fertile. T1 seeds germinate normally and have a uniformly wild-type inbred phenotype. This new system represents a rapid, user-friendly transformation process that can potentially facilitate high-throughput production of transgenic T0 plants in B73, Mo17, and the recently developed Fast-Flowering Mini-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. These differentially expressed genes belonged to various functional categories such as membrane proteins, circadian rhythm, signaling, response to stimulus, regulation of plant hypersensitive response, sequence-specific DNA binding transcription factor activity and transcription regulatory region binding. In addition to regulating genes involved in Agrobacterium-mediated plant transformation, AtVIP2 overexpressor line showed differential expression of genes involved in abiotic stresses. The majority of the genes involved in abscisic acid (ABA) response pathway, containing the Abscisic Acid Responsive Element (ABRE) element within their promoters, were down-regulated in AtVIP2 overexpressor lines. Consistent with this observation, AtVIP2 overexpressor lines were more susceptible to ABA and other abiotic stresses. Based on the above findings, we hypothesize that VIP2 not only plays a role in Agrobacterium-mediated plant transformation but also acts as a general transcriptional regulator in plants.

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. Tomato plants inoculated with the CFA- CMA- mutant DB29 showed elevated transcript levels of SlICS, which encodes isochorismate synthase, an enzyme involved in SA biosynthesis in S. lycopersicum. Furthermore, expression of genes encoding SA-mediated defense proteins were elevated in DB29-inoculated plants compared with plants inoculated with DC3000, suggesting that COR suppresses SlICS-mediated SA responses. Sequence analysis of SlICS revealed that it encodes a protein that is 55 and 59.6% identical to the A. thaliana ICS-encoded proteins AtICS1 and AtICS2, respectively. Tomato plants silenced for SlICS were hypersusceptible to DC3000 and accumulated lower levels of SA after infection with DC3000 compared with inoculated wild-type tomato plants. Unlike what has been shown for A. thaliana, the COR- mutant DB29 was impaired for persistence in SlICS-silenced tomato plants; thus, COR has additional roles in virulence that are SA independent and important in the latter stages of disease development. In summary, the infection assays, metabolic profiling, and gene expression results described in this study indicate that the intact COR molecule is required for both suppression of SA-mediated defense responses and full disease symptom development in tomato.

Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing.

Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.

Electroelution of intact proteins from SDS-PAGE gels and their subsequent MALDI-TOF MS analysis.

A method has been developed for the extraction of intact proteins from SDS-PAGE gels and for subsequent MALDI-TOF MS analysis to determine precise molecular mass. The method consists of an electroelution step and a subsequent low-temperature matrix-analyte cocrystallization step prior to MALDI-TOF MS. Proteins were first extracted from polyacrylamide gels using electroelution (ProteoPLUS). Following electroelution, salts, SDS, and dye were removed by dialysis using the same electroelution tubes. The proteins were then concentrated in a vacuum centrifuge. Using bovine serum albumin (BSA) and phosphorylase B (Phos B) as standards, it was found that electroelution was more effective than passive elution in extracting intact proteins from gels. Optimized protein recoveries were determined to be 89% for BSA and 58% for Phos B, supporting the hypothesis of an inverse relationship between recovery and protein size. The traditional practice of "fixing gels" using methanol and acetic acid was found to reduce protein recoveries significantly and should be avoided if possible. A subsequent sample preparation method involving the low-temperature cocrystallization of whole proteins with MALDI matrices (trans-3,5-dimethoxy-4-hydroxycinnamic acid, sinapinic acid, 10 mg/mL) was also developed to remove residual impurities further. Proteins and MALDI matrices were mixed, sealed, and stored at 4 degrees C to allow for overnight cocrystallization of matrix and protein. The supernatant was removed and protein-matrix crystals redissolved using the same matrix solution. MALDI-TOF MS signal intensities were found to increase three- to fourfold using the low-temperature cocrystallization method relative to the more common method of immediate analysis following matrix-analyte mixing and "dried droplet" deposition. The reported method is illustrated using two beta-1,3-endoglucanase isoforms from wheat apoplast extracts.

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. Errors in the consensus two-directional (sense and antisense) "2D" sequences were first characterized by way of comparison with an internal reference assembly. Both Illumina-based correction and self-correction were performed and the resulting corrected reads assembled into high-quality hybrid and non-hybrid assemblies. Corrected read datasets and assemblies were subsequently compared. The results shown here indicate that both hybrid and non-hybrid methods can be used to assemble Oxford Nanopore reads into informative multi-chromosome assemblies, each with slightly different outcomes in terms of contiguity and accuracy.