The role of OUTER ENVELOPE PROTEIN 40 in starch-dependent pathogen susceptibility of Arabidopsis thaliana
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Anthracnose is a major yield-reducing plant disease caused by fungal pathogens of the Colletotrichum genus. The model pathogen Colletotrichum higginsianum shares a highly similar infection strategy with agriculturally important Colletotrichum species and establishes a compatible interaction with the model plant Arabidopsis thaliana (Arabidopsis). A deeper understanding of both fungal pathogenicity and plant defence mechanisms could contribute to the development of strategies to mitigate anthracnose-associated crop losses. The starch-deficient Arabidopsis mutant plastidic phosphoglucomutase (pgm) displays hypersusceptibility to C. higginsianum compared to the wild type (WT). Previous studies suggested that this phenotype results from alterations in cell wall matrix composition and impaired accumulation of defence-associated metabolites due to carbohydrate depletion during the dark phase. In a forward genetic suppressor screen targeting pgm hypersusceptibility, a premature stop codon in OUTER ENVELOPE PROTEIN 40 (OEP40) was identified as the causal mutation for the suppression. OEP40 functions as a glucose and glucose-phosphate permeable channel in the outer chloroplast envelope. However, the mechanism by which loss of OEP40 suppresses pgm hypersusceptibility remained elusive.
This thesis therefore investigated the mechanisms underlying OEP40-mediated suppression of pgm hypersusceptibility, with particular focus on defence responses, carbohydrate metabolism, and cell wall composition. To dissect the contribution of individual pathways, loss-of-function mutants affecting salicylic acid (SA), camalexin (CA), and cell wall biosynthesis were introduced into the pgm oep40-3 background and subsequently characterised in susceptibility assays and biochemical analyses, in addition to transcriptomic profiling.
In the WT background, oep40-3 exhibited enhanced resistance to C. higginsianum and altered cell wall matrix composition, while overall soluble sugar levels remained largely unchanged. Compared to pgm, the pgm oep40-3 double mutant showed strongly reduced susceptibility to C. higginsianum, accompanied by elevated accumulation of glycosylated SA, dihydroxybenzoic acids, and camalexin under mock conditions, as well as enhanced reactive oxygen species (ROS) production, indicating constitutive defence priming. Genetic analysis further demonstrated that suppression of pgm hypersusceptibility was partially dependent on SA signalling but independent of CA accumulation.
Cell wall analyses revealed that loss of OEP40 restored the pgm-associated cell wall arabinose defect, situated in the rhamnogalacturonan. Furthermore, pgm oep40-3 displayed restored nucleotide sugar pools compared to pgm, suggesting increased allocation of metabolic resources towards cell wall biosynthesis. Reconstitution of reduced arabinose levels in pgm oep40-3 using arabinan biosynthesis mutants did not conclusively link restored arabinose content to suppression of hypersusceptibility, likely because the resulting cell wall architecture differed from that of pgm.
Transcriptome analysis supported these findings and demonstrated extensive reprogramming of pgm oep40-3 gene expression. Under mock conditions, the transcriptomic profile of pgm oep40-3 clustered more closely with WT and oep40-3 than with pgm, suggesting that loss of OEP40 broadly counteracts pgm-associated transcriptional alterations. Compared to WT, pgm oep40-3 showed enrichment of upregulated genes associated with starvation responses and downregulation of genes linked to cell cycle regulation. In comparison to pgm, defence associated genes were significantly enriched among upregulated transcripts, whereas downregulated genes were associated with cell wall- and thylakoid-related processes.
Taken together, this study establishes OEP40 as an important regulator of Arabidopsis defence responses by demonstrating that the loss of OEP40 enhances resistance in multiple plant-pathogen interactions. The results further show that suppression of pgm hypersusceptibility is partially dependent on SA signalling but independent of CA induction. In addition, loss of OEP40 substantially restores metabolic defects associated with starch deficiency, including altered nucleotide sugar availability and cell wall composition.
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