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Plants have a dynamic immune system to protect themselves against pathogens. Recognition of a pathogen leads to the activation of this immune system, steered by different phytohormones. One of these, salicylic acid, regulates plant responses to biotrophic pathogens that thrive on living host tissue. Typical SA-mediated responses are the production of antimicrobial proteins and metabolites and the strengthening of the cell wall. These responses can occur both locally and systemically. Due to their nature, these responses cost energy and resources that cannot be invested in plant growth and development. This suppression also works the other way around: active growth and developmental processes suppress immunity. This mutual inhibition is called the growth-immunity tradeoff and is actively regulated by the plant. The research in this thesis is focused on the model plant Arabidopsis thaliana (thale cress), in which the SA content is controlled at the level of synthesis and catabolism. Two enzymes that catabolize SA through hydroxylation are the paralogous 2-oxoglutarate Fe(II)-dependent oxygenases DMR6/S5H (DOWNY MILDEW RESISTANT 6/SA 5-HYDROXYLASE) and DLO1/S3H (DMR6-LIKE OXYGENASE 1/SA 3-HYDROXYLASE). dmr6/dlo1 mutants have enhanced SA levels and therefore a constitutively active immune system and enhanced disease resistance. The high SA levels of double mutant plants cause hyperresistance but also dwarfism. On the other hand, overexpression of DMR6 or DLO1 leads to depletion of SA and thus enhanced susceptibility. We use the perturbed SA catabolism of the dmr6/dlo1 mutants and DMR6/DLO1 overexpression lines to investigate the SA-mediated growth-immunity tradeoff. We found that altered expression of DMR6 and/or DLO1 had major effects on the transcriptome that correlated to the plant’s SA content. We found that enhanced SA levels in dmr6/dlo1 mutants caused reduced photosynthetic efficiency and a sped up development and reduced SA levels in DMR6/DLO1 overexpression lines led to reduced pathogen-associated molecular pattern (PAMP)-triggered immune responses. We further identified a significant effect of the genetic background to perturbations in DMR6/DLO1 expression in different Arabidopsis accessions: several Arabidopsis accessions had a reduced growth tradeoff but retained high disease resistance. To systematically investigate the genetics behind regulation of the growth-immunity tradeoff, we further employed a forward genetics screen for restored growth and high resistance in dmr6-3 dlo1 double mutants and identified seven unique zund mutants. These plants carried mutations in known regulators of SA synthesis (ICS1, ISOCHORISMATE SYNTHASE 1), perception (NPR1, NONEXPRESSOR OF PR GENES 1), or signaling (PAD4, PHYTOALEXIN DEFICIENT 4), indicating that a suppression of SA synthesis or SA responses reduces the growth tradeoff in high immunity dmr6-3 dlo1 mutants. Three other zund mutants carried a mutation in the same gene: MED15a (MEDIATOR 15a), a component of the MEDIATOR complex for transcription initiation. We investigate the function of MED15a in the growth-immunity tradeoff in detail, and find that around 80% of dmr6-3 dlo1-induced gene expression changes are genetically dependent on the integrity of two amino acids in the MED15a kinase-inducible (KIX) domain. I discuss the implications of this research for applications to breed for sustainable disease resistance in crops. more...

Pesticides, mostly fungicides, are used in large quantities in viticulture to contain the spread of fungal and fungal-like diseases such as powdery mildew (PM) and downy mildew (DM), toward which cultivated grapevine (Vitis vinifera) is highly susceptible. Such consumption of fungicides is costly and deleterious for human health and environment. As an alternative, such diseases have been successfully controlled by inactivation of plant susceptibility genes in crops. The knocking out of DMR6 genes was demonstrated to be very effective in controlling DM in Arabidopsis thaliana and some cultivated species, but its efficacy toward DM resistance has yet to be demonstrated in grapevine. In addition, silencing of MLO genes resulted in resistance to PM in grapevine. Introgression of nonfunctional copies of susceptibility genes can be achieved via traditional breeding, but crossing is not always desirable in grapevine, since maintenance of clonal genetic integrity is commercially important for wine grapes. The advent of genome editing now offers revolutionary tools to edit and completely knock out susceptibility genes in many crops while maintaining their cultivar and clonal genetic backgrounds. In the present work, CRISPR/Cas9 technology was used to edit DM and PM susceptibility genes in different grapevine clones. Several plants edited in DMR6 and MLO genes were obtained and are currently being screened for DM and PM resistance. This work will establish whether full knockout of DMR6 and MLO genes provides durable resistance to DM and PM, respectively, and will hopefully deliver mildew-resistant grapevine plants. more...

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. more...

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. more...