Xue-Chen Wang

A CRISPR/Cas9 toolkit for multiplex genome editing in plants

BackgroundTo accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.ResultsWe developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.ConclusionsWe developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

A novel drought-inducible gene, ATAF1, encodes a NAC family protein that negatively regulates the expression of stress-responsive genes in Arabidopsis

NAC proteins are plant-specific transcriptional regulators. ATAF1 was one of the first identified NAC proteins in Arabidopsis. In present study, we characterized the ATAF1 expression and biological function in response to water deficit stress. ATAF1 mRNA expression was strongly induced by dehydration and abscisic acid (ABA) treatment, but inhibited by water treatment, suggesting a general role in drought stress responses. Transient expression analysis in onion epidermal cells indicated the nuclear localization for the ATAF1::GFP fusion protein. Yeast transactivation analysis showed that ATAF1 had ability to activate reporter gene expression. Furthermore, domain deletion analysis revealed that the ATAF1 transactivation activity was conferred by its C-terminal domain. When ATAF1 gene was knocked out by T-DNA insertions, Arabidopsisataf1-1 and ataf1-2 mutants displayed a recovery rate about seven times higher than wild-type plants in drought response test. This ataf1 phenotype was coincident with the enhanced expression of stress responsive marker genes, such as COR47, ERD10, KIN1, RD22 and RD29A under drought stress. Above evidences suggest that ATAF1, as a transcriptional regulator, negatively regulates the expression of stress responsive genes under drought stress in Arabidopsis.

Transcriptional Modulation of Ethylene Response Factor Protein JERF3 in the Oxidative Stress Response Enhances Tolerance of Tobacco Seedlings to Salt, Drought, and Freezing

Abiotic stresses such as drought, cold, and salinity affect normal growth and development in plants. The production and accumulation of reactive oxygen species (ROS) cause oxidative stress under these abiotic conditions. Recent research has elucidated the significant role of ethylene response factor (ERF) proteins in plant adaptation to abiotic stresses. Our earlier functional analysis of an ERF protein, JERF3, indicated that JERF3-expressing tobacco (Nicotiana tabacum) adapts better to salinity in vitro. This article extends that study by showing that transcriptional regulation of JERF3 in the oxidative stress response modulates the increased tolerance to abiotic stresses. First, we confirm that JERF3-expressing tobacco enhances adaptation to drought, freezing, and osmotic stress during germination and seedling development. Then we demonstrate that JERF3-expressing tobacco imparts not only higher expression of osmotic stress genes compared to wild-type tobacco, but also the activation of photosynthetic carbon assimilation/metabolism and oxidative genes. More importantly, this regulation of the expression of oxidative genes subsequently enhances the activities of superoxide dismutase but reduces the content of ROS in tobacco under drought, cold, salt, and abscisic acid treatments. This indicates that JERF3 also modulates the abiotic stress response via the regulation of the oxidative stress response. Further assays indicate that JERF3 activates the expression of reporter genes driven by the osmotic-responsive GCC box, DRE, and CE1 and by oxidative-responsive as-1 in transient assays, suggesting the transcriptional activation of JERF3 in the expression of genes involved in response to oxidative and osmotic stress. Our results therefore establish that JERF3 activates the expression of such genes through transcription, resulting in decreased accumulation of ROS and, in turn, enhanced adaptation to drought, freezing, and salt in tobacco.

Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

Tomato stress-responsive factor TSRF1 interacts with ethylene responsive element GCC box and regulates pathogen resistance to Ralstonia solanacearum

Ethylene responsive factors (ERFs) are important in regulating plant pathogen resistance, abiotic stress tolerance and plant development. Recent studies have greatly enlarged the ERF protein family and revealed more important roles of ERFs in plants. Here, we report our finding of a tomato ERF protein TSRF1, which is transcriptionally up-regulated by ethylene, salicylic acid, or Ralstonia solanacearum strain BJ1057 infection. Biochemical analysis indicates that TSRF1 specifically interacts in vitro with the GCC box, an element present in the promoters of many pathogenesis-related (PR) genes. Further investigation evidences that TSRF1 activates in vivothe expression of reporter β-glucuronidase gene controlled by GCC box. More importantly, overexpressing TSRF1 in tobacco and tomato constitutively activates the expression of PR genes, and subsequently enhancing transgenic plant resistance to the bacterial wilt caused by Ralstonia solanacearum strain BJ1057. Therefore our investigation not only extends the functions of ERF proteins in plant resistance to R. solanacearum, but also provides further clues to understanding the mechanism of host regulatory proteins in response to the infection of pathogens.

Transcriptional regulation of the ethylene response factor LeERF2 in the expression of ethylene biosynthesis genes controls ethylene production in tomato and tobacco.

Fine-tuning of ethylene production plays an important role in developmental processes and in plant responses to stress, but very little is known about the regulation of ethylene response factor (ERF) proteins in ethylene biosynthesis genes and ethylene production. Identifying cis-acting elements and transcription factors that play a role in this process, therefore, is important. Previously, a tomato (Solanum lycopersicum [f. sp. Lycopersicon esculentum]) ERF protein, LeERF2, an allele of TERF2, was reported to confer ethylene triple response on plants. This paper reports the transcriptional modulation of LeERF2/TERF2 in ethylene biosynthesis in tomato and tobacco (Nicotiana tabacum). Using overexpressing and antisense LeERF2/TERF2 transgenic tomato, we found that LeERF2/TERF2 is an important regulator in the expression of ethylene biosynthesis genes and the production of ethylene. Expression analysis revealed that LeERF2/TERF2 is ethylene inducible, and ethylene production stimulated by ethylene was suppressed in antisense LeERF2/TERF2 transgenic tomato, indicating LeERF2/TERF2 to be a positive regulator in the feedback loop of ethylene induction. Further research showed that LeERF2/TERF2 conservatively modulates ethylene biosynthesis in tobacco and that such regulation in tobacco is associated with the elongation of the hypocotyl and insensitivity to abscisic acid and glucose during germination and seedling development. The effects on ethylene synthesis were similar to those of another ERF protein, TERF1, because TERF1 and LeERF2/TERF2 have overlapping roles in the transcriptional regulation of ethylene biosynthesis in tobacco. Biochemical analysis showed that LeERF2/TERF2 interacted with GCC box in the promoter of NtACS3 and with dehydration-responsive element in the promoter of LeACO3, resulting in transcriptional activation of the genes for ethylene biosynthesis in tomato and tobacco, which is a novel regulatory function of ERF proteins in plant ethylene biosynthesis.

Extracellular Calmodulin-Induced Stomatal Closure Is Mediated by Heterotrimeric G Protein and H2O2

Extracellular calmodulin (ExtCaM) exerts multiple functions in animals and plants, but the mode of ExtCaM action is not well understood. In this paper, we provide evidence that ExtCaM stimulates a cascade of intracellular signaling events to regulate stomatal movement. Analysis of the changes of cytosolic free Ca2+ ([Ca2+]cyt) and H2O2 in Vicia faba guard cells combined with epidermal strip bioassay suggests that ExtCaM induces an increase in both H2O2 levels and [Ca2+]cyt, leading to a reduction in stomatal aperture. Pharmacological studies implicate heterotrimeric G protein in transmitting the ExtCaM signal, acting upstream of [Ca2+]cyt elevation, and generating H2O2 in guard cell responses. To further test the role of heterotrimeric G protein in ExtCaM signaling in stomatal closure, we checked guard cell responses in the Arabidopsis (Arabidopsis thaliana) Gα-subunit-null gpa1 mutants and cGα overexpression lines. We found that gpa1 mutants were insensitive to ExtCaM stimulation of stomatal closure, whereas cGα overexpression enhanced the guard cell response to ExtCaM. Furthermore, gpa1 mutants are impaired in ExtCaM induction of H2O2 generation in guard cells. Taken together, our results strongly suggest that ExtCaM activates an intracellular signaling pathway involving activation of a heterotrimeric G protein, H2O2 generation, and changes in [Ca2+]cyt in the regulation of stomatal movements.

Over-expression of a vacuolar Na+/H+ antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na+/H+ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T2 generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na+ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.

NADK2, an Arabidopsis Chloroplastic NAD Kinase, Plays a Vital Role in Both Chlorophyll Synthesis and Chloroplast Protection

As one of terminal electron acceptors in photosynthetic electron transport chain, NADP receives electron and H+ to synthesize NADPH, an important reducing energy in chlorophyll synthesis and Calvin cycle. NAD kinase (NADK), the catalyzing enzyme for the de novo synthesis of NADP from substrates NAD and ATP, may play an important role in the synthesis of NADPH. NADK activity has been observed in different sub-cellular fractions of mitochondria, chloroplast, and cytoplasm. Recently, two distinct NADK isoforms (NADK1 and NADK2) have been identified in Arabidopsis. However, the physiological roles of NADKs remain unclear. In present study, we investigated the physiological role of Arabidiposis NADK2. Sub-cellular localization of the NADK2–GFP fusion protein indicated that the NADK2 protein was localized in the chloroplast. The NADK2 knock out mutant (nadk2) showed obvious growth inhibition and smaller rosette leaves with a pale yellow color. Parallel to the reduced chlorophyll content, the expression levels of two POR genes, encoding key enzymes in chlorophyll synthesis, were down regulated in the nadk2 plants. The nadk2 plants also displayed hypersensitivity to environmental stresses provoking oxidative stress, such as UVB, drought, heat shock and salinity. These results suggest that NADK2 may be a chloroplast NAD kinase and play a vital role in chlorophyll synthesis and chloroplast protection against oxidative damage.

A signaling pathway linking nitric oxide production to heterotrimeric G protein and hydrogen peroxide regulates extracellular calmodulin induction of stomatal closure in Arabidopsis.

Extracellular calmodulin (ExtCaM) regulates stomatal movement by eliciting a cascade of intracellular signaling events including heterotrimeric G protein, hydrogen peroxide (H2O2), and Ca2+. However, the ExtCaM-mediated guard cell signaling pathway remains poorly understood. In this report, we show that Arabidopsis (Arabidopsis thaliana) NITRIC OXIDE ASSOCIATED1 (AtNOA1)-dependent nitric oxide (NO) accumulation plays a crucial role in ExtCaM-induced stomatal closure. ExtCaM triggered a significant increase in NO levels associated with stomatal closure in the wild type, but both effects were abolished in the Atnoa1 mutant. Furthermore, we found that ExtCaM-mediated NO generation is regulated by GPA1, the Gα-subunit of heterotrimeric G protein. The ExtCaM-dependent NO accumulation was nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGα). In addition, cGα Atnoa1 and gpa1-2 Atnoa1 double mutants exhibited a similar response as did Atnoa1. The defect in gpa1 was rescued by overexpression of AtNOA1. Finally, we demonstrated that G protein activation of NO production depends on H2O2. Reduced H2O2 levels in guard cells blocked the stomatal response of cGα lines, whereas exogenously applied H2O2 rescued the defect in ExtCaM-mediated stomatal closure in gpa1 mutants. Moreover, the atrbohD/F mutant, which lacks the NADPH oxidase activity in guard cells, had impaired NO generation in response to ExtCaM, and H2O2-induced stomatal closure and NO accumulation were greatly impaired in Atnoa1. These findings have established a signaling pathway leading to ExtCaM-induced stomatal closure, which involves GPA1-dependent activation of H2O2 production and subsequent AtNOA1-dependent NO accumulation.