Plant Growth Regulation

, Volume 85, Issue 2, pp 231–242 | Cite as

Both epiphytic and endophytic strains of Rhodococcus fascians influence transporter gene expression and cytokinins in infected Pisum sativum L. seedlings

  • Pragatheswari Dhandapani
  • Jiancheng Song
  • Ondrej Novak
  • Paula E. JamesonEmail author
Original paper


Some strains of the soil bacterium Rhodococcus fascians maintain an epiphytic life style while others become endophytic. Virulent, endophytic strains cause multiple shoot growth and inhibit root growth of seed-inoculated Pisum sativum L. We were interested in assessing, at the molecular level, the impact of strains of contrasting niche on the emerging shoots and roots of inoculated seeds. The presence of R. fascians was monitored microscopically, endogenous cytokinin and chlorophyll levels were measured, and the expression of genes monitored by RT-qPCR. The expression of the pea sugar transporter genes (SWEET and SUT), amino acid (AAP) transporters and cell wall invertase gene family members, as well as expression of plant and bacterial cytokinin biosynthesis (IPT), activation (LOG) and degradation (CKX) genes were monitored. Both the virulent strain and the epiphytic strain affected the expression of the transporter genes, with less obvious differences between the strains on the shoot compared with the effect on the root. Strong expression of the R. fascians genes, RfIPT, RfLOG and RfCKX, in pea seedlings at 15 days post inoculation was mirrored by increased expression of transporter gene family members in the plant. However, the elevated levels of isopentenyl adenine-type and zeatin-type cytokinins were not consistently associated with the virulent strain. In conclusion, while both the virulent strain and the epiphytic strain impacted the expression of transporter genes in the shoots and roots, only the virulent strain affected morphology. The inhibited root growth, the greening of the roots, and the expression of the pea response regulators in the infected roots are indicative of a response to cytokinin, but a role for the ‘classical’ cytokinins as virulence determinants was not established.


Amino acid transporter Cell wall invertase Cytokinin Pea Rhodococcus fascians Sucrose transporter Sugar Will Eventually be Exported Transporter (SWEET) 



A UC scholarship to PD is gratefully acknowledged. Thanks to Graeme Bull, Jan McKenzie and Neil Andrews for assistance with microscopy, and to the anonymous referees for their constructive comments. O.N. was funded by the Czech Science Foundation (Nr. 17-06613S).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Antoniadi I, Plačková L, Simonovik B, Doležal K, Turnbull C, Ljung K, Novák O (2015) Cell-type-specific cytokinin distribution within the Arabidopsis primary root apex. Plant Cell 27:1955–1967CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bezrutczyk M, Yang J, Eom J-S et al (2018) Sugar flux and signalling in plant-microbe interactions. Plant J. PubMedGoogle Scholar
  3. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefPubMedGoogle Scholar
  4. Carletom HM, Druvy RAB (1957) Histological technique: for normal pathological tissues and the identification of parasites. Oxford University Press, LondonGoogle Scholar
  5. Chen L-Q (2014) SWEET sugar transporters for phloem transport and pathogen nutrition. New Phytol 201(4):1150–1155CrossRefPubMedGoogle Scholar
  6. Chen H-Y, Huh J-H, Yu Y-Chi, Ho L-H, Chen L-Q, Tholl D, Frommer WB, Guo W-J (2015) The Arabidopsis vacuolar sugar transporter SWEET2 limits carbon sequestration from roots and restricts Pythium infection. Plant J 83:1046–1058CrossRefPubMedGoogle Scholar
  7. Cortleven A, Schmülling T (2015) Regulation of chloroplast development and function by cytokinin. J Exp Bot 66:4999–5013CrossRefPubMedGoogle Scholar
  8. Cortleven A, Marg I, Yamburenko MV, Schlicke H, Hill K, Grimm B, Schaller GE, Schmülling T (2016) Cytokinin regulates etioplast-chloroplast transition through activation of chloroplast-related genes. Plant Physiol 172:464–478CrossRefPubMedPubMedCentralGoogle Scholar
  9. Creason AL, Vandeputte OM, Savory EA, Davis EW II, Putnam ML, Hu E, Swader-Hines D, Mol A, Baucher M, Prinsen E, Zdanowska M, Givan SA, Jaziri ME, Loper JE, Mahmud T, Chang JH (2014) Analysis of genome sequences from plant pathogenic Rhodococcus reveals genetic novelties in virulence loci. PLoS ONE 9(7):e101996. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Crespi M, Messens E, Caplan AB, Van Montagu M, Desomer J (1992) Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene. EMBO J 11:795–804PubMedPubMedCentralGoogle Scholar
  11. Crespi M, Vereecke D, Temmerman W, Van Montagu M, Desomer J (1994) The fas operon of Rhodococcus fascians encodes new genes required for efficient fasciation of host plants. J Bacteriol 176:2492–2501CrossRefPubMedPubMedCentralGoogle Scholar
  12. Depuydt S, Doležal K, Van Lijsebettens M, Moritz T, Holsters M, Vereecke D (2008) Modulation of the hormone setting by Rhodococcus fascians results in ectopic KNOX activation in Arabidopsis. Plant Physiol 146:1267–1281CrossRefPubMedPubMedCentralGoogle Scholar
  13. Depuydt S, Trenkamp S, Fernie AR, Elftieh S, Renou J-P, Vuylsteke M, Holsters M, Vereecke D (2009) An integrated genomics approach to define niche establishment by Rhodococcus fascians. Plant Physiol 149:1366–1386CrossRefPubMedPubMedCentralGoogle Scholar
  14. Dhandapani P (2014) Rhodococcus fascians-plant interactions: microbiological and molecular aspects. Unpublished PhD thesis, University of Canterbury, Christchurch, New ZealandGoogle Scholar
  15. Dhandapani P, Song J, Novak O, Jameson PE (2017) Infection by Rhodococcus fascians maintains cotyledons as a sink tissue for the pathogen. Ann Bot 119:841–852PubMedGoogle Scholar
  16. Eason JR, Jameson PE, Bannister P (1995) Virulence assessment of Rhodococcus fascians strains on pea cultivars. Plant Pathol 44:141–147Google Scholar
  17. Eason JR, Morris RO, Jameson PE (1996) The relationship between virulence and cytokinin production by Rhodococcus fascians (Tilford 1936) Goodfellow 1984. Plant Pathol 45:323–331CrossRefGoogle Scholar
  18. Evans T, Song J, Jameson PE (2012) Micro-scale chlorophyll analysis and developmental expression of a cytokinin oxidase/dehydrogenase gene during leaf development and senescence. Plant Growth Regul 66:95–99CrossRefGoogle Scholar
  19. Francis IM, Stes E, Zhang Y, Rangel D, Audenaert K, Vereecke D (2016) Mining the genome of Rhodococcus fascians, a plant growth-promoting bacterium gone astray. New Biotechnol. Google Scholar
  20. Gális I, Bilyeu K, Wood G, Jameson PE (2005a) Rhodococcus fascians: shoot proliferation without elevated cytokinins? Plant Growth Regul 46:109–115CrossRefGoogle Scholar
  21. Gális I, Bilyeu KD, Godinho MJG, Jameson PE (2005b) Expression of three Arabidopsis cytokinin oxidase/dehydrogenase promoter::GUS chimeric constructs in tobacco: response to developmental and biotic factors. Plant Growth Regul 45:173–182CrossRefGoogle Scholar
  22. Guo Q, Love J, Song J, Roche J, Turnbull MH, Jameson PE (2017) Insights into the functional relationship between cytokinin-induced root system phenotypes and nitrate uptake in Brassica napus L. Funct Plant Biol 44:832–844CrossRefGoogle Scholar
  23. Hwang I, Sheen J, Muller B (2012) Cytokinin signaling networks. Annu Rev Plant Biol 63:353–380CrossRefPubMedGoogle Scholar
  24. Jameson PE (2000) Cytokinins and auxins in plant-pathogen interactions—an overview. Plant Growth Regul 32:369–380CrossRefGoogle Scholar
  25. Jameson PE, Song J (2016) Cytokinin: a key driver of seed yield. J Exp Bot 67:593–606CrossRefPubMedGoogle Scholar
  26. Jameson PE, Dhandapani P, Novak O, Song J (2016) Cytokinins and expression of SWEET, SUT, CWINV and AAP genes increase as pea seeds germinate. Int J Mol Sci 17:2013. CrossRefPubMedCentralGoogle Scholar
  27. Kobayashi K, Ohnishi A, Sasaki D, Fujii S, Iwase A, Sugimoto K, Masuda T, Wada H (2017) Shoot removal induces chloroplast development in roots via cytokinin signalling. Plant Physiol 173:2340–2355CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kurakawa T, Ueda N, Maekawa M, Kobayashi K, Kojima M, Nagato Y, Sakakibara H, Kyozuka J (2007) Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445:652–655CrossRefPubMedGoogle Scholar
  29. Kuroha T, Tokunaga H, Kojima M, Ishida T, Nagawa S, Fukuda H, Sugimoto K, Sakakibara H (2009) Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation pathway in Arabidopsis. Plant Cell 21:3152–3169CrossRefPubMedPubMedCentralGoogle Scholar
  30. Lawson E, Gantotti B, Starr M (1982) A 78-megadalton plasmid occurs in avirulent strains as well as virulent strains of Corynebacterium fascians. Curr Microbiol 7:327–332CrossRefGoogle Scholar
  31. Matsubara S, Armstrong DJ, Skoog F (1968) Cytokinins in tRNA of Corynebacterium fascians. Plant Physiol 43:451–453CrossRefPubMedPubMedCentralGoogle Scholar
  32. Morris RO (1987) Molecular aspects of hormone synthesis and action genes specifying auxin and cytokinin biosynthesis in prokaryotes. In: Davies PJ (ed) Plant hormones. Kluwer Academic Publishers, Dordrecht, pp 318–339Google Scholar
  33. Pertry I, Vaclavikova K, Depuydt S, Galuszka P, Spichal L, Temmerman W, Stes E, Schmulling T, Kakimoto T, Van Montagu MCE, Strnad M, Holsters M, Tarkowski P, Vereecke D (2009) Identification of Rhodococcus fascians cytokinins and their modus operandi to reshape the plant. Proc Natl Acad Sci 106(3):929–934CrossRefPubMedPubMedCentralGoogle Scholar
  34. Pertry I, Václavíková K, Gemrotová M, Spíchal L, Galuszka P, Depuydt S, Temmerman W, Stes E, De Keyser A, Riefler M, Biondi S, Novák O, Schmülling T, Strnad M, Tarkowski P, Holsters M, Vereecke D (2010) Rhodococcus fascians impacts plant development through the dynamic fas-mediated production of a cytokinin mix. Mol Plant Microbe Interact 23:1164–1174CrossRefPubMedGoogle Scholar
  35. Radhika V, Ueda N, Tsuboi Y, Kojima M, Kikuchi J, Kudo K, Sakakibara H (2015) Methylated cytokinins from the phytopathogen Rhodococcus fascians mimic plant hormone activity. Plant Physiol 169:1118–1126CrossRefPubMedPubMedCentralGoogle Scholar
  36. Savory EA et al (2017) Evolutionary transitions between beneficial and phytopathogenic Rhodococcus challenge disease management. eLIFE. PubMedPubMedCentralGoogle Scholar
  37. Song J, Jiang L, Jameson PE (2012) Co-ordinate regulation of cytokinin gene family members during flag leaf and reproductive development in wheat. BMC Plant Biol 12:78CrossRefPubMedPubMedCentralGoogle Scholar
  38. Stange RR, Jeffares D, Young C, Scott DB, Eason JR, Jameson PE (1996) PCR amplification of the fas-1 gene for the detection of virulent strains of Rhodococcus fascians. Plant Pathol 45:407–417CrossRefGoogle Scholar
  39. Svačinová J, Novák O, Plačková L, Lenobel R, Holík J, Strnad M, Doležal K (2012) A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction. Plant Methods 18:17CrossRefGoogle Scholar
  40. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefPubMedGoogle Scholar
  41. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res 25:4876–4882CrossRefPubMedPubMedCentralGoogle Scholar
  42. Vereecke D, Burssens S, Simón-Mateo C et al (2000) The Rhodococcus fascians-plant interaction: morphological traits and biotechnological applications. Planta 210:241–251CrossRefPubMedGoogle Scholar
  43. Vereecke D, Temmerman W, Jaziri M, Holsters M, Goethals K (2003) Towards an understanding of the Rhodococcus fascians-plant interaction. In: Stacey G, Kean N (eds) Molecular plant microbe interactions, vol 6. American Phytopathological Society, St. PaulGoogle Scholar
  44. Werner T, Motyka V, Laucou V et al (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Pragatheswari Dhandapani
    • 1
  • Jiancheng Song
    • 1
    • 2
  • Ondrej Novak
    • 3
  • Paula E. Jameson
    • 1
    Email author
  1. 1.School of Biological SciencesUniversity of CanterburyChristchurchNew Zealand
  2. 2.School of Life SciencesYantai UniversityYantaiChina
  3. 3.Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural ResearchInstitute of Experimental Botany CAS & Faculty of Science of Palacký UniversityOlomoucCzech Republic

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