Suitability of genes in Arabidopsis as above-ground fluorescent reporters to screen for clubroot infection.

Abstract

Clubroot is a soil-borne disease that affects plants in the Brassicaceae family caused by the obligate parasite Plasmodiophora brassicae. Clubroot infection begins in the roots and leads to gall formation, leading to an overall decrease in plant health. In particular, this can be economically devastating with regards to the Brassica species, which includes major commercial crops such as broccoli, cauliflower, and canola. Currently there are no reliable methods for detecting clubroot infection without digging up the plant to inspect its roots, which is inefficient and impractical when dealing with large numbers of individuals. The objective of this research was to identify potential reporter genes which could be used to screen for early clubroot infection in the shoot on a large-scale basis using a fluorescent non-destructive method. 19 genes in Arabidopsis thaliana, also belonging to the Brassicaceae family, which were either up- or down-regulated during infection according to RNA-Seq data were chosen for testing. A time course consisting of 0, 2, 5, 7, 14, 21, and 28 days post infection was established and gene expression on these days was observed with RT-PCR and RT-qPCR. 8 genes were shown to have coinciding expression trends between the RNA-Seq, RT-PCR, and initial RT-qPCR data, and their promoters were selected to be cloned into reporter vectors. tdTomato and mOrange2 were chosen as fluorescent reporters for their brightness and photostability. A final promoter-reporter construct, GASA6::mOrange2, was transformed into Arabidopsis and T1 seeds generated transgenic lines ginger1 and ginger2. Continued RT-qPCR investigation and cloning were conducted concurrently. Unfortunately, final RT-qPCR data revealed that there was no significant difference in expression between control and infected plants for any of the potential reporter genes. The functions of these genes were discussed to evaluate their connection to clubroot disease, and as possible indicators for other potential infection reporter genes. Though inconclusive, results of this research provide insight into the gene expression dynamics in shoot tissue during clubroot infection.