{"title":"Straightforward and affordable agroinfiltration with RUBY accelerates RNA silencing research.","authors":"Midori Tabara, Ayumi Matsumoto, Yuriko Kibayashi, Atsushi Takeda, Kazuki Motomura","doi":"10.1007/s11103-024-01463-8","DOIUrl":null,"url":null,"abstract":"<p><p>Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.</p>","PeriodicalId":20064,"journal":{"name":"Plant Molecular Biology","volume":null,"pages":null},"PeriodicalIF":3.9000,"publicationDate":"2024-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11102880/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Molecular Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s11103-024-01463-8","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.
期刊介绍:
Plant Molecular Biology is an international journal dedicated to rapid publication of original research articles in all areas of plant biology.The Editorial Board welcomes full-length manuscripts that address important biological problems of broad interest, including research in comparative genomics, functional genomics, proteomics, bioinformatics, computational biology, biochemical and regulatory networks, and biotechnology. Because space in the journal is limited, however, preference is given to publication of results that provide significant new insights into biological problems and that advance the understanding of structure, function, mechanisms, or regulation. Authors must ensure that results are of high quality and that manuscripts are written for a broad plant science audience.