An unwanted guest: Metabolic and physical accommodations to powdery mildew infection by the host plant Arabidopsis thaliana

Adapted plant biotrophic organisms feed on the living tissue of their plant hosts. The success of colonization and reproduction relies on navigating the narrow path between limiting detrimental impact on the host that can trigger immune responses and maximizing nutrient acquisition. One common strategy among such interactors is to influence host tissue to better suit the needs of the interacting microbe, either through increased metabolic output, provision of specific compounds required by the biotroph, and in some cases to accommodate the feeding structures of fungi. Powdery mildew is one such fungus that causes reduced yield and loss in quality of harvest across many agronomically important crop species. Research into this pathogen has revealed that it has an extensive effector repertoire to manipulate host defenses and it establishes a metabolic sink at the sites of infection, rerouting carbon flux within host tissues to fuel its growth and reproduction. This thesis presents the responses in Arabidopsis thaliana leaf tissue during challenge by Golovinomyces orontii that are infection-specific metabolic changes and identify susceptibility factors of the host that do not affect host defense signaling.In the first two chapters plant lipid metabolism and the Arabidopsis-powdery mildew pathosystem is introduced, as well as providing a review of adapted biotrophs, both pathogenic and mutualist, that alter host cell cycle during colonization and nutrient acquisition from their host details the molecular mechanisms underpinning changes in endoploidy. Other examples of cell cycle control manipulations include host cell hypertrophy, acytokinetic mitosis, and increased cell division. A common strategy for hijacking the progression through cell cycle is effector-mediated interactions with promoters of the endocycle and G2-M transition. Chapter 3 presents the role of the host PDH bypass during infection. Its induction is specific to the cells surrounding the site of infection, highlighting a change modulated by the establishment of a metabolic sink through nutrient uptake by the fungus. This pathway serves to connect the elevated glycolytic activity to fatty acid biosynthesis. Capacity of the pathway to mobilize pyruvate in the cytosol was shown to be linked to reproductive output of the fungus. Using isotopic labeling experiments, the carbon flux passing through the PDH bypass was shown to contribute to host lipids, as well as lipids accumulating in fungal asexual conidia. This demonstrates that the pathway provides precursors for fungal energy reserves. To determine what changes in lipid metabolism occur in Arabidopsis leaves during infection, lipid content and its fatty acid profile in uninfected and infected leaves was analyzed. An increase in triacylglycerols and very long chain fatty acids was observed, occurring at the expense of membrane lipids. In particular, lipids in the thylakoid membrane showed the greatest decrease in abundance. Host plants deficient or overexpressing transcription factors promoting embryogenesis and seed maturation were phenotyped to look for changes in powdery mildew reproduction. In addition, mutant plants with altered flux into specific lipid pools were analyzed for their susceptibility to infection. This led to the development of a model that identified the flux of acyl chains from the chloroplast to the cytosol as a promoter of fungal spore production, while flux into the acyl-CoA pool of the endoplasmic reticulum and TAG synthesis reduced spore output. Powdery mildew infection requires accommodation of the feeding structure in the epidermal cells that mediates effector release into host tissue and nutrient uptake. Chapter 5 identifies the host gene ACYL-COA BINDING PROTEIN4 (ACBP4) as a susceptibility factor during infection by G. orontii. Compared to the other 6 single mutants of the ACBP gene family, acbp4 plants exhibit the greatest reduction in fungal spore production. The altered susceptibility of the mutant was determined not to be a result of altered lipid content or defense signaling. One striking difference was the lack of very long chain fatty acids in lipid from uninfected tissues in the mutant relative to wild type leaves, though they accumulated at wild type levels in infected leaves, suggesting the mutant affects acyl chain trafficking dynamics that affect the plant’s suitability as a host prior to infection. Further, the cells in the leaf epidermis had half the area of wild type leaves, suggesting that establishment of the haustorium could be impeded. This mutant identifies a mechanism of susceptibility to biotrophic pathogens, while decreasing resistance to necrotrophs, suggesting it may coordinate processes central to leaf development and nutrient availability for plant pathogens.

Advisor: Wildermuth, Mary C