Transposase-assisted target-site integration for efficient plant genome engineering.

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops1,2. Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3-5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6-9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria10-12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans.

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.

Novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress.

KEY MESSAGE: Agrobacterium tumefaciens caused tissue browning leading to subsequent cell death in plant transformation and novel anti-oxidative compounds enhanced Agrobacterium -mediated plant transformation by mitigating oxidative stress. Browning and death of cells transformed with Agrobacterium tumefaciens is a long-standing and high impact problem in plant transformation and the agricultural biotechnology industry, severely limiting the production of transgenic plants. Using our tomato cv. MicroTom transformation system, we demonstrated that Agrobacterium caused tissue browning (TB) leading to subsequent cell death by our correlation study. Without an antioxidant (lipoic acid, LA) TB was severe and associated with high levels of GUS transient expression and low stable transformation frequency (STF). LA addition shifted the curve in that most TB was intermediate and associated with the highest levels of GUS transient expression and STF. We evaluated 18 novel anti-oxidative compounds for their potential to enhance Agrobacterium-mediated transformation, by screening for TB reduction and monitoring GUS transient expression. Promising compounds were further evaluated for their effect on MicroTom and soybean STF. Among twelve non-antioxidant compounds, seven and five significantly (P < 0.05) reduced TB and increased STF, respectively. Among six antioxidants four of them significantly reduced TB and five of them significantly increased STF. The most efficient compound found to increase STF was melatonin (MEL, an antioxidant). Optimal concentrations and stages to use MEL in transformation were determined, and Southern blot analysis showed that T-DNA integration was not affected by MEL. The ability of diverse compounds with different anti-oxidative mechanisms can reduce Agrobacterium-mediated TB and increase STF, strongly supporting that oxidative stress is an important limiting factor in Agrobacterium-mediated transformation and the limiting factor can be controlled by these compounds at different levels.

Cytokinins secreted by Agrobacterium promote transformation by repressing a plant myb transcription factor.

Agrobacterium-mediated transformation is the most widely used technique for generating transgenic plants. However, many crops remain recalcitrant. We found that an Arabidopsis myb family transcription factor (MTF1) inhibited plant transformation susceptibility. Mutating MTF1 increased attachment of several Agrobacterium strains to roots and increased both stable and transient transformation in both susceptible and transformation-resistant Arabidopsis ecotypes. Cytokinins from Agrobacterium tumefaciens decreased the expression of MTF1 through activation of the cytokinin response regulator ARR3. Mutating AHK3 and AHK4, genes that encode cytokinin-responsive kinases, increased the expression of MTF1 and impaired plant transformation. Mutant mtf1 plants also had increased expression of AT14A, which encodes a putative transmembrane receptor for cell adhesion molecules. Plants overexpressing AT14A exhibited increased susceptibility to transformation, whereas at14a mutant plants exhibited decreased attachment of bacteria to roots and decreased transformation, suggesting that AT14A may serve as an anchor point for Agrobacteria. Thus, by promoting bacterial attachment and transformation of resistant plants and increasing such processes in susceptible plants, treating roots with cytokinins may help engineer crops with improved features or yield.

Constitutive expression exposes functional redundancy between the Arabidopsis histone H2A gene HTA1 and other H2A gene family members.

The Arabidopsis thaliana histone H2A gene HTA1 is essential for efficient transformation of Arabidopsis roots by Agrobacterium tumefaciens. Disruption of this gene in the rat5 mutant results in decreased transformation. In Arabidopsis, histone H2A proteins are encoded by a 13-member gene family. RNA encoded by these genes accumulates to differing levels in roots and whole plants; HTA1 transcripts accumulate to levels up to 1000-fold lower than do transcripts of other HTA genes. We examined the extent to which other HTA genes or cDNAs could compensate for loss of HTA1 activity when overexpressed in rat5 mutant plants. Overexpression of all tested HTA cDNAs restored transformation competence to the rat5 mutant. However, only the HTA1 gene, but not other HTA genes, could phenotypically complement rat5 mutant plants when expressed from their native promoters. Expression analysis of HTA promoters indicated that they had distinct but somewhat overlapping patterns of expression in mature plants. However, only the HTA1 promoter was induced by wounding or by Agrobacterium infection of root segments. Our data suggest that, with respect to Agrobacterium-mediated transformation, all tested histone H2A proteins are functionally redundant. However, this functional redundancy is not normally evidenced because of the different expression patterns of the HTA genes.

Binding of sulfonylurea by AtMRP5, an Arabidopsis multidrug resistance-related protein that functions in salt tolerance.

Recently, a new member of the ABC transporter superfamily of Arabidopsis, AtMRP5, was identified and characterized. In the present work, we found that AtMRP5 can bind specifically to sulfonurea when it is expressed in HEK293 cells. We also present evidence for a new role of AtMRP5 in the salt stress response of Arabidopsis. We used reverse genetics to identify an Arabidopsis mutant (atmrp5-2) in which the AtMRP5 gene was disrupted by transferred DNA insertion. In root-bending assays using Murashige and Skoog medium supplemented with 100 mm NaCl, root growth of atmrp5-2 was substantially inhibited in contrast to the almost normal growth of wild-type seedlings. This hypersensitive response of the atmrp5-2 mutant was not observed during mannitol treatment. The root growth of the wild-type plant grown in Murashige and Skoog medium supplemented with the MRP inhibitor glibenclamide and NaCl was inhibited to a very similar extent as the root growth of atmrp5-2 grown in NaCl alone. The Na(+)-dependent reduction of root growth of the wild-type plant in the presence of glibenclamide was partially restored by diazoxide, a known K+ channel opener that reverses the inhibitory effects of sulfonylureas in animal cells. Moreover, the atmrp5-2 mutant was defective in 86Rb+ uptake. When seedlings were treated with 100 mm NaCl, atmrp5-2 seedlings accumulated less K+ and more Na+ than those of the wild type. These observations suggest that AtMRP5 is a putative sulfonylurea receptor that is involved in K+ homeostasis and, thus, also participates in the NaCl stress response.

Identification of Arabidopsis rat mutants.

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation.

Transformation of plant cells by Agrobacterium tumefaciens involves both bacterial virulence proteins and host proteins. We have previously shown that the Arabidopsis thaliana gene H2A-1 (RAT5), which encodes histone H2A-1, is involved in T-DNA integration into the plant genome. Mutation of RAT5 results in a severely decreased frequency of transformation, whereas overexpression of RAT5 enhances the transformation frequency (Mysore et al., 2000b). We show here that the expression pattern of RAT5 correlates with plant root cells most susceptible to transformation. As opposed to a cyclin-GUS fusion gene whose expression is limited to meristematic tissues, the H2A-1 gene is expressed in many non-dividing cells. Under normal circumstances, the H2A-1 gene is expressed in the elongation zone of the root, the region that is most susceptible to Agrobacterium transformation. In addition, when roots are incubated on medium containing phytohormones, a concomitant shift in H2A-1 expression and transformation susceptibility to the root base is observed. Inoculation of root segments with a transfer-competent, but not a transformation-deficient Agrobacterium strain induces H2A-1 expression. Furthermore, pre-treatment of Arabidopsis root segments with phytohormones both induces H2A-1 expression and increases the frequency of Agrobacterium transformation. Our results suggest that the expression of the H2A-1 gene is both a marker for, and a predictor of, plant cells most susceptible to Agrobacterium transformation.