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

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 MesorhizobiumL. japonicus symbiosis.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Abbreviations

ER:

Endoplasmic reticulum

GFP:

Green fluorescent protein

IT:

Infection thread

References

  1. Bergersen FJ (1961) The growth of rhizobia in synthetic media. Aust J Biol Sci 14:349–360

    CAS  Google Scholar 

  2. Bhuvaneswari TV, Turgeon BG, Bauer WD (1980) Early events in the infection of soybean (Glycine max L. Merr) by Rhizobium japonicum I. Localization of infectible root cells. Plant Physiol 66(6):1027–1031

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Boevink P, Cruz SS, Hawes C, Harris N, Oparka KJ (1996) Virus-mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plant cells. Plant J 10:935–941

    CAS  Article  Google Scholar 

  4. Boevink P, Oparka K, Cruz SS, Martin B, Betteridge A, Hawes C (1998) Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J 15:441–447

    CAS  Article  PubMed  Google Scholar 

  5. Brandizzi F, Irons SL, Johansen J, Kotzer A, Neumann U (2004) GFP is the way to glow: bioimaging of the plant endomembrane system. J Microsc 214(2):138–158

    CAS  Article  PubMed  Google Scholar 

  6. Bush DS (1995) Calcium regulation in plant cells and its role in signalling. Annu Rev Plant Physiol Plant Mol Biol 46:95–122

    CAS  Article  Google Scholar 

  7. Callaham DA, Torrey JG (1981) The structural basis for infection of root hairs of Trifolium repens by Rhizobium. Can J Bot 59:1647–1664

    Article  Google Scholar 

  8. Cárdenas L, Holdaway-Clarke TL, Sánchez F, Quinto C, Feijó JA, Kunkel JG, Hepler PK (2000) Ion changes in legume root hairs responding to Nod factors. Plant Physiol 123:443–451

    Article  PubMed  PubMed Central  Google Scholar 

  9. Chen J, Doyle C, Qi X, Zheng H (2012) The endoplasmic reticulum: a social network in plant cells. J Integr Plant Biol 54(11):840–850

    CAS  PubMed  Google Scholar 

  10. Essl D, Dirnberger D, Gomord V, Strasser R, Faye L, Gloessl J, Steinkellner H (1999) The N-terminal 77 amino acids from tobacco N-acetylglucosaminyltransferase I are sufficient to retain a reporter protein in the Golgi apparatus of Nicotiana benthamiana cells. FEBS Lett 453:169–173

    CAS  Article  PubMed  Google Scholar 

  11. Fahraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16:374–381

  12. Farquhar ML, Peterson RL (1996) Root hairs: specialized tubular cells extending root surfaces. Bot Rev 62(1):1–40

    Article  Google Scholar 

  13. 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 148:1985–1995

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Gage DJ (2004) Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiol Mol Biol Rev 68(2):280–300

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Genre A, Bonfante P (2007) Check-in procedures for plant entry by biotrophic microbes. Mol Plant Microbe Interact 20(9):1023–1030

    CAS  Article  PubMed  Google Scholar 

  16. Genre A, Chabaud M, Timmers T, Bonfante P, Barker DG (2005) Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection. Plant Cell 17:3489–3499

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Genre A, Chabaud M, Faccio A, Barker DG, Bonfante P (2008) Prepenetration apparatus assembly preceded and predicts the colonization patterns of arbuscular mycorrhizal fungi within the root cortex of both Medicago truncatula and Daucus carota. Plant Cell 20:1407–1420

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Griffing LR (2010) Networking in the endoplasmic reticulum. Biochem Soc Trans 38:747–753

    CAS  Article  PubMed  Google Scholar 

  19. Hardham AR, Daigo Takemoto D, White RG (2008) Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack. BMC Plant Biol 8:63

    Article  PubMed  PubMed Central  Google Scholar 

  20. 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 94:2122–2127

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Hawes C, Saint-Jore C, Martin B, Zheng HJ (2001) ER confirmed as the location of mystery organelles in Arabidopsis plants expressing GFP. Trends Plant Sci 6:245–246

    CAS  Article  PubMed  Google Scholar 

  22. Handberg K, Stougaard J (1992) Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J 2(4):487–496

  23. 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 38:871–881

  24. Irons SL, Evans DE, Brandizzi F (2003) The first 238 amino acids of the human lamin B receptor are targeted to the nuclear envelope in plants. J Exp Bot 54:943–950

    CAS  Article  PubMed  Google Scholar 

  25. Lipka V, Panstruga R (2005) Dynamic cellular responses in plant–microbe interactions. Curr Opin Plant Biol 8(6):625–631

    CAS  Article  PubMed  Google Scholar 

  26. Matsushima R, Hayashi Y, Kondo M, Shimada T, Nishimura M, Hara-Nishimura I (2002) An endoplasmic reticulum-derived structure that is induced under stress conditions in Arabidopsis. Plant Physiol 130(4):1807–1814

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Matsushima R, Kondo M, Nishimura M, Hara-Nishimura I (2003) A novel ER-derived compartment, the ER body, selectively accumulates a beta-glucosidase with an ER-retention signal in Arabidopsis. Plant J 33(3):493–502

    CAS  Article  PubMed  Google Scholar 

  28. Meusser B, Hirsch C, Jarosch E, Sommer T (2005) ERAD: The long road to destruction. Nat Cell Biol 7:766–772

  29. Nebenführ A, Gallagher LA, Dunahay TG, Frohlick JA, Mazurkiewicz AM, Meehl JB, Staehelin LA (1999) Stop-and-go movements of the plant Golgi stacks are mediated by the acto-myosin system. Plant Physiol 121:1127–1141

    Article  PubMed  PubMed Central  Google Scholar 

  30. Pagny S, Lerouge P, Faye L, Gomord V (1999) Signals and mechanisms for protein retention in the endoplasmic reticulum. J Exp Bot 50(331):157–164

    CAS  Article  Google Scholar 

  31. Pagny S, Cabanes-Macheteau M, Gillikin JW, Leborgne-Castel N, Lerouge P, Boston RS, Faye L, Gomord V (2000) Protein recycling from the Golgi apparatus to the endoplasmic reticulum in plants and its minor contribution to calreticulin retention. Plant Cell 12(5):739–756

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Pagny S, Bouissonnie F, Sarkar M, Follet-Gueye ML, Driouich A, Schachter H, Faye L, Gomord V (2003) Structural requirements for Arabidopsis beta 1,2 xylosyltransferase activity and targeting to the Golgi. Plant J 33(1):189–203

    CAS  Article  PubMed  Google Scholar 

  33. Pickard WE (2003) The role of cytoplasmic streaming in symplastic transport. Plant Cell Environ 26:1–15

    CAS  Article  Google Scholar 

  34. Quader H (1990) Formation and disintegration of cisternae of the endoplasmic reticulum visualized in live cells by conventional fluorescence and confocal laser scanning microscopy: evidence for the involvement of calcium and the cytoskeleton. Protoplasma 155(1–3):166–175

    Article  Google Scholar 

  35. Quader H, Hofmann A, Schnepf E (1989) Reorganization of the endoplasmic reticulum in epidermal cells of onion bulb scales after cold stress: involvement of cytoskeletal elements. Planta 177(2):273–280

    CAS  Article  PubMed  Google Scholar 

  36. Ridge RW (1988) Freeze-substitution improves the ultrastructural presenvation of legume root hairs. Bot Mag Tokyo 101:427–441

    Article  Google Scholar 

  37. Ridge RW (1993) A model of legume root hair-growth and Rhizobium infection. Symbiosis 14(1–3):359–373

  38. Ridge RW (1995) Micro-vesicles, pyriform vesicles and macro-vesicles associated with the plasma membrane in the root hairs of Vicia hirsuta after freeze-substitution. J Plant Res 108:363–368

    Article  Google Scholar 

  39. Ridge RW, Uozumi Y, Plazinski J, Hurley UA, Williamson RE (1999) Developmental transitions and dynamics of the cortical ER of Arabidopsis cells seen with green fluorescent protein. Plant Cell Physiol 40(1):1253–1261

    CAS  Article  PubMed  Google Scholar 

  40. Roth LE, Stacey G (1989) Bacterium release into host cells of nitrogen-fixing soybean nodules: the symbiosome membrane comes from three sources. Eur J Cell Biol 49:13–23

    CAS  PubMed  Google Scholar 

  41. Staehelin LA (1997) The plant ER: a dynamic organelle composed of a large number of discrete functional domains. Plant J 11(6):1151–1165

    CAS  Article  PubMed  Google Scholar 

  42. Stiller J, Martirani L, Tuppale S, Chian R-J, Chiurazzi M, Gresshoff PM (1997) High frequency transformation and regeneration of transgenic plants in the model legume Lotus japonicus. J Exp Bot 48:1357–1365

  43. Takemoto D, Jones DA, Hardham AR (2003) GFP-tagging of cell components reveals the dynamics of subcellular re-organization in response to infection of Arabidopsis by oomycete pathogens. Plant J 2003(33):775–792

    Article  Google Scholar 

  44. Vassileva VN, Kouchi H, Ridge RW (2005) Microtubule dynamics in living root hairs: transient slowing by lipochitin oligosaccharide nodulation signals. Plant Cell 17:1777–1787

  45. Verchot-Lubicz J, Goldstein RE (2010) Cytoplasmic streaming enables the distribution of molecules and vesicles in large plant cells. Protoplasma 240:99–107

    Article  PubMed  Google Scholar 

  46. Wee EG-T, Sherrier DJ, Prime TA, Dupree P (1998) Targeting of active sialyltransferase to the plant Golgi apparatus. Plant Cell 10:1759–1768

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

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

Affiliations

Authors

Corresponding author

Correspondence to F. M. Perrine-Walker.

Additional information

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Keywords

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