Gene editing of plant DNA has the potential to produce crops with increased performance and resilience, but it can take a long time to achieve these gains. In order to shorten this process, scientists often use screening tools to determine where and how edits to the plant genome can be most effective.
Researchers from the University of Illinois Urbana-Champaign have expanded the toolkit available for studying small regulatory elements called upstream open reading frames (uORFs). The new transient expression assay, a type of screening tool, developed by the researchers uses fluorescent protein reporters linked to uORFs to quickly analyze how changes to uORFs sequences affect gene regulation in vivo.
uORFs are DNA sequence elements that help regulate the translation of messenger RNA (mRNA) into proteins. Since uORFs are prevalent in plant genomes, they present a powerful target for researchers to control gene expression. Previous research has shown that uORFs often reduce the amount of protein produced from an mRNA molecule. When uORF DNA sequences are removed or altered using gene-editing approaches such as CRISPR-Cas9, this inhibitory effect can be lifted, resulting in increased production of proteins from specific genes.
Existing methods for studying uORFs typically attach these regulatory elements to so-called reporter genes and temporarily introduce them into model plants or specialized cell systems for evaluation. One commonly used reporter is luciferase, the enzyme responsible for the glow of fireflies, which produces a measurable light signal during chemical reactions. However, these approaches often require additional sample preparation and specialized reagents, particularly in experimentally demanding systems such as protoplasts—plant cells with their cell walls removed.
This method developed by the Illinois team aims to simplify this process by using intact leaf tissue and fluorescent proteins, enabling rapid analysis of uORF-mediated gene regulation while reducing sample handling and consumable materials.
The development of this method was spearheaded by Ben Haas, as part of the Stephen Long lab in the Realizing Increased Photosynthetic Efficiency (RIPE) project.
"The big goal of RIPE is to engineer photosynthesis for the benefit of agriculture and small-holder farmers. Many RIPE researchers aim to modulate gene products in that pathway," said Haas. "And gene editing can be a very powerful tool to achieve that in a variety of crop species."
The method works by linking natural or modified uORFs to fluorescent reporter proteins that emit measurable light signals in plant leaf tissue. The researchers then temporarily introduce these genetic constructs into leaves using Agrobacterium, a bacterium commonly used in plant biotechnology to transfer the DNA into plants. After several days, the fluorescent signals can be measured using a standard laboratory instrument. By comparing fluorescence levels between natural and modified uORFs, the researchers can determine whether changes to the regulatory elements alter gene expression.
"We compare the signal from the original sequence to the signal from the modified version," said Haas. "If the fluorescence changes significantly, it indicates that the uORF is affecting gene expression."
Although fluorescent proteins have long been used as reporters in molecular biology, their application for study uORF-mediated gene regulation in plants is more recent. Measuring fluorescence directly from intact leaf tissue avoids much of the additional sample preparation required for many current uORF assays.
Using this method, the researchers analyzed sequences from soybean and cowpea–the two crops at the center of the RIPE project. The authors identified uORFs for genes involved in plant photoprotection and observed the consequential changes in fluorescence intensity.
The team hopes the tool will be useful to other researchers and has deposited the reporter plasmid described in the study with Addgene, where it is publicly available to order.
"This tool could be useful to any plant researcher who's interested in measuring the translational strength of their transcript leader; it really doesn't have to be particularly limited to uORFs," said Haas. "Researchers can put in their own sequences and have the first results in as little as a month."
This work was supported by the Gates Foundation , the Foundation for Food & Agriculture Research , UK Foreign, Commonwealth & Development Office , Gates Agricultural Innovations and the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) at the University of Illinois.
