Martin H. Spalding

High-efficiency TALEN-based gene editing produces disease-resistant rice

volume 30 number 5 may 2012 nature biotechnology 24 repeat units for recognition of a specific set of 24 contiguous nucleotides at the target sites (Supplementary Fig. 1). For each pair of TALEN genes, one TALEN gene (half of the pair) was under the control of the 35S promoter of cauliflower mosaic virus and the other gene was driven by the maize ubiquitin 1 promoter, comprising a specific TALEN pair in a single plasmid (Supplementary Fig. 2). Each plasmid also contained a marker gene for hygromycin resistance. These constructs were introduced pathogen’s nutritional needs and enhance its persistence2,14. The Os11N3 promoter contains an effector-binding element (EBE) for AvrXa7, overlapping with another EBE for PthXo3 and with the TATA box (Fig. 1a and Supplementary Fig. 1). We deployed two pairs of designer TALENs (pair 1 and pair 2) independently to induce mutations in these overlapping EBEs of the Os11N3 promoter and thus to interfere with the virulence function of AvrXa7 and PthXo3, but not the developmental function of Os11N3 (Supplementary Fig. 1 and Supplementary Note). The TALE repetitive regions used for nuclease fusions included the native AvrXa7 and three designer TALE repetitive regions custom synthesized using a modular assembly method8. Each designer TALEN contained To the Editor: Transcription activator–like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo) contribute to pathogen virulence by transcriptionally activating specific rice disease-susceptibility (S) genes1,2. TAL effector nucleases (TALENs)—fusion proteins derived from the DNA recognition repeats of native or customized TAL effectors and the DNA cleavage domains of FokI3–5—have been used to create site-specific gene modifications in plant cells6,7, yeast8, animals9–12 and even human pluripotent cells13. Here, we exploit TALEN technology to edit a specific S gene in rice to thwart the virulence strategy of X. oryzae and thereby engineer heritable genome modifications for resistance to bacterial blight, a devastating disease in a crop that feeds half of the world’s population. We targeted the rice bacterial blight susceptibility gene Os11N3 (also called OsSWEET14) for TALEN-based disruption. This rice gene encodes a member of the SWEET sucrose-efflux transporter family and is hijacked by X. oryzae pv. oryzae, using its endogenous TAL effectors AvrXa7 or PthXo3, to activate the gene and thus divert sugars from the plant cell so as to satisfy the High-efficiency TALEN-based gene editing produces disease-resistant rice

Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice

The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust technology for targeted gene editing in various organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at single cleavage sites have been reported in transient and stable transformations, although genetic transmission of edits has been reported only in Arabidopsis and rice. Large chromosomal excision between two remote nuclease-targeted loci has been reported only in a few non-plant species. Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the inheritance of genome edits in multiple generations and construction of a set of facile vectors for high-efficiency, multiplex gene targeting. Four sugar efflux transporter genes were modified in rice at high efficiency; the most efficient system yielding 87–100% editing in T0 transgenic plants, all with di-allelic edits. Furthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several genome-edited but transgene-free rice plants. We also demonstrated proof-of-efficiency of Cas9/sgRNAs in producing large chromosomal deletions (115–245 kb) involving three different clusters of genes in rice protoplasts and verification of deletions of two clusters in regenerated T0 generation plants. Together, these data demonstrate the power of our Cas9/sgRNA platform for targeted gene/genome editing in rice and other crops, enabling both basic research and agricultural applications.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand

The world’s crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

Quantification of Compartmented Metabolic Fluxes in Developing Soybean Embryos by Employing Biosynthetically Directed Fractional 13C Labeling, Two-Dimensional [13C, 1H] Nuclear Magnetic Resonance, and Comprehensive Isotopomer Balancing

Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis tool that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-13C Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [13C, 1H] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 13C isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain sds. A high flux was found through the oxidative pentose phosphate pathway (19.99 ± 4.39 μmol d−1 cotyledon−1, or 104.2 carbon mol ± 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 ± 5.00 μmol d−1 cotyledon−1, or 113.2 carbon mol ±26.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.

Elevated CO2 Effects during Leaf Ontogeny (A New Perspective on Acclimation).

For many plants growth in elevated CO2 leads to reduced rates of photosynthesis. To examine the role that leaf ontogeny plays in the acclimation response, we monitored photosynthesis and some related parameters at short intervals throughout the ontogenetic development of tobacco (Nicotiana tabacum L.) leaves under ambient (350 [mu]L L-1)- and high (950 [mu]L L-1)-CO2 conditions. The pattern of photosynthetic rate over time was similar between the two treatments and consistent with the expected pattern for a typical dicot leaf. However, the photosynthesis pattern in high-CO2-grown tobacco was shifted temporally to an earlier maximum and subsequent senescent decline. Ribulose-1,5-biphosphate carboxylase/oxygenase activity appeared to be the main factor regulating photosynthetic rates in both treatments. Therefore, we propose a new model for interpreting the acclimation response. Lowered photosynthetic rates observed during acclimation appear to be the result of a shift in the timing of the normal photosynthetic stages of leaf ontogeny to an earlier onset of the natural decline in photosynthetic rates associated with senescence.

Transcriptome-Wide Changes in Chlamydomonas reinhardtii Gene Expression Regulated by Carbon Dioxide and the CO2-Concentrating Mechanism Regulator CIA5/CCM1

Using powerful tools to investigate the regulation of global gene expression in the model microalga Chlamydomonas reinhardtii, we observed an impact of CO2 and CIA5, a key transcription regulator, on expression of almost 25% of all the genes. We also discovered an array of gene clusters with distinctive expression patterns that provide insight into the regulatory interaction between CIA5 and CO2. We used RNA sequencing to query the Chlamydomonas reinhardtii transcriptome for regulation by CO2 and by the transcription regulator CIA5 (CCM1). Both CO2 and CIA5 are known to play roles in acclimation to low CO2 and in induction of an essential CO2-concentrating mechanism (CCM), but less is known about their interaction and impact on the whole transcriptome. Our comparison of the transcriptome of a wild type versus a cia5 mutant strain under three different CO2 conditions, high CO2 (5%), low CO2 (0.03 to 0.05%), and very low CO2 (<0.02%), provided an entry into global changes in the gene expression patterns occurring in response to the interaction between CO2 and CIA5. We observed a massive impact of CIA5 and CO2 on the transcriptome, affecting almost 25% of all Chlamydomonas genes, and we discovered an array of gene clusters with distinctive expression patterns that provide insight into the regulatory interaction between CIA5 and CO2. Several individual clusters respond primarily to either CIA5 or CO2, providing access to genes regulated by one factor but decoupled from the other. Three distinct clusters clearly associated with CCM-related genes may represent a rich source of candidates for new CCM components, including a small cluster of genes encoding putative inorganic carbon transporters.

Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture

Many microalgae are capable of acclimating to CO2 limited environments by operating a CO2 concentrating mechanism (CCM), which is driven by various energy-coupled inorganic carbon (Ci; CO2 and HCO3−) uptake systems. Chlamydomonas reinhardtii (hereafter, Chlamydomonas), a versatile genetic model organism, has been used for several decades to exemplify the active Ci transport in eukaryotic algae, but only recently have many molecular details behind these Ci uptake systems emerged. Recent advances in genetic and molecular approaches, combined with the genome sequencing of Chlamydomonas and several other eukaryotic algae have unraveled some unique characteristics associated with the Ci uptake mechanism and the Ci-recapture system in eukaryotic microalgae. Several good candidate genes for Ci transporters in Chlamydomonas have been identified, and a few specific gene products have been linked with the Ci uptake systems associated with the different acclimation states. This review will focus on the latest studies on characterization of functional components involved in the Ci uptake and the Ci-recapture in Chlamydomonas.

Use of designer nucleases for targeted gene and genome editing in plants.

The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single-celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double-stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technologys longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production.

The CO2 concentrating mechanism and photosynthetic carbon assimilation in limiting CO2: how Chlamydomonas works against the gradient

The CO2 concentrating mechanism (CCM) represents an effective strategy for carbon acquisition that enables microalgae to survive and proliferate when the CO2 concentration limits photosynthesis. The CCM improves photosynthetic performance by raising the CO2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), simultaneously enhancing carbon fixation and suppressing photorespiration. Active inorganic carbon (Ci) uptake, Rubisco sequestration and interconversion between different Ci species catalyzed by carbonic anhydrases (CAs) are key components in the CCM, and an array of molecular regulatory elements is present to facilitate the sensing of CO2 availability, to regulate the expression of the CCM and to coordinate interplay between photosynthetic carbon metabolism and other metabolic processes in response to limiting CO2 conditions. This review intends to integrate our current understanding of the eukaryotic algal CCM and its interaction with carbon assimilation, based largely on Chlamydomonas as a model, and to illustrate how Chlamydomonas acclimates to limiting CO2 conditions and how its CCM is regulated.

Knockdown of limiting-CO2–induced gene HLA3 decreases HCO3− transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii

The CO2-concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon (Ci; CO2 and HCO3−) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-CO2–inducible, putative ABC-type transporter HLA3 might function as a HCO3− transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [14C]-Ci uptake in very low CO2 conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. Although knockdown of HLA3 mRNA alone resulted in only modest but high-pH–dependent decreases in photosynthetic Ci affinity and Ci uptake, the combination of nearly complete knockdown of HLA3 mRNA with mutations in LCIB (which encodes limiting-Ci–inducible plastid-localized protein required for normal Ci uptake or accumulation in low-CO2 conditions) and/or simultaneous, apparently off-target knockdown of LCIA mRNA (which encodes limiting-Ci–inducible plastid envelope protein reported to transport HCO3−) resulted in dramatic decreases in growth, Ci uptake, and photosynthetic Ci affinity, especially at pH 9, at which HCO3− is the predominant form of available Ci. Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HCO3− transport, along with additional evidence supporting a role for LCIA in chloroplast envelope HCO3− transport and a role for LCIB in chloroplast Ci accumulation.