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Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis

Abstract

Nuclear migration during infection thread (IT) development in root hairs is essential for legume-Rhizobium symbiosis. However, little is known about the relationships between IT formation, nuclear migration, and microtubule dynamics. To this aim, we used transgenic Lotus japonicus expressing a fusion of the green fluorescent protein and tubulin-α6 from Arabidopsis thaliana to visualize in vivo dynamics of cortical microtubules (CMT) and endoplasmic microtubules (EMTs) in root hairs in the presence or absence of Mesorhizobium loti inoculation. We also examined the effect of microtubule-depolymerizing herbicide, cremart, on IT initiation and growth, since cremart is known to inhibit nuclear migration. In live imaging studies of M. loti-treated L. japonicus root hairs, EMTs were found in deformed, curled, and infected root hairs. The continuous reorganization of the EMT array linked to the nucleus appeared to be essential for the reorientation, curling, and IT initiation and the growth of zone II root hairs which are susceptible to rhizobial infection. During IT initiation, the EMTs appeared to be linked to the root hair surface surrounding the M. loti microcolonies. During IT growth, EMTs dissociated from the curled root hair tip, remained linked to the nucleus, and appeared to surround the IT tip. Lack or disorganized EMT arrays that were no longer linked to the nucleus were observed only in infection-aborted root hairs. Cremart affected IT formation and nodulation in a concentration-dependent manner, suggesting that the microtubule (MT) organization and successive nuclear migration are essential for successful nodulation in L. japonicus by M. loti.

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Abbreviations

DMSO:

Dimethylsulphoxide

GFP:

Green fluorescent protein

IT(s):

Infection thread(s)

MT(s):

Microtubule(s)

CMT(s):

Cortical microtubule(s)

EMT(s):

Endoplasmic microtubule(s)

TUA6:

Tubulin-α6

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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 declare that they have no conflict of interest.

Author information

Correspondence to F. M. Perrine-Walker.

Additional information

Handling editor: Anne-Catherine Schmit

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1
figure7

Lack of EMT array in zone III root hair of uninoculated transgenic GFP-TUA6 L. japonicus. (a) Light transmission image of zone III root hair lacking the nucleus at the tip region; (c) Confocal image of same root hair highlighting the lack of an EMT array within the root hair tip region. Bars = 10 μm (JPEG 1,826 kb)

Fig. S2
figure8

Co-localization between EMT arrays and thick active cytoplasm in M. Loti inoculated transgenic GFP-TUA6 L. japonicus root hairs. (a) EMT array of curled root hair with nucleus near the tip; (b) Light transmission image of same root hair; (c) Composite image of EMT array (in green) and light transmission image of the same root hair; (d) EMT array of infected root hair with nucleus in close proximity to an infection thread; (e) Light transmission image of same infected root hair and (f) Composite image of EMT array (in green) and light transmission image of the same infected root hair. Note arrow heads show co-localization of GFP labeling to active thick cytoplasm between nucleus and root hair tip and infection thread tip respectively in root hairs. n, nucleus; IT, infection thread; Bars = 15 μm (JPEG 1,871 kb)

In vivo EMT dynamics in uninoculated Zone II root hair cells of transgenic L. japonicus expressing GFP-TUA6. Time-lapse series of images showing in vivo EMT dynamics in Zone II root hair cells of 4-d old transgenic L. japonicus seedlings. Bundled EMTs appeared linked the nucleus and other organelles. EMTs were present within the endoplasm of the root hair (sub-apical region). Blue arrow in Zone II root hair cell numbered 1 highlights changes in bundled EMTs which disappeared after 53 frames. Green arrow in Zone II root hair cell numbered 2 highlights changes in bundled EMTs linked to the nucleus which disappeared after 24 frames. Red arrowhead in Zone II root hair cell numbered 2 highlights changes in EMTs within the endoplasm between frames number 19 to 38. Yellow and red arrows in Zone II root hair cell numbered 3 highlight the changes in EMTs which appeared linked to a vacuole. Images of the root hairs were acquired every 4 seconds and the movie consists of 50 frames. n, nucleus; Bars = 10 μm (AVI 27,772 kb)

In vivo EMT dynamics in a deformed root hair cell of transgenic L. japonicus expressing GFP-TUA6 inoculated with M. loti in an early stage of root hair curling. Time-lapse series of images showing in vivo EMT dynamics (α-tubulin; in green) and the active cytoplasm in the deforming root hair tip (light transmission/grayscale) in 4-d post inoculated transgenic L. japonicus root hair cell at early stage of root hair curling. Bundled EMTs linked to the bending focus near the tip extended to the curling region of the root hair. The images were acquired every 4 seconds and the movie consists of 18 frames. GFP labeling co-localized to the active cytoplasm and its motility. The composite stack images of the root hair were acquired every 4 seconds and the movie consists of 18 frames. Bars = 15 μm (AVI 1,407 kb)

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Movie S1

In vivo EMT dynamics in uninoculated Zone II root hair cells of transgenic L. japonicus expressing GFP-TUA6. Time-lapse series of images showing in vivo EMT dynamics in Zone II root hair cells of 4-d old transgenic L. japonicus seedlings. Bundled EMTs appeared linked the nucleus and other organelles. EMTs were present within the endoplasm of the root hair (sub-apical region). Blue arrow in Zone II root hair cell numbered 1 highlights changes in bundled EMTs which disappeared after 53 frames. Green arrow in Zone II root hair cell numbered 2 highlights changes in bundled EMTs linked to the nucleus which disappeared after 24 frames. Red arrowhead in Zone II root hair cell numbered 2 highlights changes in EMTs within the endoplasm between frames number 19 to 38. Yellow and red arrows in Zone II root hair cell numbered 3 highlight the changes in EMTs which appeared linked to a vacuole. Images of the root hairs were acquired every 4 seconds and the movie consists of 50 frames. n, nucleus; Bars = 10 μm (AVI 27,772 kb)

Movie S2

In vivo EMT dynamics in a deformed root hair cell of transgenic L. japonicus expressing GFP-TUA6 inoculated with M. loti in an early stage of root hair curling. Time-lapse series of images showing in vivo EMT dynamics in 3-d post inoculated transgenic L. japonicus root hair cell at early stage of root hair curling. Bundled EMTs linked the nucleus to the root hair tip. Images of the root hairs were acquired every 4 seconds and the movie consists of 20 frames. False-color Green Fire Blue was used to show EMTs in green and outline of the root hair cell in blue. n, nucleus; Bars = 10 μm (AVI 15,364 kb)

Movie S3

In vivo EMT dynamics in a deformed root hair cell of transgenic L. japonicus expressing GFP-TUA6 inoculated with M. loti in an early stage of root hair curling. Time-lapse series of images showing in vivo EMT dynamics (α-tubulin; in green) and the active cytoplasm in the deforming root hair tip (light transmission/grayscale) in 4-d post inoculated transgenic L. japonicus root hair cell at early stage of root hair curling. Bundled EMTs linked to the bending focus near the tip extended to the curling region of the root hair. The images were acquired every 4 seconds and the movie consists of 18 frames. GFP labeling co-localized to the active cytoplasm and its motility. The composite stack images of the root hair were acquired every 4 seconds and the movie consists of 18 frames. Bars = 15 μm (AVI 1,407 kb)

Movie S4

In vivo EMT dynamics in a deformed root hair cell of transgenic L. japonicus expressing GFP-TUA6 inoculated with M. loti in an early stage of root hair curling. Time-lapse series of images showing in vivo EMT dynamics in 3-d post inoculated L. japonicus root hair in a more advanced stage of root hair curling. Bundled EMTs linked to the nucleus and the bending focus near the root hair tip, display dynamic stability and instability in the curling region of the tip. Some EMTs curved following the cell membrane and once at the root hair tip retracted towards the nucleus. The images were acquired every 4 seconds and the movie consists of 19 frames. False-color Green Fire Blue was used to show EMTs in green and outline of the root hair cell in blue. N, nucleus; Bars = 5 μm (AVI 7,003 kb)

Movie S5

In vivo EMT dynamics in an infected root hair of transgenic L. japonicus expressing GFP-TUA6 inoculated with M. loti strain TONO. Time-lapse Z series of images showing in vivo EMT dynamics in 4-d post inoculated L. japonicus root hairs. Bundled EMTs linked to the nucleus, extend toward the infection thread surrounding the IT tip. The images were acquired every 4 seconds and the movie consists of 16 frames. False-color Green Fire Blue was used to show EMTs in green and outline of the root hair cell in blue. Bars = 5 μm (AVI 10,110 kb)

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Perrine-Walker, F.M., Lartaud, M., Kouchi, H. et al. Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis. Protoplasma 251, 1099–1111 (2014). https://doi.org/10.1007/s00709-014-0618-z

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Keywords

  • Endoplasmic microtubule
  • Cremart
  • Rhizobia
  • Tubulin
  • Infection thread
  • Green fluorescent protein