Browsing by Author "Chaya, Timothy"
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Item A Bacillus velezensis strain shows antimicrobial activity against soilborne and foliar fungi and oomycetes(Frontiers in Fungal Biology, 2024-02-23) Wockenfuss, Anna; Chan, Kevin; Cooper, Jessica G.; Chaya, Timothy; Mauriello, Megan A.; Yannarell, Sarah M.; Maresca, Julia A.; Donofrio, Nicole M.Biological control uses naturally occurring antagonists such as bacteria or fungi for environmentally friendly control of plant pathogens. Bacillus spp. have been used for biocontrol of numerous plant and insect pests and are well-known to synthesize a variety of bioactive secondary metabolites. We hypothesized that bacteria isolated from agricultural soil would be effective antagonists of soilborne fungal pathogens. Here, we show that the Delaware soil isolate Bacillus velezensis strain S4 has in vitro activity against soilborne and foliar plant pathogenic fungi, including two with a large host range, and one oomycete. Further, this strain shows putative protease and cellulase activity, consistent with our prior finding that the genome of this organism is highly enriched in antifungal and antimicrobial biosynthetic gene clusters. We demonstrate that this bacterium causes changes to the fungal and oomycete hyphae at the inhibition zone, with some of the hyphae forming bubble-like structures and irregular branching. We tested strain S4 against Magnaporthe oryzae spores, which typically form germ tubes and penetration structures called appressoria, on the surface of the leaf. Our results suggest that after 12 hours of incubation with the bacterium, fungal spores form germ tubes, but instead of producing appressoria, they appear to form rounded, bubble-like structures. Future work will investigate whether a single antifungal molecule induces all these effects, or if they are the result of a combination of bacterially produced antimicrobials.Item Analysis of reactive oxygen species kinetics in pathogenic fungi of barley and maize using the HyPer sensor(University of Delaware, 2019) Chaya, TimothyMagnaporthe oryzae and Cochliobolus heterostrophus represent two important fungal pathogens that pose a threat to global food production. The hemi-biotroph M. oryzae has a broad host range with the ability to infect rice, barley, millet, and other grasses. C. heterostrophus on the other hand is a necrotroph with a host range of maize, soybeans, millet, wheat and teosinte. The fungal infection in host plant tissue is a complex process that is in part defined by regulation of Reactive Oxygen Species (ROS). It is well established that plants use ROS as a key component of innate immunity, growth and development. Analogously, fungi need ROS for development and production of necessary infection structures. In both organisms, ROS production must be carefully regulated to prevent deleterious effects. This research is exploring the ROS dynamics in both fungal systems. It is hypothesized that these pathogens will utilize and regulate ROS in a different manner during infection. ☐ To aid in the understanding of this process, I am utilizing a genetically encoded reporter for hydrogen peroxide called HyPer. Three different microscopy approaches for ROS kinetics were conducted. The first was an in-situ analysis of conidia in a perfusion chamber, characterizing the response kinetics of each fungus when treated with oxidative (hydrogen peroxide) or reductive (dithiothreitol) solutions. The second approach was imaging in planta ROS levels through select infection stages. The last approach was the generation of a high throughput imaging workflow to analyze HyPer sensor mutants in future forward genetic screens. With a baseline of how each fungus responds to ROS, we plan to analyze HyPer mutant libraries under development for forward genetic screens, to identify genes related to ROS generation and attenuation. These genes can provide targets for the development of new fungicides or breeding of more resistant plants.Item The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved(Plant Physiology, 2023-12-04) Chaya, Timothy; Banerjee, Aparajita; Rutter, Brian D.; Adekanye, Deji; Ross, Jean; Hu, Guobin; Innes, Roger W.; Caplan, Jeffrey L.Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.