Gene Gun-Mediated Transient Gene Expression for Functional Studies in Plant Immunity.

Abstract

One major threat to plant cultivation are fungal pathogens, which can cause substantial yield losses in agriculture. As an example, cereal powdery mildew fungi such as the barley (Hordeum vulgare) pathogen, Blumeria graminis f. sp. hordei (Bgh), are among the ten most relevant fungal plant pathogens in molecular plant pathology and can lead to yield losses of up to 30%. Plant Mildew resistance Locus O (MLO) genes are required for successful colonization of plants by powdery mildew fungi. Accordingly, loss-of-function mlo mutants confer durable resistance against powdery mildew fungi in many plant species. In the case of barley, mlo-based resistance has been used for more than 40years in agriculture without powdery mildew fungi effectively overcoming this kind of immunity. However, the molecular basis of mlo resistance and function(s) of the transmembrane Mlo protein(s) are still incompletely understood. The generation of transgenic barley plants to study the plant immune response and the involvement of Mlo therein is time-consuming and challenging. Therefore, transient gene expression via gene gun-mediated particle bombardment became a popular, easy, and efficient tool to investigate different aspects of plant defense responses in barley. Since Bgh fails to penetrate leaf epidermal cells of mlo mutants, single-cell complementation upon biolistic transformation resulting in (over-)expression of Mlo can be used to characterize the Mlo protein functionally in vivo. In this chapter, we describe in detail the gene gun-mediated transient expression of Mlo in barley leaf epidermal cells followed by powdery mildew inoculation and the subsequent microscopic evaluation. However, gene gun-mediated transient gene expression may be also used to address other research questions or to transform the epidermal tissues of other plant organs and/or species.