Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens

The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity.In nature, plants are constantly exposed to a large number of pathogens including fungi, bacteria, and viruses. In response, plants have evolved multiple layers of defense mechanisms to resist the pathogen attack (1). The first line of plant defense against fungi is penetration resistance that is achieved by localized cell wall appositions (CWAs), also called papillae, on an inner surface of cell walls at the site of fungal penetration (2). CWAs consist primarily of callose (β-1,3-glucan), lignin, cell wall proteins, and reactive oxygen species (2–4). The focal deposition of these elements in papillae appears to be an early and essential factor in plant penetration resistance (5).Studies on the genetic basis of penetration resistance have revealed that entry control of A. thaliana against nonadapted powdery mildews largely depends on several PENETRATION (PEN) genes (PEN1, PEN2, and PEN3). All three PEN proteins are also recruited to attempted fungal penetration sites (6–11). Intriguing findings show that the focal accumulation of PEN1 and PEN3 occurs outside the plasma membrane and within papillae or haustorial encasements (3, 11, 12). Disruption of actin cytoskeleton by pharmacological inhibitors blocks PEN3–GFP accumulation at most penetration sites, but has a lesser effect on the recruitment of GFP–PEN1 to these sites (11), suggesting that transport pathways mediating PEN1 and PEN3 recruitment and export to the apoplastic papillae are distinct.Accumulation of dynamically moving cytoplasm near the pathogen penetration site is the most striking and microscopically visible early response in epidermal cells (2). The secretory vesicles and organelles, including peroxisomes, Golgi, mitochondria, and the nucleus, also move toward penetration sites (7, 13). In addition to the deposition of cell wall reinforcements and focal accumulation of penetration-related proteins such as PEN3, the accretion of cytoplasm and organelles at sites of attempted fungal penetration involves reorganization of actin cytoskeleton, which forms a radial array focused on penetration site (10, 11, 14–18). Consistent with this finding, disruption of AFs hampers penetration resistance, leading to increased penetration frequency by various fungal and oomycete pathogens (15–17, 19). However, the mechanisms that drive AF dynamics and active transport of cellular components toward sites of attempted pathogen penetration remain elusive. Myosins are molecular motors responsible for AF-based motility (20). Recently, plant class XI myosins were implicated in the organization of actin cytoskeleton, organelle and vesicle transport, cell expansion, and plant growth (21–27). Although none of the individual myosin gene knockouts produces plant growth defects (22), progressive elimination of two to four highly expressed myosins results in concomitant reduction in cell and plant size (23, 24). However, relatively little is known about the functions of myosins in plant–pathogen interactions (28, 29).Using pharmacological and genetic approaches to disrupt myosin function in Arabidopsis, we show that transient assembly and polarization of actin filament (AF) bundles toward the fungal penetration site are regulated by myosin motors. Furthermore, we demonstrate that plant myosins contribute to focal aggregation of a battery of cellular defense activities at the infection site and papillary deposition of cell wall appositions of lignin-like compounds, callose and PEN1, and are required for plant penetration resistance.