Comparative transcriptional profiling of Arabidopsis arr6-3 mutant and wild-type (Col-0) plants mock-treated and after infection with Plectosphaerella cucumerina BMM

Cytokinin signaling pathway, that is mediated by Arabidopsis Response Regulators (ARRs) proteins, has gained functional relevance in the modulation of plant disease resistance responses. We describe here a novel function of ARR6 in the regulation of Arabidopsis immunity and cell wall composition. arr6 mutants were found to be more susceptible and resistant, respectively, to the vascular bacteria Ralstonia solanacearum GMI1000 and the necrotrophic fungus Plectosphaerella cucumerina BMM. In contrast, transgenic Arabidopsis lines overexpressing ARR6 showed the opposite phenotypes, further supporting a role of ARR6 in regulating disease resistance responses. Transcriptomics and metabolomics analyses of arr6 revealed that canonical immune pathways, like those modulated by defensive phytohormones or triggered by Microbe-Associated Molecular Patterns, are not altered in arr6 plants, suggesting that novel, uncharacterized defensive pathways explain arr6-mediated resistance. We identified alterations in the composition of arr6 cell walls in comparison to that of wild-type plants walls. Remarkably, pectin-enriched cell wall fractions extracted from arr6 walls trigger more intense immune responses than those activated by the corresponding wild-type fractions. Our data suggest that arr6 pectin wall fraction contained specific pentose-based oligosaccharides that would function as non-yet described wall-related Damage-Associated Molecular Patterns (DAMPs). These data supported a novel function of ARR6 in the regulation of cell-wall mediated immunity and reinforce the relevance of plant cell wall composition in the modulation of specific disease resistance responses. Eighteen-day-old wild-type (Col-0) and arr6-3 plants were mock-treated or Plectosphaerella cucumerina BMM-inoculated, and samples were collected at 1 day post infection (dpi). Each sample represented a pool of 25 rosettes from plants grown under short-day conditions. Three biological replicates were obtained for each combination of genotype and treatment were independently hybridized for each transcriptomic comparison. Fluorophore-marked (Cy3 and Cy5) complementary RNA was prepared and the product was used to hybridize Arabidopsis Oligonucleotide Microarrays (Qiagen-Operon Version 3.0.; Arizona University, USA) at the Genomic Unit of CNB-CSIC (Madrid). After hybridization, the array was scanned and the intensities were used to generate the array images. These images were then used to correct and normalize the data. The expression levels of the genes were visualized with the FIESTA Viewer.