Plant Science

Gene expression analysis is a fundamental technique to elucidate the regulatory mechanisms of genes of interest or to reveal the patterns of plant response to environmental stimuli. Traditionally, gene expression analyses have required RNA extraction, followed by cDNA synthesis and qPCR analyses. However, this conventional method is costly and time-consuming, limiting the amount of data collected. The protocol outlined in this study, which utilizes a chemiluminescence system, offers a cost-effective and rapid method for assessing the expression of Arabidopsis (Arabidopsis thaliana) genes, exemplified by analyzing the nitrate-inducible expression of a major nitrate transporter gene, nitrate transporter 2.1 (NRT2.1). A reporter construct, containing the NRT2.1 promoter fused to the firefly luciferase gene, was introduced into wild-type and mutant Arabidopsis plants. Seeds obtained from the transgenic lines were grown for 3 days in 96-well microplates containing a nitrate-free nutrient solution. After 3 days, the nutrient solution was replaced with a fresh batch, which was supplemented with luciferin potassium. One hour later, nitrate was added at various concentrations, and the temporal expression pattern of NRT2.1 was analyzed by monitoring the chemiluminescence signals. This method allowed for the cost-effective, quantitative, and high-throughput analysis of NRT2.1 expression over time under the effects of various nutrient conditions and genetic backgrounds.

Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery.

Yield losses attributed to plant pathogens pose a serious threat to plant productivity and food security. Botrytis cinerea is one of the most devastating plant pathogens, infecting a wide array of plant species; it has also been established as a model organism to study plant–pathogen interactions. In this context, development of different assays to follow the relative success of B. cinerea infections is required. Here, we describe two methods to quantify B. cinerea development in Arabidopsis thaliana genotypes through measurements of lesion development and quantification of fungal genomic DNA in infected tissues. This provides two independent techniques that are useful in assessing the susceptibility or tolerance of different Arabidopsis genotypes to B. cinerea.

Key features

• Protocol for the propagation of the necrotrophic plant pathogen fungus Botrytis cinerea and spore production.

• Two methods of Arabidopsis thaliana infection with the pathogen using droplet and spray inoculation.

• Two readouts, either by measuring lesion size or by the quantification of fungal DNA using quantitative PCR.

• The two methods are applicable across plant species susceptible the B. cinerea.

Graphical overview

A simplified overview of the droplet and spray infection methods used for the determination of B. cinerea growth in different Arabidopsis genotypes

Virus-mediated transient gene overexpression and gene expression silencing can be used to screen gene functions in plants. Sugarcane mosaic virus (SCMV) is a positive strand RNA virus in the Potyviridae family that has been modified to be used as vector to infect monocots, including maize (Zea mays), for transient gene overexpression and gene expression silencing. Relative to stable transformation, SCMV-mediated transient expression in maize has the advantages of being faster and less expensive. Here, we describe a protocol for cloning constructs into the plasmid vector pSCMV-CS3. After maize seedlings are transformed with pSCMV-CS3 constructs by particle bombardment, the virus replicates and spreads systemically in the plants. Subsequent infections of maize seedlings can be accomplished by rub inoculation with sap from SCMV-infested plants. As an example of a practical application of the method, we also describe virus-induced gene silencing (VIGS) of fall armyworm (Spodoptera frugiperda) gene expression. Transgenic viruses are created by cloning a segment of the fall armyworm target gene into pSCMV-CS3 prior to maize transformation. Caterpillars are fed on the virus-infected maize plants, which make dsRNA to silence the expression of the fall armyworm target gene after ingestion. This use of SCMV for plant-mediated VIGS in insects allows rapid screening of gene functions when caterpillars are feeding on their host plants.

Graphical overview

Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants.

Graphical overview

Workflow for autolysin production from Chlamydomonas reinhardtii

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

Plant genomes are pronouncedly enriched in repeat elements such as transposons. These repeats are epigenetically regulated by DNA methylation. Whole genome high-depth sequencing after bisulfite treatment remains an expensive and laborious method to reliably profile the DNA methylome, especially when considering large genomes such as in crops. Here, we present a simple reproducible Southern hybridisation–based assay to obtain incontrovertible methylation patterns from targeted regions in the rice genome. By employing minor but key modifications, we reliably detected transposon copy number variations over multiple generations. This method can be regarded as a gold standard for validation of epigenetic variations at target loci, and the consequent proliferation of transposons, or segregation in several plant replicates and genotypes.

Graphical abstract:

Gene expression depends on the binding of transcription factors with DNA regulatory sequences. The level of accessibility for these sequences varies between cells and cell types. Until recently, using the Tn5 assay for transposase-accessible chromatin for sequencing (ATAC-seq) technology allowed assessing the profiles of chromatin from an entire organ or, when coupled with the isolation of nuclei tagged in specific cell types (INTACT) method, from a cell-type. Recently, ATAC-seq experiments were conducted at the level of individual plant nuclei. Applying single nuclei ATAC-seq (sNucATAC-seq) technology to thousands of individual cells revealed more finely tuned profiles of chromatin accessibility. In this manuscript, we describe a method to isolate nuclei fom plant roots and green tissues, permeabilize the nuclear membrane using detergent to allow the penetration of the Tn5 transposase, and re-suspend them in a nuclei resuspension buffer compatible with the construction of sNucATAC-seq libraries using the 10× Genomic’s Chromium technology. This protocol was successfully applied on Arabidopsis thaliana and Glycine max root nuclei.

Transgenic plants are produced both to investigate gene function and to confer desirable traits into crops. Transgene copy number is known to influence expression levels, and consequently, phenotypes. Similarly, knowledge of transgene zygosity is desirable for making quantitative assessments of phenotype and tracking the inheritance of transgenes in progeny generations. Since the first transgenic plants were produced, several methods for determining copy number have been applied, including Southern blotting, quantitative real-time PCR, and more recently, sequencing methods; however, each method has specific disadvantages, compromising throughput, accuracy, or expense. Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of occurrence of specific sequences to be accurately estimated. Confidence increases with the number of partitions; therefore, the availability of emulsion technologies that enable reactions to be divided into tens of thousands of nanodroplets allows accurate determination of copy number in what has become known as digital droplet PCR (ddPCR). ddPCR offers similar benefits of low costs and scalability as other PCR techniques but with superior accuracy and reliability.

Graphic abstract:

Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of transgene copy number to be accurately assessed.