植物科学

Cloning systems like Gateway and Golden Gate/Braid are known because of their efficiency and accuracy. While the main drawback of Gateway is the expensive cost of the enzymes used in its two-step (LR and BP) reaction, Golden Gate requires non-reusable components due to their specific restriction sites. We present the Brick into the Gateway (BiG) protocol as a new cloning strategy, faster and more economic method that combines (i) reusable modules or bricks assembled by the GoldenBraid approach, and (ii) Gateway LR reactions [recombination of attachment sites: attL (L from left) and attR (R from right)] avoiding the BP reaction [recombination of attachment sites: attP (P from phage) and attB (B from bacteria)] usually necessary in the Gateway cloning. The starting point is to perform a PCR reaction to add type IIS restriction sites into DNA fragments generating specific fusion sites. Then, this PCR product is used to design GoldenBraid bricks, including the attL Gateway recombination sites. Using the Golden Gate method, these bricks are assembled to produce an attL1–gene of interest–attL2 fragment, which is integrated into a compatible vector producing a Gateway entry vector. Finally, the fragment containing the target gene is recombined by LR reaction into the Gateway destination vector.

Graphical abstract

Gene knock-down in plants is a useful approach to study genotype-phenotype relationships, render disease resistance to crops, and enable efficient biosynthesis of molecules in plants. Small interfering RNA (siRNA)-mediated gene silencing is one of the most common ways to achieve gene knock-down in plants. Traditionally, siRNA is delivered into intact plant cells by coding the siRNA sequences into DNA vectors, which are then delivered through viral and/or bacterial methods. In this protocol, we provide an alternative direct delivery method of siRNA molecules into intact plant cells for efficient transient gene knock-down in model tobacco plant, Nicotiana benthamiana, leaves. Our approach uses one dimensional carbon-based nanomaterials, single-walled carbon nanotubes (SWNTs), to deliver siRNA, and does not rely on viral/bacterial delivery. The distinct advantages of our method are i) there is no need for DNA coding of siRNA sequences, ii) this abiotic method could work in a broader range of plant species than biotic methods, and iii) there are fewer regulatory complications when using abiotic delivery methods, whereby gene silencing is transient without permanent modification of the plant genome.

Graphic abstract

DNA damage is one of the common consequences of exposure to various stress conditions. Different methods have been developed to accurately assess DNA damage and fragmentation in cells and tissues exposed to different stress agents. However, owing to the presence of firm cellulosic cell wall and phenolics, plant cells and tissues are not easily amenable to be subjected to these assays. Here, we describe an optimized TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay-based protocol to determine the extent of DNA fragmentation and programmed cell death in plant root cells subjected to various stress conditions. The method described here has the advantages of simplicity, reliability and reproducibility.

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using traditional co-transfection of single gRNA expression vectors, the likelihood of delivering several gRNAs into the same cell decreases in accordance with the number of gRNAs. Thus, we have developed a method to efficiently assemble gRNA expression cassettes (2-30 gRNAs) into one single vector using a Golden-Gate assembly method (Vad-Nielsen et al., 2016). In this protocol, we describe the detailed step-by-step instructions for assembly of the multiplexed gRNA expression array. The gRNA scaffold used in our expression array is the gRNA 1.0 system for the Cas9 protein from Streptococcus pyogenes driven by the human U6 promoter.

Visualization of nuclei in S-phase cells in tissues is important for not only cell cycle research but also developmental research because morphogenesis is usually achieved by a combination of cell proliferation and cell expansion. Recently, DNA labeling with 5-ethynyl-2′-deoxyuridine (EdU), which is an analog of thymidine, has been used to visualize nuclei in S-phase cells to assess the activity of cell proliferation during development of plants. EdU is efficiently incorporated into newly synthesized DNA, and detection of EdU is based on the covalent reaction between EdU and Alexa Fluor^® dye, which is one of useful fluorescent dyes; this allows us to use mild conditions for the assay without any DNA denaturation. This method could be easily applicable, and, indeed, has been used for various model and non-model plant species. Here, we have described a protocol developed for the detection of nuclei in S-phase cells in leaves.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) is a method for detecting DNA fragmentation by labelling the 3' terminal end of nucleic acids. This method can be used both in animal and plant tissues. In animal tissues, the use of Proteinase K is sufficient for permeabilizing the cells and to obtain optimal labelling, but in plant tissues, the presence of the cell wall, does not allow proper labelling. For this reason, we carried out several modifications to the original TUNEL protocol (ApoAlert^® DNA Fragmentation Assay Kit, Clontech) to obtain an optimal labelling. These modifications were additional treatments with cellulase, Triton X-100 and Proteinase K. Also, we describe the optimization of the positive controls by adjusting the units of DNase used. The PI concentration for counterstaining has been also specifically adjusted to avoid excessive background noise and hence to correctly observe both labeled and unlabelled nuclei. This work also describes an additional protocol to collect, store and include samples (specifically stigmatic arms) in such a way that they do not interfere with the TUNEL labelling.

DNA fragmentation with length corresponding to multiple integer of approximately 180 base pairs is a distinct feature of apoptosis in animals and programmed cell death in plants. This feature can simply be detected by DNA gel electrophoresis followed by ethidium bromide staining, although in some cases it is difficult to distinguish the DNA laddering. We herein describe a protocol to detect a programmed cell death-associated DNA laddering of plant tissues. After agarose-gel electrophoresis of genomic DNA, Southern hybridization using DIG-labeled genomic DNA probe is performed, that improves detection of DNA laddering.

Transposable elements (TEs) are a major component of all genomes, thus the epigenetic mechanisms controlling their activity is an important field of study. Cytosine methylation is one of the factors regulating the transcription and transposition of TEs, alongside Histone modifications and small RNAs. Adapter PCR-based methods [such as Amplified Fragment Length Polymorphism (AFLP)] have been successfully used as high-throughput methods to genotype un-sequenced genomes. Here we use methylation-sensitive restriction enzymes, in combination with PCR on adaptor-ligated restriction fragments, to evaluate epigenetic changes in TEs between genomic DNA samples.