Gene Activity In Plants Research Articles

Boosting plant genome editing with a versatile CRISPR-Combo system.

CRISPR-Cas9, its derived base editors and CRISPR activation systems have greatly aided genome engineering in plants. However, these systems are mostly used separately, leaving their combinational potential largely untapped. Here we develop a versatile CRISPR-Combo platform, based on a single Cas9 protein, for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants. We showcase the powerful applications of CRISPR-Combo for boosting plant genome editing. First, CRISPR-Combo is used to shorten the plant life cycle and reduce the efforts in screening transgene-free genome-edited plants by activation of a florigen gene in Arabidopsis. Next, we demonstrate accelerated regeneration and propagation of genome-edited plants by activation of morphogenic genes in poplar. Furthermore, we apply CRISPR-Combo to achieve rice regeneration without exogenous plant hormones, which is established as a new method to predominately enrich heritable targeted mutations. In conclusion, CRISPR-Combo is a versatile genome engineering tool with promising applications in crop breeding.

Strong and tunable anti-CRISPR/Cas activities in plants.

SummaryCRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti‐CRISPR proteins as tools to prevent CRISPR/Cas‐mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti‐CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site‐directed mutagenesis in leaves when transiently co‐expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a‐mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas‐based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose‐dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin‐regulated activation of a downstream reporter gene. The strong anti‐Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.

Multiplex and optimization of dCas9-TV-mediated gene activation in plants.

Synthetic gene activators consisting of nuclease-dead Cas9 (dCas9) for single-guide RNA (sgRNA)-directed promoter binding and a transcriptional activation domain (TAD) represent new tools for gene activation from endogenous genomic locus in basic and applied plant research. However, multiplex gene coactivation by dCas9-TADs has not been demonstrated in whole plants. There is also room to optimize the performance of these tools. Here, we report that our previously developed gene activator, dCas9-TV, could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold, with one sgRNA targeting to each promoter. The gene coactivation could persist to at least the fourth generation. Astonishingly, the polycistronic tRNA-sgRNA expression under the maize ubiquitin promoter, a Pol II promoter, could cause enormous activation of these genes by up to >40,000-fold in rice. Moreover, the yeast GCN4 coiled coil-mediated dCas9-TV dimerization appeared to be promising for enhancing gene activation. Finally, we successfully introduced a self-amplification loop for dCas9-TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2, a previously characterized dCas9-TV-refractory gene with considerable basal expression. Collectively, this work illustrates the robustness of dCas9-TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9-TADs in plants.

Isolation of Plant Root Nuclei for Single Cell RNA Sequencing.

The characterization of the transcriptional similarities and differences existing between plant cells and cell types is important to better understand the biology of each cell composing the plant, to reveal new molecular mechanisms controlling gene activity, and to ultimately implement meaningful strategies to enhance plant cell biology. To gain a deeper understanding of the regulation of plant gene activity, the individual transcriptome of each plant cell needs to be established. Until recently, single cell approaches were mostly limited to bulk transcriptomic studies on selected cell types. Accessing specific cell types required the development of labor-intensive strategies. Recently, single cell sequencing strategies were successfully applied on isolated Arabidopsis thaliana root protoplasts. However, this strategy relies on the successful isolation of viable protoplasts upon the optimization of the enzymatic cocktails required to digest the cell wall and on the compatibility of fragile plant protoplasts with the use of microfluidic systems to generate single cell transcriptomic libraries. To overcome these difficulties, we present a simple and fast alternative strategy: the isolation and use of plant nuclei to access meaningful transcriptomic information from plant cells. This protocol was specifically developed to enable the use of the plant nuclei with 10× Genomics' Chromium technology partitions technology. Briefly, the plant nuclei are released from the root by chopping into a nuclei isolation buffer before purification by filtration then nuclei sorting. Upon sorting, the nuclei are resuspended in a low divalent ion buffer compatible with the Chromium technology in order to create single nuclei ribonucleic acid-sequencing libraries (sNucRNA-seq). © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis seed sterilization and planting Basic Protocol 2: Nuclei isolation from Arabidopsis roots Basic Protocol 3: Fluorescent-activated nuclei sorting (FANS) purification Support Protocol: Estimation of nuclei density using Countess II automated cell counter Alternate Protocol 1: Proper growth conditions for Medicago truncatula and Sorghum bicolor Alternate Protocol 2: Estimation of nuclei density using sNucRNA-seq technology.

The repurposing of type I-E CRISPR-Cascade for gene activation in plants

CRISPR-Cas systems are robust and facile tools for manipulating the genome, epigenome and transcriptome of eukaryotic organisms. Most groups use class 2 effectors, such as Cas9 and Cas12a, however, other CRISPR-Cas systems may provide unique opportunities for genome engineering. Indeed, the multi-subunit composition of class 1 systems offers to expand the number of domains and functionalities that may be recruited to a genomic target. Here we report DNA targeting in Zea mays using a class 1 type I-E CRISPR-Cas system from S. thermophilus. First, we engineer its Cascade complex to modulate gene expression by tethering a plant transcriptional activation domain to 3 different subunits. Next, using an immunofluorescent assay, we confirm Cascade cellular complex formation and observe enhanced gene activation when multiple subunits tagged with the transcriptional activator are combined. Finally, we examine Cascade mediated gene activation at chromosomal DNA targets by reprogramming Zea mays cells to change color.

Macro optical projection tomography for large scale 3D imaging of plant structures and gene activity.

Optical projection tomography (OPT) is a well-established method for visualising gene activity in plants and animals. However, a limitation of conventional OPT is that the specimen upper size limit precludes its application to larger structures. To address this problem we constructed a macro version called Macro OPT (M-OPT). We apply M-OPT to 3D live imaging of gene activity in growing whole plants and to visualise structural morphology in large optically cleared plant and insect specimens up to 60 mm tall and 45 mm deep. We also show how M-OPT can be used to image gene expression domains in 3D within fixed tissue and to visualise gene activity in 3D in clones of growing young whole Arabidopsis plants. A further application of M-OPT is to visualise plant-insect interactions. Thus M-OPT provides an effective 3D imaging platform that allows the study of gene activity, internal plant structures and plant-insect interactions at a macroscopic scale.

Role of phenolic acids and enzymes of phenylpropanoid pathway in resistance of chayote fruit (Sechium edule) against infestation by melon fly, Bactrocera cucurbitae

AbstractMelon fly is a serious pest of cucurbits all over the world causing huge losses to yield. However, the only exception is the chayote fruit (Sechium edule) that shows resistance to melon fly infestation. Studies on culture of melon fly indicated the absence of plant traits resisting oviposition on chayote fruit. However, the melon fly was unable to complete its life cycle successfully on chayote showing that factors inhibiting larval development in melon fly could be attributed to biochemical constituents. Studies were, therefore, carried out to compare the biochemical responses of chayote, a melon fly resistant species and bitter gourd, a susceptible species to melon fly infestation with regard to the levels of phenolic acids and activities of the enzymes of phenylpropanoid pathway (PPP) leading to synthesis of lignin. The resistant chayote exhibited significantly higher accumulation of lignin associated with higher activities of phenylalanine ammonia‐lyase (PAL), tyrosine ammonia‐lyase (TAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD). On the contrary, the susceptible bitter gourd recorded lower activities of PAL, CAD and POD and a decreasing trend of TAL during infestation associated with a lower lignin content. The monomer composition of lignin in the resistant chayote showed twofold higher level of guaiacyl (G) and syringyl (S) units compared to susceptible bitter gourd and the G/S ratio during infestation increased in chayote while decreasing in bitter gourd. The levels of PPP intermediates, p‐coumaric acid was higher in chayote while p‐hydroxy benzoic acid, a chemo‐attractant, was higher in bitter gourd. Incorporation of p‐coumaric acid in the larval diet strongly inhibited larval growth even as p‐hydroxy benzoic acid promoted growth confirming the direct role of p‐coumaric acid in conferring resistance to chayote. The level of salicylic acid, a signal molecule involved in induction of defence response, was higher in chayote compared to bitter gourd. Chayote also exhibited higher level of activity of POD in the phloem exudates compared to bitter gourd. The higher concentration of sugars in exudates of chayote might act like signalling molecules causing activation of plant genes, especially of the phenylpropanoid biosynthesis pathway and possibly produce an osmotic effect inducing resistance against the melon fly. Thus, the study revealed that the resistance in chayote to melon fly infestation is a complex, multi‐layered process in which the activities of PPP enzymes generating phenolic intermediates leading to lignin biosynthesis and the composition of exudates appear to play significant roles. Besides, the study also indicated that different forms of lignin might play a role in the resistance of chayote against melon fly infestation.

MIGS: miRNA‐induced gene silencing

Gene silencing is an important tool in the study of gene function. Virus-induced gene silencing (VIGS) and hairpin RNA interference (hpRNAi), both of which rely on small interfering RNAs, together with artificial microRNAs (amiRNA), are amongst the most popular methods for reduction of gene activity in plants. However, all three approaches have limitations. Here, we introduce miRNA-induced gene silencing (MIGS). This method exploits a special 22-nucleotide miRNA of Arabidopsis thaliana, miR173, which can trigger production of another class of small RNAs called trans-acting small interfering RNAs (tasiRNAs). We show that fusion of gene fragments to an upstream miR173 target site is sufficient for effective silencing of the corresponding endogenous gene. MIGS can be reliably used for the knockdown of a single gene or of multiple unrelated genes. In addition, we show that MIGS can be applied to other species by co-expression of miR173.

Use of the fluorescent timer DsRED-E5 as reporter to monitor dynamics of gene activity in plants.

Fluorescent proteins, such as green fluorescent protein and red fluorescent protein (DsRED), have become frequently used reporters in plant biology. However, their potential to monitor dynamic gene regulation is limited by their high stability. The recently made DsRED-E5 variant overcame this problem. DsRED-E5 changes its emission spectrum over time from green to red in a concentration independent manner. Therefore, the green to red fluorescence ratio indicates the age of the protein and can be used as a fluorescent timer to monitor dynamics of gene expression. Here, we analyzed the potential of DsRED-E5 as reporter in plant cells. We showed that in cowpea (Vigna unguiculata) mesophyll protoplasts, DsRED-E5 changes its fluorescence in a way similar to animal cells. Moreover, the timing of this shift is suitable to study developmental processes in plants. To test whether DsRed-E5 can be used to monitor gene regulation in plant organs, we placed DsRED-E5 under the control of promoters that are either up- or down-regulated (MtACT4 and LeEXT1 promoters) or constitutively expressed (MtACT2 promoter) during root hair development in Medicago truncatula. Analysis of the fluorescence ratios clearly provided more accurate insight into the timing of promoter activity.

Differential activation of H + -ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco

In arbuscular mycorrhizas, H+-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526-S532). The proton-pumping H+-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H+-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H+-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of beta-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H+-ATPase activity in the periarbuscular membrane results from selective induction of two H+-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface.

Genetically modified plants and the 35S promoter: assessing the risks and enhancing the debate

The 35S promoter, derived from the common plant virus, cauliflower mosaic virus (CaMV), is a component of transgenic constructs in more than 80% of genetically modified (GM) plants. Alarming reports have suggested that the 35S promoter might cause accidental activation of plant genes or endogenous viruses, promote horizontal gene transfer, or might even recombine with mammalian viruses such as HIV, with unexpected consequences. In this article, we discuss the properties of CaMV and the 35S promoter and the potential risks associated with the use of the promoter in GM plants, concluding that any risks are no greater than those encountered in conventional plant breeding. Keywords: Cauliflower mosaic virus, 35S promoter, risk assessment.

Genetically modified plants and the 35S promoter: assessing the risks and enhancing the debate

The 35S promoter, derived from the common plant virus, cauliflower mosaic virus (CaMV), is a component of transgenic constructs in more than 80% of genetically modified (GM) plants. Alarming reports have suggested that the 35S promoter might cause accidental activation of plant genes or endogenous viruses, promote horizontal gene transfer, or might even recombine with mammalian viruses such as HIV, with unexpected consequences. In this article, we discuss the properties of CaMV and the 35S promoter and the potential risks associated with the use of the promoter in GM plants, concluding that any risks are no greater than those encountered in conventional plant breeding.

Antisense-mediated depletion of potato leaf omega3 fatty acid desaturase lowers linolenic acid content and reduces gene activation in response to wounding.

Fatty acid omega3 desaturases act on membrane lipids to catalyse the formation of trienoic fatty acids, the most abundant in plant tissues being alpha-linolenic acid. This fatty acid is a precursor of jasmonic acid, a plant growth regulator involved in the control of wound-induced gene activation in plants and in the induction of tuberization in potato. We isolated a potato omega3 desaturase cDNA, possibly encoding a plastidial isoform, and used it to investigate its expression pattern throughout plant development and in response to wounding. Plastidial omega3 desaturase gene transcripts accumulate rapidly upon wounding, preceding the jasmonate-dependent induction of the wound-responsive proteinase inhibitor II gene. We generated transgenic potato plants constitutively expressing an antisense RNA to this plastidial omega3 desaturase. Selected transgenic lines in which the cognate omega3 desaturase mRNA is largely depleted show a marked reduction, of up to 60%, in trienoic acids in leaves and tubers. In these lines, a corresponding reduction in jasmonate content and proteinase inhibitor II expression is observed upon wounding. Our results indicate that a reduction in omega3 desaturase mRNA levels compromises the wound-induced activation of proteinase inhibitor II, suggesting that wound-induced synthesis of linolenic acid is required for jasmonic acid production. The antisense-mediated depletion of fatty acid omega3 desaturases is a viable alternative for reducing trienoic fatty acid content in plant species in which a mutant screening approach is not applicable.

The tef1 box, a ubiquitous cis-acting element involved in the activation of plant genes that are highly expressed in cycling cells.

In Arabidopsis thaliana, the tef1 box is a cis-acting promoter element of the EF-1 alpha A1 gene involved in the activation of transcription in meristematic tissues. The initiation of root calli in transgenic Arabidopsis by 2,4-D shows that the tef1-dependent expression of the GUS reporter gene is not restricted to meristematic regions but involves all of the cycling cells. Hybridization experiments conducted using Arabidopsis cDNA clones organized in a dense array on filters, and cDNA probes prepared from cells in various states of growth, or blocked at different steps of the cell cycle, indicate that the enhanced expression of EF-1 alpha genes occurs in cycling cells at the point of entry into the cell cycle and remains constant during transit through the cycle. The analysis of several promoters of genes, other than EF-1 alpha, which are overexpressed in growing cells and involved in the processes of translation or redox regulation, reveals the presence of sequences showing partial homologies with the tef1 box. The Arabidopsis ribosomal gene srp18 and the tobacco gene thioh2, encoding a thioredoxin h, contain such sequences. Gel retardation experiments suggest that these sequences are targets for the same proteins as those that interact with the tef1 box of the Arabidopsis EF-1 alpha A1 gene. In transfected Arabidopsis protoplasts, the putative tef1 sequence thioh2 partially restores the activity of a tef1 box-less EF-1 alpha A1 promoter. These data demonstrate that the tef1 box is a ubiquitous cis-acting element involved in the transcriptional activation of plant genes that are overexpressed in cycling cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced stable expression of aVibrio luciferase under the control of the ?-translational enhancer in transgenic plants

A fusion gene usingluxA andluxB genes ofVibrio species has been designed to express light autonomously in plants.LuxA:luxB was introduced into plants by a high-efficiency transformation system consisting of a high-copy virulence helper plasmid pUCD2614 and T-vector pUCD2715 containingluxA:luxB. The expression ofluxA:luxB fusion gene was optimized by adjusting the spacing between the genes and by placing the translational efficiency of its mRNA under the control of the Ω-3 translational enhancer. The resulting transgenic plants synthesized luciferase at levels greater than 1% of the total leaf protein. These plants produced light autonomously and light intensity was enhanced by the addition of aldehyde. That theluxA:luxB fusion has been optimized enables its use as a reporter for gene activity in plants during development and under various stress-inducing conditions. These results show that a specific protein from an introduced foreign gene can be produced with high efficiency in cultivated plants and such a system is therefore amenable for production of desired proteins through conventional farming methods.

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants. Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.

New patterns of gene activity in plants detected using an Agrobacterium vector

A vector has been designed that contains a truncated CaMV (cauliflower mosaic virus) 35S promoter fused to a receptor gene encoding beta-glucuronidase (GUS), placed adjacent to the left border sequence of an Agrobacterium vector. In potato plants transformed with this vector, different patterns of transcription were detected at high frequency using in situ assays for GUS activity. Previous studies in Drosophila using analogous vectors have shown that the new patterns of transcription in many cases reflect the patterns of expression of genes adjacent to the site of vector insertion. If this is also the case in plants, the vector described here will be useful in identifying the activity of genes in different cell types and will assist in determining their function.

Control of gene activity in plants: a genetical view

Control of gene activity in plants: a genetical view

Quantitative Regulation of Gene Activity in Plants

The regulation of activity of ribosomal RNA genes has been studied in euploid and aneuploid varieties of hyacinth. The gene/gene product relationship was determined by measuring the specific gene product (stable rRNA) and the total gene product (protein) in varieties with different dosages of rRNA genes. The results show that the cellular rRNA content is positively regulated, that maximum gene utilization is not normally employed, and that the degree of utilization responds to the total genome rather than to the specific rRNA gene dosage. The mechanism of control of this regulation appears to be at the transcriptional level.