AUTHOR=Wang Yongqin , Chai Chenglin , Khatabi Behnam , Scheible Wolf-Rüdiger , Udvardi Michael K. , Saha Malay C. , Kang Yun , Nelson Richard S. 

TITLE=An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.)

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.685187

DOI=10.3389/fpls.2021.685187

ISSN=1664-462X

ABSTRACT=<p>Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The <italic>Brome mosaic virus</italic> (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes <italic>PHYTOENE DESATURASE</italic> (<italic>TaPDS</italic>) and <italic>PHOSPHATE2</italic> (<italic>TaPHO2</italic>) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of <italic>TaPDS</italic> transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For <italic>TaPHO2</italic>, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed <italic>enhanced GREEN FLUORESCENT PROTEIN</italic> (<italic>eGFP</italic>) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of <italic>eGFP</italic> and <italic>TaPHO2</italic> mRNA expression in roots at 14 and 21 dpi, and <italic>TaPHO2</italic> silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated <italic>TaPDS</italic> silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased <italic>TaPDS</italic> transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.</p>