Methods to Study PAMP-Triggered Immunity in Brassica Species

The first layer of active defence in plants is based on the perception of pathogen-associated molecular patterns (PAMPs) leading to PAMP-triggered Immunity (PTI). PTI is increasingly being investigated in crop plants where it may have potential to provide durable disease resistance in the field. Limiting this work however is an absence of reliable bioassays to investigate PAMP responses in some species. Here, we present a series of methods to investigate PTI in Brassica napus. The assays allow measuring early responses such as the oxidative burst, MAPK phosphorylation and PAMP-induced marker gene expression. Illumina-based RNA sequencing analysis produced a genome-wide survey of transcriptional changes upon PAMP treatment seen in both the A and C genomes of the allotetraploid B. napus. Later responses characterised include callose deposition and lignification at the cell wall, seedling growth inhibition and PAMP-induced resistance (PIR) to the fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Furthermore, using these assays we demonstrate substantial variation in PAMP responses within a collection of diverse B. napus cultivars. The assays reported here could have widespread application in B. napus breeding and mapping programs to improve selection for broad-spectrum disease resistance.

9