Jiejie Li

The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.

Capping Protein Modulates the Dynamic Behavior of Actin Filaments in Response to Phosphatidic Acid in Arabidopsis

Capping protein (CP) binds to the fast-growing ends of actin filaments and is negatively regulated by signaling lipids in vitro. This study demonstrates that the end-capping activity of CP is inhibited by membrane signaling phospholipids in living epidermal cells from Arabidopsis thaliana and that CP remodels the actin cytoskeleton in response to changes in these phospholipids. Remodeling of actin filament arrays in response to biotic and abiotic stimuli is thought to require precise control over the generation and availability of filament ends. Heterodimeric capping protein (CP) is an abundant filament capper, and its activity is inhibited by membrane signaling phospholipids in vitro. How exactly CP modulates the properties of filament ends in cells and whether its activity is coordinated by phospholipids in vivo is not well understood. By observing directly the dynamic behavior of individual filament ends in the cortical array of living Arabidopsis thaliana epidermal cells, we dissected the contribution of CP to actin organization and dynamics in response to the signaling phospholipid, phosphatidic acid (PA). Here, we examined three cp knockdown mutants and found that reduced CP levels resulted in more dynamic activity at filament ends, and this significantly enhanced filament-filament annealing and filament elongation from free ends. The cp mutants also exhibited more dense actin filament arrays. Treatment of wild-type cells with exogenous PA phenocopied the actin-based defects in cp mutants, with an increase in the density of filament arrays and enhanced annealing frequency. These cytoskeletal responses to exogenous PA were completely abrogated in cp mutants. Our data provide compelling genetic evidence that the end-capping activity of CP is inhibited by membrane signaling lipids in eukaryotic cells. Specifically, CP acts as a PA biosensor and key transducer of fluxes in membrane signaling phospholipids into changes in actin cytoskeleton dynamics.

ACTIN DEPOLYMERIZING FACTOR4 Regulates Actin Dynamics during Innate Immune Signaling in Arabidopsis

Disruption of the host-cell actin cytoskeleton promotes the susceptibility of plants to diverse microbes. However, the signaling cascades and regulatory proteins linking the perception of microbes to cytoskeletal remodeling remain largely uncharacterized. This study implicates a key actin binding protein, ACTIN DEPOLYMERIZING FACTOR4, in plant innate immune signaling. Conserved microbe-associated molecular patterns (MAMPs) are sensed by pattern recognition receptors (PRRs) on cells of plants and animals. MAMP perception typically triggers rearrangements to actin cytoskeletal arrays during innate immune signaling. However, the signaling cascades linking PRR activation by MAMPs to cytoskeleton remodeling are not well characterized. Here, we developed a system to dissect, at high spatial and temporal resolution, the regulation of actin dynamics during innate immune signaling in plant cells. Within minutes of MAMP perception, we detected changes to single actin filament turnover in epidermal cells treated with bacterial and fungal MAMPs. These MAMP-induced alterations phenocopied an ACTIN DEPOLYMERIZING FACTOR4 (ADF4) knockout mutant. Moreover, actin arrays in the adf4 mutant were unresponsive to a bacterial MAMP, elf26, but responded normally to the fungal MAMP, chitin. Together, our data provide strong genetic and cytological evidence for the inhibition of ADF activity regulating actin remodeling during innate immune signaling. This work is the first to directly link an ADF/cofilin to the cytoskeletal rearrangements elicited directly after pathogen perception in plant or mammalian cells.

Regulation of cytoskeletal dynamics by phospholipase D and phosphatidic acid

Plants respond to diverse biotic and abiotic stimuli as well as to endogenous developmental cues. Many of these stimuli result in altered activity of phospholipase D (PLD), an enzyme that hydrolyzes structural phospholipids producing phosphatidic acid (PA). PA is a key signaling intermediate in animals, but its targets in plants are relatively uncharacterized. Recent studies have demonstrated that the cytoskeleton is a major target of PLD-PA signaling and identified a positive feedback loop between actin turnover and PLD activity. Moreover, two cytoskeletal proteins, capping protein and MAP65-1, have been identified as PA-binding proteins regulating actin and microtubule organization and dynamics. In this review, we highlight the role of the PLD-PA module as an important hub for housekeeping and stress-induced regulation of membrane-associated cytoskeletal dynamics.

Actin dynamics in the cortical array of plant cells.

The actin cytoskeleton changes in organization and dynamics as cellular functions are reprogrammed following responses to diverse stimuli, hormones, and developmental cues. How this is choreographed and what molecular players are involved in actin remodeling continues to be an area of intense scrutiny. Advances in imaging modalities and fluorescent fusion protein reporters have illuminated the strikingly dynamic behavior of single actin filaments at high spatial and temporal resolutions. This led to a model for the stochastic dynamic turnover of actin filaments and predicted the actions and responsibilities of several key actin-binding proteins. Recently, aspects of this model have been tested using powerful genetic strategies in both Arabidopsis and Physcomitrella. Collectively, the latest data emphasize the importance of filament severing activities and regulation of barbed-end availability as key facets of plant actin filament turnover.

Signaling to Actin Stochastic Dynamics

Advances in microscopy techniques applied to living cells have dramatically transformed our view of the actin cytoskeleton as a framework for cellular processes. Conventional fluorescence imaging and static analyses are useful for quantifying cellular architecture and the network of filaments that support vesicle trafficking, organelle movement, and response to biotic stress. However, new imaging techniques have revealed remarkably dynamic features of individual actin filaments and the mechanisms that underpin their construction and turnover. In this review, we briefly summarize knowledge about actin and actin-binding proteins in plant systems. We focus on the quantitative properties of the turnover of individual actin filaments, highlight actin-binding proteins that participate in actin dynamics, and summarize the current genetic evidence that has been used to dissect specific aspects of the stochastic dynamics model. Finally, we describe some signaling pathways in which recent data implicate changes in actin filament dynamics and the associated cytoplasmic responses.

Capping protein integrates multiple MAMP signalling pathways to modulate actin dynamics during plant innate immunity

Plants and animals perceive diverse microbe-associated molecular patterns (MAMPs) via pattern recognition receptors and activate innate immune signalling. The actin cytoskeleton has been suggested as a target for innate immune signalling and a key transducer of cellular responses. However, the molecular mechanisms underlying actin remodelling and the precise functions of these rearrangements during innate immunity remain largely unknown. Here we demonstrate rapid actin remodelling in response to several distinct MAMP signalling pathways in plant epidermal cells. The regulation of actin dynamics is a convergence point for basal defence machinery, such as cell wall fortification and transcriptional reprogramming. Our quantitative analyses of actin dynamics and genetic studies reveal that MAMP-stimulated actin remodelling is due to the inhibition of capping protein (CP) by the signalling lipid, phosphatidic acid. In addition, CP promotes resistance against bacterial and fungal phytopathogens. These findings demonstrate that CP is a central target for the plant innate immune response.

Plk1‐dependent microtubule dynamics promotes androgen receptor signaling in prostate cancer

The androgen receptor (AR) signaling continues to be essential in castrate‐resistant prostate cancer (CRPC). Taxel‐based chemotherapy is the current standard treatment for CRPC patients. Unfortunately, almost all patients eventually develop resistance toward this chemotherapy. Significantly, it was recently found that the anti‐tumor effect of paclitaxel in CRPC is due to its inhibition of AR activity via its inhibition of microtubule dynamics. Polo‐like kinase 1 (Plk1), a critical regulator in many cell cycle events, is elevated in prostate cancer (PCa) and linked to tumor grades. Of note, we have previously shown that Plk1 phosphorylates CLIP‐170 and p150Glued, two important regulators of microtubule dynamics.

The availability of filament ends modulates actin stochastic dynamics in live plant cells

To test the role of filament barbed ends in actin dynamics, a system is established to modulate the levels of capping protein (CP) genetically. CP levels correlate with changes in actin array architecture, availability of filament ends, and axial cell expansion.

Capping Protein Modulates Actin Remodeling in Response to ROS during Plant Innate Immunity

Actin filament capping protein transduces ROS signaling into changes of actin dynamics during plant innate immunity. Plants perceive microbe-associated molecular patterns and damage-associated molecular patterns to activate innate immune signaling events, such as bursts of reactive oxygen species (ROS). The actin cytoskeleton remodels during the first 5 min of innate immune signaling in Arabidopsis (Arabidopsis thaliana) epidermal cells; however, the immune signals that impinge on actin cytoskeleton and its response regulators remain largely unknown. Here, we demonstrate that rapid actin remodeling upon elicitation with diverse microbe-associated molecular patterns and damage-associated molecular patterns represent a conserved plant immune response. Actin remodeling requires ROS generated by the defense-associated NADPH oxidase, RBOHD. Moreover, perception of flg22 by its cognate receptor complex triggers actin remodeling through the activation of RBOHD-dependent ROS production. Our genetic studies reveal that the ubiquitous heterodimeric capping protein transduces ROS signaling to the actin cytoskeleton during innate immunity. Additionally, we uncover a negative feedback loop between actin remodeling and flg22-induced ROS production.