Functional Characterization of Putative Effector Genes of Basil Downy Mildew Pathogen Peronospora belbahrii

Abstract

Peronospora belbahrii, the causal agent of the devastating downy mildew disease on basil, is an obligate biotrophic oomycete. Similar to other oomycete pathogens, P. belbahrii is believed to secrete effectors to facilitate host colonization. To this end, we did the functional characterization of 10 P. belbahrii effector candidate genes, which encode predicted secreted proteins with a translocation motif RXLR (or RXLR-EER) and/or nuclear localization signals (NLS). First, we determined their gene expression patterns during infection using reverse transcription quantitative PCR (RT-qPCR). Five genes were induced during infection and the functionality of their predicted signal peptides was confirmed using a yeast invertase secretion system, suggesting that these genes likely encode bona fide effectors that play significant roles in manipulating host cellular processes to cause disease. Their roles in pathogenicity are currently being tested through overexpression and host-induced gene silencing (HIGS). To facilitate the genetic analysis of these and additional effector candidate genes, we developed a transient expression system in basil for utilizing overexpression and HIGS in a transient manner. To better analyze the effect resulted from the in planta expression of overexpression and HIGS construct of PbEC2 on pathogen growth, a quantitative PCR approach was developed to quantify the pathogen biomass. In addition, we generated transgenic basil to express double-stranded RNAs of one selected effector gene to determine its function in pathogenicity using HIGS. This study is expected to facilitate the understanding of P. belbahrii pathogenesis and help develop tools to control this pathogen.