Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants.

Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 5'-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants.

Potato (Solanum tuberosum L.).

Agrobacterium-mediated transformation is the most common method for the incorporation of foreign genes into the genome of potato as well as many other species in the Solanaceae family. This chapter describes protocols for the genetic transformation of three species of potato: Solanum tuberosum subsp. tuberosum (Desiréé), S. tuberosum subsp. andigenum (Blue potato), and S. tuberosum subsp. andigena using internodal segments as explants.

Tomato (Solanum lycopersicum).

Agrobacterium-mediated transformation is the most common method for the incorporation of foreign genes into the genome of tomato as well as many other species in the Solanaceae family. This chapter describes a protocol for the genetic transformation of tomato cultivar Micro-Tom using cotyledons as explants. Detailed procedures are also included for determining gene-copy number using a duplex qPCR TaqMan assay, and the histochemical analysis of GUS expression.

Leucine aminopeptidase regulates defense and wound signaling in tomato downstream of jasmonic acid.

Leucine aminopeptidase A (LapA) is a late wound-response gene of tomato (Solanum lycopersicum). To elucidate the role of LapA, transgenic plants that overexpressed or abolished LapA gene expression were used. The early wound-response gene RNA levels were similar in wild-type and Lap-silenced (LapA-SI), -antisense (LapA-AS), and -overexpressing (LapA-OX) plants. By contrast, late wound-response gene RNA levels and protection against Manduca sexta damage were influenced by LapA RNA and protein levels. While LapA-OX plants had elevated levels of LapA RNAs and protein, ectopic expression of LapA was not sufficient to induce Pin (Ser proteinase inhibitor) or PPO (polyphenol oxidase) transcripts in nonwounded leaves. M. sexta larvae damaged less foliage and displayed delays in growth and development when feeding on LapA-OX plants. By contrast, LapA-SI and LapA-AS lines had lower levels of Pin and PPO RNAs than wild-type controls. Furthermore, larvae consumed more foliage and attained larger masses when feeding on LapA-SI plants. Jasmonic acid (JA) did not complement the wound-signaling phenotype of LapA-SI plants. Based on root elongation in the presence of JA, JA perception appeared to be intact in LapA-SI lines. Collectively, these data suggested that LAP-A has a role in modulating essential defenses against herbivores by promoting late wound responses and acting downstream of JA biosynthesis and perception.

Targeting and localization of wound-inducible leucine aminopeptidase A in tomato leaves.

The constitutive and wound-inducible leucine aminopeptidases (LAP-N and LAP-A, respectively) of tomato encode 60-kDa proteins with 5-kDa presequences that resemble chloroplast-targeting peptides. Cell fractionation studies and immunoblot analyses of chloroplast and total proteins have suggested a dual location of the mature LAP-A proteins in the cytosol and the plastids. In this study, the subcellular localization of tomato LAPs was further investigated using in vitro chloroplast-targeting assays and immunocytochemical techniques at the light and TEM levels. In vitro-translated LAP-A1 and LAP-N preproteins were readily transported into pea chloroplasts and processed into mature proteins of 55 kDa indicating the presence of a functional chloroplast-targeting signal in the LAP-A1 and LAP-N protein precursors. In addition, a LAP polyclonal and a LAP-A-specific antisera were used to immunolocalize LAP proteins in leaves from healthy, wounded and methyl jasmonate (MeJA)-treated plants. Low levels of LAPs and/or LAP-like proteins were detected in leaves from unwounded plants. The LAP polyclonal antiserum, which detected LAP-A, LAP-N and LAP-like proteins, and the LAP-A specific antibodies, which detected only LAP-A, showed that LAP levels increased in leaf sections after wounding and MeJA treatments. LAP-A proteins were primarily detected within the chloroplasts of spongy and palisade mesophyll cells. The localization of LAP-A was distinct from the location of early wound-response proteins that are important in the biosynthesis of jasmonic acid or systemin and more similar to the late wound-response proteins with primary roles in defense. The importance of these findings relative to the potential roles of LAP-A in defense is discussed.

Systemic wound signaling in tomato leaves is cooperatively regulated by systemin and hydroxyproline-rich glycopeptide signals.

Hydroxyproline-rich glycopeptides (HypSys peptides) have been isolated recently from tobacco and tomato leaves that are powerful activators of protease inhibitor synthesis. The peptides are processed from polyprotein precursors, two from a single tobacco precursor and three from a single tomato precursor. The precursor genes are expressed in response to wounding and methyl jasmonate, similar to the expression of the systemin precursor prosystemin in tomato leaves. Here we investigate the relationships between systemin and the tomato HypSys peptides in regulating wound signaling in tomato plants. Analysis of transgenic tomato plants over-expressing sense and antisense constructs of the tomato HypSys precursor under the 35S CaMV promoter show that the transgenic plants regulate protease inhibitor gene expression in response to wounding in a manner similar to prosystemin. The evidence indicates that the expression of both the tomato HypSys precursor gene and the prosystemin gene in response to wounding are necessary for strong systemic signaling. The data supports a role for both genes in an amplification loop that up-regulates the octadecanoid pathway and the synthesis of jasmonates to effect strong systemic signaling of defense genes. This report provides the first demonstration of the involvement of two plant peptides derived from two unrelated genes in regulating long distance wound signaling in plants.

The plant cell wall matrix harbors a precursor of defense signaling peptides.

Proteins of plant cell walls serve as structural macromolecules and play important roles in morphogenesis and development but have not been reported to be the origins of peptide signals that activate genes for plant defense. We report here that the mRNA coding the tomato leaf polyprotein precursor of three hydroxyproline-rich glycopeptide defense signals (called LeHypSys I, II, and III) is synthesized in phloem parenchyma cells in response to wounding, systemin, and methyl jasmonate, and the nascent protein is sequestered in the cell wall matrix. These findings indicate that the plant cell wall can play an active role in defense as a source of peptide signals for systemic wound signaling.

The cellular localization of prosystemin: a functional role for phloem parenchyma in systemic wound signaling.

The systemin precursor, prosystemin, has been previously shown to be sequestered in vascular bundles of tomato ( Lycopersicon esculentum Mill.) plants, but its subcellular compartmentalization and association with a specific cell type has not been established. We present in situ hybridization and immunocytochemical evidence at the light, confocal, and transmission electron microscopy levels that wound-induced and methyl jasmonate-induced prosystemin mRNA and protein are exclusively found in vascular phloem parenchyma cells of minor veins and midribs of leaves, and in the bicollateral phloem bundles of petioles and stems of tomato. Prosystemin protein was also found constitutively in parenchyma cells of various floral organs, including sepals, petals and anthers. At the subcellular level, prosystemin was found compartmentalized in the cytosol and the nucleus of vascular parenchyma cells. The cumulative data indicate that vascular phloem parenchyma cells are the sites for the synthesis and processing of prosystemin as a first line of defense signaling in response to herbivore and pathogen attacks.

The systemin precursor gene regulates both defensive and developmental genes in Solanum tuberosum.

Transformation of Solanum tuberosum, cv. Desiree, with the tomato prosystemin gene, regulated by the 35S cauliflower mosaic virus promoter, resulted in constitutive increase in defensive proteins in potato leaves, similar to its effects in tomato plants, but also resulted in a dramatic increase in storage protein levels in potato tubers. Tubers from selected transformed lines contained 4- to 5-fold increases in proteinase inhibitor I and II proteins, >50% more soluble and dry weight protein, and >50% more total nitrogen and total free amino acids than found in wild-type tubers. These results suggest that the prosystemin gene plays a dual role in potato plants in regulating proteinase inhibitor synthesis in leaves in response to wounding and in regulating storage protein synthesis in potato tubers in response to developmental cues. The results indicated that components of the systemin signaling pathway normally found in leaves have been recruited by potato plants to be developmentally regulated to synthesize and accumulate large quantities of storage proteins in tubers.