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. Presented here is a series of methods to investigate PTI in Brassica napus. The assays allow measuring early cell signalling responses, gene expression changes, cell wall reinforcement, metabolome changes and scoring PAMP-Induced resistance. 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.
Using these assays substantial variation in PAMP-responsiveness was observed amongst elite varieties of B. napus. Taking this further, a genome wide association study (GWAS) of the flg22 and elf18 triggered oxidative bursts, resistance to Pseudomonas syringae and Botrytis cinerea was carried out in a population of B. napus. A substantial number of molecular markers, covering both sequence and expression variation, have been identified as having significant association with these four traits.
QTL mapping of the flg22 triggered oxidative burst in the ADxGD double haploid cross identified a major quantitative trait loci (QTL) on C9 of B. oleracea. mRNA-seq of the parents led to a non-synonymous single nucleotide polymorphism (SNP) list and enabled fine mapping through the addition of KASPar markers to the original map. This produced a relatively small list of candidate genes including CYLIC NUCLEOTIDE GATED ION CHANNEL 4 (CNGC4) also known as DEFENCE NO DEATH 2 (DND2). An insertion in Arabidopsis thaliana DND2 showed phenotypic difference in the oxidative burst between the insertion line and Col-0, potentially indicating a role for the gene in regulating early PTI
