Identification of genes primed by chitosan
To identify genes that could be involved in chitosan-induced resistance, gene expression profiles were scrutinized. First, qRT-PCR analysis of a subset of 9 genes was done to successfully validate the expression data of the microarray (Fig S4a,b). Similar expression profiles were observed in the microarray and the qRT-PCR data, validating the data set. Priming profiles, i.e. subtle or non-detectable differential expression after chitosan treatment (i.e. Chitosan + Mock) and an increased differential expression after infection (i.e. Chitosan + B. cinerea ) were identified. Transcripts of the earliest time point during the infection (6 hpi) were chosen to identify primed genes involved in early immune responses. Expression of the subset of 260 DEGs unique for Chitosan +B. cinerea treatment at 6 hpi (Fig 3c) were analysed over Water + Mock, Chitosan + Mock and water + B. cinerea . From the subset, 203 down-regulated (Table S1) and 57 genes were found to be up-regulated (Table S2). An over-representation test was performed to investigate gene ontology categories of the primed genes (Panther 14.0).
Among the 203 genes that were repressed during infection (Table S1), eleven transcripts were associated with cysteine-type peptidase activity. Other transcripts were grouped with photosynthesis, light harvesting in photosystem I activity. Moreover, several had a response to hormone activity; nine ethylene-responsive transcription factor and receptor genes were significantly down-regulated from -2,3 to -1,1 compared to Water + B. cinerea . Other notable genes with strong priming include those with proteolysis activity, with a range between -3 to -1,7 fold repressed. Other genes with repressed expression belong to auxin hormones and one to the ABA receptor (ABAPYL4). Furthermore, two genes of the little-known LATERAL ORGAN BOUNDARIES (LOB) were identified as repressed. Additional transcripts were functionally unassigned within the list.
Among the 57 differentially up-regulated genes (Table S2), there was one transcript encoding peroxidase activity with 2 fold increase compared to Water + B. cinerea , nine transcripts with protein kinase activity with between +1,1 to +2,1 fold, five transcripts with transcription regulatory activity, including SlMYB20, SLWRKY51 and SlWRKY72. Additional transcripts were functionally unassigned within the list.
Importantly, uncharacterised genes also show primed expression patterns. Of these, Avr9/Cf-9 rapidly elicited protein 75 (ACRE75; Solyc11g010250.1) was up-regulated 1,6-fold in Chitosan + B. cinerea in comparison to water + B. cinerea at 6 hpi (Table S2). ACRE genes have been previously studied and characterised as important genes involved in R gene-mediated and ROS gene-independent early plant defence responses (Durrant et al., 2000) and in response to methyl-jasmonate (MeJA) treatment (van den Burg et al., 2008). ACRE75 molecular functions are still to be deciphered and therefore research into its role and other members of the ACRE gene family in priming of chitosan was pursued.