Defense gene responses toward necrotrophic fungi in Arabidopsis thaliana

Abstract: Leptosphaeria maculans an ascomycete in the diverse class of Dothideomycetes, is an devastating pathogen on Brassica crops. In this thesis, tools and knowledge in Arabidopsis thaliana was explored to enhance our knowledge on plant defense to this fungus. Mechanisms that to large extent are shared with other necrotrophic fungi. Plants have evolved disease resistance (R) genes encoding for nucleotide-binding site (NB) and leucine-rich repeat (LRR) proteins with N-terminals represented by either Toll/Interleukin-1 receptor (TIR) or coiled-coil (CC) domains. A genome-wide study of presence and diversification of CC-NB-LRR and TIR-NB-LRR encoding genes, and shorter domain combinations in 19 Arabidopsis thaliana accessions and Arabidopsis lyrata, Capsella rubella, Brassica rapa and Eutrema salsugineum were performed. Out of 528 R genes analyzed, 12 CC-NB-LRR and 17 TIR-NB-LRR genes were conserved among the 19 A. thaliana genotypes, while only two CC-NB-LRRs, including ZAR1, and three TIR-NB-LRRs were conserved when comparing the five species. The resistance to Leptosphaeria maculans 1 (RLM1) locus confers resistance to L. maculans and has experienced conservation and diversification events particularly in B. rapa. RLM3 is unique in comparison to other characterized R proteins due to its three brevis radix (BRX) domains adjacent to the TIR and the NB domains. Three homologs were found in Camelina sativa that diverged from Arabidopsis ~17 million years ago. The BRX domains is able to interact with RLM3 possibly signaling downstream signaling response after pathogen contact. Two A. thaliana mutants, being susceptible to L. maculans (lms1 and lms5) were further characterized. LMS1 encodes a lipid phosphate phosphatase-like protein but lacks phosphatidic acid phosphatase activity. LMS1 is localized in the plasma membrane where it interacts through the third motif of the phospatidic phosphatase domain with the salicylic acid binding protein 3 (SABP3). SABP3 mutagenesis abolishes the interaction with LMS1. The lms1 mutant displays enhanced susceptibility to diverse pathogens, and high endogenous levels of SA and JA were found upon pathogen challenge. The LMS1-SABP3 interaction is central to maintain accurate JA and SA hormone homeostasis and defense signaling to necrotrophic pathogens. Mapping and genomic resequencing of the lms5 mutant revealed a mutation in a prolyl oligopeptidase (POP1) gene. POP1 interacts with the glycolate oxidase 1 protein, a regulator of hydrogen peroxide production. Upon fungal challenge levels of callose and indole-acetic acid (IAA) were elevated in lms5 compared to wild-type. The data highlight the importance of a balanced redox and auxin homeostasis to counteract invasion of L. maculans. In conclusion, this thesis work has revealed new defense genes, intricate interactions and signaling forming an important platform for future research and application in molecular breeding.