Powdery mildew fungi are obligate biotrophs that obtain all nutrients from their plant host. This widespread pathogen infects agriculturally important crops like wheat, barley, cucurbits, and grapevine which can require numerous fungicide treatments to control. The powdery mildew Golovinomyces orontii infects the model plant Arabidopsis thaliana. Endoreduplication, or DNA replication without division, is induced in plant cells underlying its fungal feeding structure. This resulting enhanced DNA content supports its asexual reproduction. Fungal asexual reproductive structures contain chains of spores filled with lipid bodies. Elevated ploidy has long been associated with enhanced metabolic capacity, yet the induction of and metabolic changes resulting in this phenomenon remain underexplored. Here, metabolic and cell cycle contributors from the host and pathogen side are explored.

First, we show that a local ploidy-associated shift in plant primary metabolism to use the pyruvate dehydrogenase (PDH) bypass acts in a dose-dependent manner to support fungal reproduction. Not only are plant PDH bypass-derived lipids incorporated into fungal spore storage lipids, but they also act in a regulatory capacity to control the number of reproductive structures per colony, ensuring the fitness of spores that are formed. Evolutionarily distant plant and animal biotrophs require host lipids for their growth and/or reproduction. We provide a mechanism by which energy-dense host lipids are increased for microbial utilization. Next, spray-induced gene silencing (SIGS) methods were developed for powdery mildew fungi to investigate powdery mildew genetic contributors of disease. SIGS is an emerging tool being developed to protect crops from disease. It utilizes exogenously applied RNA designed to reduce expression of critical genes in plant pests using endogenous RNA interference machinery. For grape powdery mildew, dominant varieties are susceptible to powdery mildew and extensive resistance to fungicides has caused a need for new disease management strategies. G. orontii, was found to uptake RNA directly from its environment, independent of the host. SIGS methods using long double-stranded (dsRNA) and small interfering RNA (siRNA) were developed using A. thaliana whole plant and detached leaf assays. Methods were optimized against the azole-fungicide target, CYP51. Ten additional powdery mildew targets were screened identifying five genes that contribute to fungal proliferation: lipase a, lipase 1, β-carotene 15,15'-dioxygenase, apoptosis-antagonizing transcription factor (AATF), and effector candidate 2; four of which represent novel powdery mildew virulence targets. Translation of these methods to the Erysiphe necator-Vitis vinifera pathosystem showed similar reduction in disease with SIGS against CYP51 and the novel target AATF indicating high throughput screening of conserved powdery mildew targets in the G. orontii-A. thaliana pathosystem could prioritize targets for testing in grapevine powdery mildew and other systems. SIGS may be the future of controlling powdery mildew growth on crops because of its flexibility, resilience to resistance development, reduced environmental and health risks, and rapid transition from the bench to the field.

Lastly, powdery mildew effectors and host targets were examined in the context of powdery mildew-induced endoreduplication. Plant TCP transcription factors which regulate growth and development are heavily targeted by effectors of diverse plant pathogens; many of these TCPs have described roles in regulating cell expansion, proliferation, or endoreduplication. In a yeast-two hybrid screen between A. thaliana proteins and Golovinomyces orontii MPIPZ effectors, 34% of the interactions identified involved TCP proteins. In this study the contribution of TCPs and their targeting effectors to powdery mildew infection is ascertained. Four of the five TCPs targeted by effectors were found to suppress G. orontii MGH1 growth. Adult A. thaliana T-DNA mutants tcp13, tcp14, tcp15, and tcp20 were more susceptible to G. orontii MGH1. Using SIGS, homologs of G. orontii effector candidates (OECs) in G. orontii MGH1 were targeted. SIGS against OEC14, OEC60/OEC61, and OEC70 resulted in significantly reduced spore production compared to controls. With previous SIGS results above, a total of 7 novel powdery mildew targets have been identified. Homolog OEC60/OEC61 was investigated further. It encodes a short N-terminal domain originally annotated in G. orontii MPIPZ OEC60 and OEC61 coupled to a larger glycosyl hydrolase family 17 domain (GH17). To date, OEC60 and OEC61 homologs across all sequenced powdery mildews include the GH17 domain, suggesting the lack of GH17 domain in the MPIPZ isolate may be due to the incomplete nature of that genome. GH17 domain-containing proteins are found widely in plants and fungi. When homolog of OEC60/OEC61 is targeted in the E. necator-grapevine system, significantly less spores were produced, providing evidence this is a conserved powdery mildew virulence factor. Using Agrobacterium-mediated transient expression assays in Nicotiana benthamiana, G. orontii MGH1 homolog of OEC60/OEC61 is shown to be nuclear-targeted and co-localizes with TCP13 and TCP14. Co-immunoprecipitation further shows OEC60/OEC61 interacts with TCP13 in planta. Additionally, tcp14 mutants have enhanced endoreduplication at the site of powdery mildew infection which is known to correlate with enhanced powdery mildew asexual reproduction. Together, a role for OEC60/OEC61 in limiting TCP13/14 function to promote endoreduplication at the infection site is indicated. To further elucidate this interaction node, proteomic analysis (2-D LC MS/MS) of the MYC-purified protein complex from G. orontii-infected and uninfected plants expressing G. orontii MGH1 OEC60/OEC61-MYC was performed. These findings and tools may be used to further probe into powdery mildew-induced endoreduplication to uncover mechanisms of the complex transformations that occur in host tissue associated with biotrophs.