Threat alert: Actin enzyme inhibition mediates innate immune signaling in Arabidopsis

Research Areas:

Plant biology, Arabidopsis thaliana, Actin, Cytoskeletal remodeling, Innate immune response, Green fluorescent protein

Imaging Needs:

High spatial and temporal resolution

Imaging System (VAEM/TIRF):
  • Green fluorescent protein GFP-fABD2
  • Olympus IX-71 microscope
  • X60 1.45-numerical aperture PlanApo TIRF objective lens
  • Illumination: 488nm solid-state 50-mW laser (Intelligent Imaging Innovations) at 10% power
  • Hamamatsu ImagEM C9100-12 EM-CCD
  • Intelligent Imaging Innovations Slidebook software and Fiji software
Imaging System (epifluorescence microscopy):
  • Nikon E600 microscope
  • 100-W Hg lamp
  • x40 0.75–numerical aperture PlanFluor objective lens
  • 360/340-nm filter
  • Hamamatsu ORCA-ER CCD camera
  • Metamorph software
Fine resolution, wide view

Learn out how the Hamamatsu ImagEM camera's enhanced low-light capability, precise localization and wide field of view of help Ashley Cadby and colleagues to visualize the synthesis of cell walls in living bacteria. Read now.

THE QUESTION

How do cells activate a response to pathogen detection?

Plant and animal cells recognize potential invaders in part by detecting highly conserved markers, called microbe-associated molecular patterns (MAMPs), on the surfaces of bacteria, fungi and other pathogens. Cells exposed to MAMPs respond with changes in lipid content, cell wall fortification, phagocytosis, and more, as part of the innate immune response. All of these actions involve alterations in the cytoskeleton—specifically affecting actin dynamics. However, the intracellular mechanisms linking pathogen detection and cytoskeletal remodeling are not well understood.

THE BARRIERS

The cytoskeletal response to innate immune signaling has been associated with destabilization of actin bundles and increased availability of actin filaments. Hundreds of actin binding proteins regulate the assembly and disassembly of actin filaments in eukaryotes. When these proteins are inhibited, plants become more susceptible to pathogens.1 Might actin binding proteins help to mediate the cellular response to pathogen detection?

THE SOLUTION

Acting Depolymerizing Factor4 Regulates Actin Dynamics during Innate Immune Signaling in Arabidopsis
Jessica L. Henty-Ridilla, Jiejie Li, Brad Day, and Christopher J. Staiger
The Plant Cell. Jan 2014; 26(2): 340–352. PMCID: PMC3963580.

To study the regulation of actin remodeling during the innate immune response, Henty-Ridilla et al.2 observed living epidermal cells from the model plant Arabidopsis thaliana after exposure to fungal and bacterial MAMPs. The team measured changes in actin arrays in cells expressing the actin reporter green fluorescent protein (GFP)-fABD2. The team used variable-angle epifluorescence microscopy (VAEM) with a Hamamatsu ImagEM C9100-12 EM-CCD camera to obtain high contrast, low background images. Within minutes of MAMP exposure, the cortical cytoskeleton showed a dose- and time-dependent increase in actin filament availability.

The authors zoomed in to examine changes in single actin filaments using time-lapse VAEM microscopy. Five minutes following exposure to the bacterial MAMP elf26, they saw a significant change in actin filament origination, together with an increase in length and lifespan, and decreased severing. The net result: increased filament abundance. This response mimicked the appearance of mutant cells lacking the gene for actin depolymerizing factor4 (ADF4). The authors propose that the cellular response to elf26 might involve inhibition or downregulation of ADF4 activity. Consistent with this idea, ADF4 knockout cells show a reduced response to elf26, but reacted with the normal increase in actin filament abundance in response to another related MAMP. The attenuated reaction to elf26 went beyond actin regulation; ADF4 knockout cells also showed decreased cell wall fortification after exposure to bacterial (but not fungal) MAMP, as revealed by epifluorescence microscopy using the Hamamatsu ORCA-ER camera.

The authors conclude that multiple, separate signaling pathways mediate different MAMP responses. Moreover, they suggest that multiple actin binding proteins may be involved even in the specific response to the bacterial MAMP elf26, as part of that response remains intact in ADF4 mutant. This study is the first to link an ADF to pathogen perception in eukaryotes.

THE POSSIBILITIES

Henty-Ridilla et al. used Hamamatsu’s ORCA-ER and ImagEM C9100-12 cameras to quantify rapid changes in actin bundles and single actin filaments in living plant cells. Learn how researchers pinpoint features as small as 35–42 nanometers within a wide area of the living cell—read Exciting Insights into Cell Growth.

References

  1. Henty-Ridilla, et al. The plant actin cytoskeleton responds to signals from microbe-associated molecular patterns. PLoS Pathog Apr 2013; 9(4): e1003290. PMCID: PMC3616984.
  2. Henty-Ridilla JL et al. Actin Depolymerizing Factor4 Regulates Actin Dynamics during Innate Immune Signaling in Arabidopsis. The Plant Cell Jan 2014; 26: 340–352. PMCID: PMC3963580.
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