Molecular analysis of Arabidopsis susceptibility to oomycete pathogens

Abstract

Plants are under constant attack by pathogens, but successful infections are more exception than rule. Plants use their defense system to fend off many attackers. On the other hand, they also contain so-called susceptibility genes (S genes), that facilitate or support the infection process. Impairment of S genes can lead to loss of susceptibility. Two groups of S gene-encoded proteins can be distinguished: negative regulators of defense and susceptibility factors. DOWNY MILDEW RESISTANT 6 (DMR6) and its paralog DMR6-LIKE OXYGENASE 1 (DLO1) belong to the group of negative regulators of defense. dmr6 and dlo1 mutants are enhanced resistant to the downy mildew causing oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), due to their increased levels of salicylic acid (SA). A dmr6-3 dlo1 double mutant shows much higher levels of SA than the single mutants, indicating partial redundancy between DMR6 and DLO1. DMR6 and DLO1 differ in their spatial expression pattern in downy mildew-infected Arabidopsis leaves; DMR6 is mostly expressed in cells that are in contact with hyphae and haustoria of H. arabidopsidis, while DLO1 is expressed mainly in the vascular tissues near infection sites. We conclude that DMR6 and DLO1 redundantly suppress plant immunity, but also have distinct activities as a result of their differential localization of expression. Plant metabolite analysis showed that DMR6 and DLO1 hydroxylate SA at the 5- and 3-position of the aromatic ring of this phenolic acid, respectively. While dmr6 and dlo1 mutant plants accumulate SA, overexpression lines of DMR6 and DLO1 have reduced levels of SA, but high levels of the enzyme products 2,5- and 2,3-dihydroxy benzoic acid (DHBA), respectively. One amino acid in the otherwise conserved proposed substrate-binding pocket differs between DMR6 (Tyr) and DLO1 (Phe), and their respective orthologs. However, this amino acid difference appeared not to be responsible for the difference in position of hydroxylation on the aromatic ring of SA mediated by DMR6 and DLO1. The difference in biological activity between DMR6 and DLO1 seems mostly determined by their promoters and not by the products of their enzymatic activity. We conclude that DMR6 and DLO1 control activated plant immunity by lowering SA levels through hydroxylation. In a second research line, I searched for novel susceptibility genes, required for infection of Arabidopsis with the oomycete pathogen Phytophthora capsici. Screening of EMS-mutagenized plants, in a highly susceptible genetic background, led to the identification of 32 P. capsici-resistant mutants. The genomes of the mutants were sequenced and analyzed to detect recessive mutations in the coding sequences of all Arabidopsis genes. When focusing on non-synonymous mutations in coding sequences, we identified 98-259 mutated genes per mutant. A shortlist was made with candidate genes that were mutated in three or more mutants. Validation of the candidate genes is currently being performed by sequencing the corresponding mutations in resistant plants of BC1S2 populations. The resulting candidate Arabidopsis S genes will form the basis for translational research to obtain disease resistant crops. The application of S genes in resistance breeding can contribute to future food security and sustainable agriculture.