A detached petal disc assay and virus-induced gene silencing facilitate the study of Botrytis cinerea resistance in rose flowers

Abstract

Fresh-cut roses (<i>Rosa hybrida</i>) are one of the most important ornamental crops worldwide, with annual trade in the billions of dollars. Gray mold disease caused by the pathogen <i>Botrytis cinerea</i> is the most serious fungal threat to cut roses, causing extensive postharvest losses. In this study, we optimized a detached petal disc assay (DPDA) for artificial <i>B. cinerea</i> inoculation and quantification of disease symptoms in rose petals. Furthermore, as the identification of rose genes involved in <i>B. cinerea</i> resistance could provide useful genetic and genomic resources, we devised a virus-induced gene silencing (VIGS) procedure for the functional analysis of <i>B. cinerea</i> resistance genes in rose petals. We used <i>RhPR10.1</i> as a reporter of silencing efficiency and found that the rose cultivar 'Samantha' showed the greatest decrease in <i>RhPR10.1</i> expression among the cultivars tested. To determine whether jasmonic acid and ethylene are required for <i>B. cinerea</i> resistance in rose petals, we used VIGS to silence the expression of <i>RhLOX5</i> and <i>RhEIN3</i> (encoding a jasmonic acid biosynthesis pathway protein and an ethylene regulatory protein, respectively) and found that petal susceptibility to <i>B. cinerea</i> was affected. Finally, a VIGS screen of <i>B. cinerea</i>-induced rose transcription factors demonstrated the potential benefits of this method for the high-throughput identification of gene function in <i>B. cinerea</i> resistance. Collectively, our data show that the combination of the DPDA and VIGS is a reliable and high-throughput method for studying <i>B. cinerea</i> resistance in rose.