Endoplasmic reticulum-targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation

Protoplasma volume 251, pages817–826(2014)Cite this article

Abstract

The endoplasmic reticulum (ER) of the model legume Lotus japonicus was visualized using green fluorescent protein (GFP) fused with the KDEL sequence to investigate the changes in the root hair cortical ER in the presence or absence of Mesorhizobium loti using live fluorescence imaging. Uninoculated root hairs displayed dynamic forms of ER, ranging from a highly condensed form to an open reticulum. In the presence of M. loti, a highly dynamic condensed form of the ER linked with the nucleus was found in deformed, curled, and infected root hairs, similar to that in uninoculated and inoculated growing zone I and II root hairs. An open reticulum was primarily found in mature inoculated zone III root hairs, similar to that found in inactive deformed/curled root hairs and infected root hairs with aborted infection threads. Co-imaging of GFP-labeled ER with light transmission demonstrated a correlation between the mobility of the ER and other organelles and the directionality of the cytoplasmic streaming in root hairs in the early stages of infection thread formation and growth. ER remodeling in root hair cells is discussed in terms of possible biological significance during root hair growth, deformation/curling, and infection in the Mesorhizobium–L. japonicus symbiosis.

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Abbreviations

ER:

Endoplasmic reticulum

GFP:

Green fluorescent protein

IT:

Infection thread

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Acknowledgments

This work was supported by a Postdoctoral Fellowship (ID no. P05458) for Foreign Researchers from the Japanese Society for the Promotion of Science to F.M. P-W.

Conflict of interest

The authors have no conflict of interest.

Author information

Author notes

  1. F. M. Perrine-Walker

    Present address: La Trobe Institute of Molecular Sciences, La Trobe University, Melbourne, 3086, Australia

Affiliations

  1. Department of Life Science, Division of Natural Sciences, International Christian University, Mitaka, 181-8585, Tokyo, Japan

    F. M. Perrine-Walker, H. Kouchi & R.W. Ridge

Authors
  1. F. M. Perrine-Walker
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  2. H. Kouchi
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  3. R.W. Ridge
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Correspondence to F. M. Perrine-Walker.

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Handling Editor: Peter Nick

Electronic supplementary material

Movie S1

ER dynamics in an uninoculated growing zone I root hair of transgenic L. japonicus expressing GFP-KDEL. Composite time-lapse series of images overlaid in grayscale for light transmission showing ER dynamics (in green) in 4-day post-uninoculated L. japonicus root hair. The ER is in a condensed form. The images were acquired every 4 s and the movie consists of 20 frames. Bar, 15 μm (AVI 15.0 MB)

Movie S2

ER dynamics in a zone III root hair of transgenic L. japonicus expressing GFP-KDEL inoculated with M. loti strain TONO. Composite time-lapse series of images showing ER dynamics (in green) and the cytoplasmic streaming in light transmission in 6-day post-inoculated L. japonicus root hair. Zone III root hair did not deform in the presence of rhizobia. The ER has an open reticulate form consisting of tubules. The images were acquired every 4 s and the movie consists of 18 frames. White arrows highlight regions of dynamic ER remodeling of ER polygon networks by the formation/disintegration of tubules. Bar, 15 μm (AVI 12.5 MB)

MOVIE S3

ER dynamics in a deformed zone II root hair of transgenic L. japonicus expressing GFP-KDEL inoculated with M. loti strain TONO. Composite time-lapse series of images overlaid in grayscale for light transmission showing ER dynamics (in green) in 3-day post-inoculated L. japonicus root hair. The ER is in a condensed form. The images were acquired every 4 s and the movie consists of 18 frames. Bar, 15 μm (AVI 13.5 MB)

MOVIE S4

ER dynamics in a curled root hair of transgenic L. japonicus expressing GFP-KDEL inoculated with M. loti strain TONO. Composite time-lapse series of images overlaid in grayscale for light transmission showing ER dynamics (in green) in 3-day post-inoculated L. japonicus root hair. The long black arrow highlights the dynamics of the ER long tubules linked to the nucleus. White arrowheads show the dynamic fusion of the GFP-labeled region of the ER along cytoplasmic strands which eventually appear to join the ER long tubular network linked to the nucleus. The images were acquired every 4 s and the movie consists of 18 frames. n nucleus. Bar, 15 μm (AVI 13.5 MB)

MOVIE S5

ER dynamics in a non-active curled root hair of transgenic L. japonicus expressing GFP-KDEL inoculated with M. loti strain TONO. Composite time-lapse series of images showing ER dynamics (in green) and the cytoplasmic streaming in light transmission in 7-day post-inoculated L. japonicus root hair. The root hair curled due to the presence of rhizobia; however, the ER has an open reticulate form consisting of tubules. The images were acquired every 4 s and the movie consists of 33 frames. The first seven frames show dynamic changes of the open reticulate ER in one focal plane; the next 17 frames show the different arrangement of the ER at different focal planes (1- to 2-μm intervals), and the remaining frames show the ER remodeling of long tubular networks in one focal plane. Bar, 20 μm (AVI 17.4 MB)

MOVIE S6

ER dynamics in an infected curled root hair of transgenic L. japonicus expressing GFP-KDEL inoculated with M. loti strain TONO. Time-lapse series of images showing ER dynamics (in green) in a 5-day post-inoculated L. japonicus root hair. Images were acquired at different focal planes to capture the dynamic ER surrounding the IT in the curled root hair tip. The ER has a condensed form with large cisternae with small holes. The images were acquired every 4 s and the movie consists of 18 frames. The first six frames show dynamic changes of the condensed form of ER in one focal plane; the next 12 frames show the different arrangement of the ER at different focal planes (1- to 2-μm intervals) surrounding the IT. Green arrows in frame 14 show the IT. Bar, 15 μm (AVI 12.7 MB)

MOVIE S7

ER dynamics in an infected curled root hair of transgenic L. japonicus expressing GFP-KDEL inoculated with M. loti strain TONO. Time-lapse series of images showing ER dynamics (in green) in 5-day post-inoculated L. japonicus root hair shown in ESM Movie S6. Images were acquired in one focal plane to capture the dynamic ER surrounding the IT in the curled root hair tip, where white arrowheads show (a) a fusion event of the ER, (b) the formation and the disintegration of an ER tubule, and (c) the formation and the disintegration of the ER polygon network. The images were acquired every 4 s and the movie consists of 18 frames. Bar, 15 μm (AVI 12.7 MB)

MOVIE S8

ER dynamics surrounding the IT in an infected curled root hair of transgenic L. japonicus expressing GFP-KDEL inoculated with M. loti strain TONO. Time-lapse series of images showing ER dynamics (in green) in 6-day post-inoculated L. japonicus root hair. Images were acquired at different focal planes to capture the dynamic ER surrounding the IT and the nucleus near the base of the infected root hair. The ER has a condensed form projecting out from the IT and the nucleus (white arrowheads). The images were acquired every 4 s and the movie consists of 48 frames. White arrows show the IT location in the infected root hair surrounded by the GFP-labeled ER. Images in different focal planes were acquired manually. n nucleus. Bar, 15 μm (AVI 36.0 MB)

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Perrine-Walker, F.M., Kouchi, H. & Ridge, R. Endoplasmic reticulum-targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation. Protoplasma 251, 817–826 (2014). https://doi.org/10.1007/s00709-013-0584-x

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Keywords

  • Cortical endoplasmic reticulum
  • Rhizobia
  • Green fluorescent protein
  • Cytoplasmic streaming
  • Root hairs