Protoplasma

, Volume 251, Issue 5, pp 1099–1111 | Cite as

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

  • F. M. Perrine-Walker
  • M. Lartaud
  • H. Kouchi
  • R. W. Ridge
Original Article

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.

Keywords

Endoplasmic microtubule Cremart Rhizobia Tubulin Infection thread Green fluorescent protein 

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

Notes

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.

Supplementary material

709_2014_618_MOESM1_ESM.doc (38 kb)
ESM 1 (DOC 38 kb)
709_2014_618_Fig7_ESM.jpg (1.8 mb)
Fig. S1

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)

709_2014_618_MOESM2_ESM.eps (1.7 mb)
High Resolution Image (EPS 1,695 kb)
709_2014_618_Fig8_ESM.jpg (1.8 mb)
Fig. S2

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)

709_2014_618_MOESM3_ESM.eps (3.3 mb)
High Resolution Image (EPS 3,371 kb)
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)

709_2014_618_MOESM5_ESM.avi (15 mb)
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)

709_2014_618_MOESM7_ESM.avi (6.8 mb)
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)
709_2014_618_MOESM8_ESM.avi (9.9 mb)
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)

References

  1. Bergersen FJ (1961) The growth of rhizobia in synthetic media. Aust J Biol Sci 14:349–360Google Scholar
  2. Bibikova TN, Blancaflor EB, Gilroy S (1999) Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana. Plant J 17:657–666PubMedCrossRefGoogle Scholar
  3. Catoira R, Timmers AC, Maillet F, Galera C, Penmetsa RV, Cook D, Denarie J, Gough C (2001) The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling. Development 128(9):1507–1518PubMedGoogle Scholar
  4. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805PubMedCrossRefGoogle Scholar
  5. Davidson AL, Newcomb W (2001) Organization of microtubules in developing pea root nodule cells. Can J Bot 79:777–786Google Scholar
  6. Doonan JH, Cove DJ, Lloyd CW (1986) Microtubules and microfilaments in tip growth: evidence that microtubules impose polarity on protonemal growth in Physcomitrella patens. J Cell Sci 89:533–540Google Scholar
  7. Emons AMC, Mulder BM (2000) How the deposition of cellulose microfibrils builds cell wall architecture. Trends Plant Sci 5(1):35–40PubMedCrossRefGoogle Scholar
  8. Esseling JJ, Lhuissier FGP, Emons AMC (2003) Nod factor-induced root hair curling: continuous growth towards the point of Nod factor application. Plant Physiol 132:1982–1988PubMedCentralPubMedCrossRefGoogle Scholar
  9. Fahraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16:374–381PubMedCrossRefGoogle Scholar
  10. Fedorova EE, de Felipe MR, Pueyo JJ, Mercedes Lucas M (2007) Conformation of cytoskeletal elements during the division of infected Lupinus albus L. nodule cells. J Exp Bot 58(8):2225–2236PubMedCrossRefGoogle Scholar
  11. Fournier J, Timmers ACJ, Sieberer BJ, Jauneau A, Chabaud M, Barker DG (2008) Mechanism of infection thread elongation in root hairs of Medicago truncatula and dynamic interplay with associated rhizobial colonization. Plant Physiol 148:1985–1995PubMedCentralPubMedCrossRefGoogle Scholar
  12. Gage DJ (2004) Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiol Mol Biol Rev 68:280–300PubMedCentralPubMedCrossRefGoogle Scholar
  13. Haseloff J, Amos B (1995) GFP in plants. Trends Genet 11:328–329PubMedCrossRefGoogle Scholar
  14. Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci U S A 94:2122–2127PubMedCentralPubMedCrossRefGoogle Scholar
  15. Imaizumi-Anraku H, Kawaguchi M, Koiwa H, Akao S, Syono K (1997) Two ineffective-nodulating mutants of Lotus japonicus: different phenotypes caused by the blockage of endocytotic bacterial release and nodule maturation. Plant Cell Physiol 38:871–881Google Scholar
  16. Ketelaar T, de Ruijter NCA, Emons AMC (2003) Unstable F-actin specifies the area and microtubule direction of cell expansion in Arabidopsis root hairs. Plant Cell 15:285–292PubMedCentralPubMedCrossRefGoogle Scholar
  17. Lloyd CW, Wells B (1985) Microtubules are at the tips of root hairs and form helical pattern corresponding to inner wall fibrils. J Cell Sci 75:225–238PubMedGoogle Scholar
  18. Lloyd CW, Pearce KJ, Rawlins DJ, Ridge RW, Shaw PJ (1987) Endoplasmic microtubules connect the advancing nucleus to the tip of legume root hairs, but F-actin is involved in basipetal migration. Cell Motil Cytoskeleton 8:27–36CrossRefGoogle Scholar
  19. Miller DD, Leferink-ten Klooster HB, Emons AMC (2000) Lipochito-oligosaccharide nodulation factors stimulate cytoplasmic polarity with longitudinal endoplasmic reticulum and vesicles at the tip in vetch root hairs. MPMI 13:1385–1390PubMedCrossRefGoogle Scholar
  20. Niwa S, Kawaguchi M, Imaizumi-Anraku H, Chechetka SA, Ishizaka M, Ikuta A, Kouchi H (2001) Responses of a model legume Lotus japonicus to lipochitin oligosaccharide nodulation factors purified from Mesorhizobium loti JRL501. MPMI 14(7):848–856PubMedCrossRefGoogle Scholar
  21. Perrine-Walker FM, Kouchi H, Ridge RW (2014) Endoplasmic reticulum targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation. Protoplasma. doi: 10.1007/s00709-013-0584-x Google Scholar
  22. Ridge RW (1990) Cytochalasin-D causes abnormal wall-ingrowths and organelle-crowding in legume root hairs. Bot Mag Tokyo 103:87–96CrossRefGoogle Scholar
  23. Ridge RW (1992) A model of legume root hair growth and Rhizobium infection. Symbiosis 14:359–373Google Scholar
  24. Sieberer BJ, Timmers ACJ, Lhuissier FGP, Emons AMC (2002) Endoplasmic microtubules configure the subapical cytoplasm and are required for fast growth of Medicago truncatula root hairs. Plant Physiol 130:977–988PubMedCentralPubMedCrossRefGoogle Scholar
  25. Sieberer BJ, Ketelaar T, Esseling JJ, Emons AMC (2005a) Microtubules guide root hair tip growth. New Phytol 167:711–719PubMedCrossRefGoogle Scholar
  26. Sieberer BJ, Timmers ACJ, Emons AMC (2005b) Nod factors alter microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair reorientation. MPMI 18:1195–1204PubMedCrossRefGoogle Scholar
  27. Timmers ACJ (2008) The role of the plant cytoskeleton in the interaction between legumes and rhizobia. J Microsc 231(2):247–256PubMedCrossRefGoogle Scholar
  28. Timmers ACJ, Auriac M-C, de Billy F, Truchet G (1998) Nod factor internalization and microtubular cytoskeleton changes occur concomitantly during nodule differentiation in alfalfa. Development 125:339–349PubMedGoogle Scholar
  29. Timmers ACJ, Auriac M-C, Truchet G (1999) Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements. Development 126:3617–3628PubMedGoogle Scholar
  30. Timmers AC, Vallotton P, Heym C, Menzel D (2007) Microtubule dynamics in root hairs of Medicago truncatula. Eur J Cell Biol 86(2):69–83PubMedCrossRefGoogle Scholar
  31. Ueda M (1975) Cremart, new organophosphorus herbicide. Jpn Pestic Inf 23:23–25Google Scholar
  32. Ueda K, Matsuyama T, Hashimoto T (1999) Visualization of microtubules in living cells of transgenic Arabidopsis thaliana. Protoplasma 206:201–206CrossRefGoogle Scholar
  33. van Batenburg FHD, Jonker R, Kijne JW (1986) Rhizobium induces marked root hair curling by redirection of tip growth: a computer simulation. Physiol Plant 66:476–480CrossRefGoogle Scholar
  34. Van Bruaene N, Joss G, Van Oostveldt P (2004) Reorganization and in vivo dynamics of microtubules during Arabidopsis root hair development. Plant Physiol 136:3905–3919PubMedCentralPubMedCrossRefGoogle Scholar
  35. van Brussel AAN, Bakhuizen R, van Spronsen PC, Spaink HP, Tak T, Lugtenberg BJJ, Kijne JW (1992) Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipo-oligosaccharides of Rhizobium. Science 257(5066):70–72PubMedCrossRefGoogle Scholar
  36. van Spronsen P, Grønlund M, Bras CP, Spaink HP, Kijne JW (2001) Cell biological changes of outer cortical root cells in early determinate nodulation. MPMI 14(7):839–847PubMedCrossRefGoogle Scholar
  37. Vassileva VN, Kouchi H, Ridge RW (2005) Microtubule dynamics in living root hairs: transient slowing by lipochitin oligosaccharide nodulation signals. Plant Cell 17:1777–1787PubMedCentralPubMedCrossRefGoogle Scholar
  38. Wasteneys GO (2000) The cytoskeleton and growth polarity. Curr Opin Plant Biol 3:503–511PubMedCrossRefGoogle Scholar
  39. Wasteneys GO (2002) Microtubule organization in the green kingdom: chaos or self order? J Cell Sci 115:1345–1354PubMedGoogle Scholar
  40. Weerasinghe RR, Collings DA, Johannes E, Allen NS (2003) The distributional changes and role of microtubules in Nod factor-challenged Medigaco sativa root hairs. Planta 218:276–289PubMedCrossRefGoogle Scholar
  41. Weerasinghe RR, McK Bird D, Allen NS (2005) Root-knot nematodes and bacterial Nod factors elicit common transduction events in Lotus japonicus. Proc Natl Acad Sci U S A 102:3147–3152PubMedCentralPubMedCrossRefGoogle Scholar
  42. Yokota K, Fukai E, Madsen LH, Jurkiewicz A, Rueda P, Radutoiu S, Held M, Hossain Md S, Szczyglowski K, Morieri G, Olroyd GED, Downie JA, Nielsen MW, Rusek AM, Sato S, Tabata S, James EK, Oyaizu H, Sandal N, Stougaard J (2009) Rearrangement of actin cytoskeleton mediates invasion of Lotus japonicus roots by Mesorhizobium loti. Plant Cell 21:267–284PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • F. M. Perrine-Walker
    • 1
    • 3
  • M. Lartaud
    • 2
  • H. Kouchi
    • 1
  • R. W. Ridge
    • 1
  1. 1.Department of Life ScienceInternational Christian UniversityMitaka-shiJapan
  2. 2.Plate-forme d’Histocytologie et d’Imagerie Cellulaire Végétale, Centre International de Recherche en Agronomie pour le DéveloppementMontpellier Cedex 5France
  3. 3.La Trobe Institute of Molecular SciencesLa Trobe UniversityMelbourneAustralia

Personalised recommendations