Role of extracellular vesicles in plant-microbe interactions

Abstract

The goal of this research is to determine the role of extracellular vesicles (EVs) in plant-microbe interactions. EVs are known to mediate intercellular transfer of small RNAs (sRNAs) in mammalian and insect systems, but very little is known about the function of EVs in plants or fungi. Our team has developed methods to purify plant EVs and has demonstrated that EVs are enriched in stress response proteins, and carry sRNAs. We hypothesize that plant EVs also mediate intercellular signaling, including trans-kingdom transfer of sRNAs. The experiments detailed in this proposal will examine whether plant and fungal EVs carry sRNAs that target each others genes, how plant EVs are produced, and how plants and fungi exchange EVs. These questions will be addressed using soybean and Arabidopsis as host plants, and Colletotrichum spp., Pseudomonas syringae, and Bradyrhizobium japonicum as microbes. We will isolate EVs from plants infected with these microbes and will quantitatively assess their protein and sRNA contents. These analyses will provide insight into how proteins and sRNAs are targeted to EVs, and how EVs differ between types of interactions. These analyses will also reveal, for the first time, the contents of EVs from a fungal plant pathogen. Most significantly, these analyses will enable us to establish a network of trans-kingdom gene regulation between plants and fungi. EV-mediated transfer of sRNAs between host cells and fungal cells will be imaged using super-resolution microscopy and RNA-PAINT. In addition, genes required for EV biosynthesis will be identified using reverse genetics. Mutants with defects in EV biogenesis will be further analyzed using 3D electron microscopy, allowing us to image plant-fungal interfaces at unprecedented resolution, capturing all stages of EV production, release, and uptake. The above objectives will be accomplished by a team of four laboratories with highly complementary expertise. The Innes lab is pioneering the study of EVs, and brings over 25 years of experience comparing soybean and Arabidopsis immune systems. The Meyers lab is an international leader in the sRNA field, and has performed extensive characterization of soybean sRNAs. The O'Connell lab is an international leader in Colletotrichum pathogenesis, and has pioneered EM methods for imaging fungal infection sites. The Thordal-Christensen lab has amassed a collection of Arabidopsis endomembrane trafficking mutants, which will be assessed for defects in EV production. With this collection of expertise and resources, our group will be able to make rapid progress toward understanding the roles of EVs in plant-microbe interactions. This research will enable development of durably disease resistant crops via improving novel disease resistance mechanisms such as host-induced gene silencing. Development of crops with durable disease resistance is a key to the creation of a sustainable and secure food supply, the ultimate goal of ERA-CAPS.