, Volume 250, Issue 6, pp 1897–1910 | Cite as

The Rpf84 gene, encoding a ribosomal large subunit protein, RPL22, regulates symbiotic nodulation in Robinia pseudoacacia

  • Zhao Feng
  • Lu Zhang
  • Yuanyuan Wu
  • Li Wang
  • Mingying Xu
  • Mo Yang
  • Yajuan Li
  • Gehong Wei
  • Minxia ChouEmail author
Original Article


Main conclusion

A homologue of the ribosomal protein L22e, Rpf84, regulates root nodule symbiosis by mediating the infection process of rhizobia and preventing bacteroids from degradation in Robinia pseudoacacia.


Ribosomal proteins (RPs) are known to have extraribosomal functions, including developmental regulation and stress responses; however, the effects of RPs on symbiotic nodulation of legumes are still unclear. Ribosomal protein 22 of the large 60S subunit (RPL22), a non-typical RP that is only found in eukaryotes, has been shown to function as a tumour suppressor in animals. Here, a homologue of RPL22, Rpf84, was identified from the leguminous tree R. pseudoacacia. Subcellular localization assays showed that Rpf84 was expressed in the cytoplasm and nucleus. Knockdown of Rpf84 by RNA interference (RNAi) technology impaired the infection process and nodule development. Compared with the control, root and stem length, dry weight and nodule number per plant were drastically decreased in Rpf84-RNAi plants. The numbers of root hair curlings, infection threads and nodule primordia were also significantly reduced. Ultrastructure analyses showed that Rpf84-RNAi nodules contained fewer infected cells with fewer bacteria. In particular, remarkable deformation of bacteroids and fusion of multiple symbiosomes occurred in infected cells. By contrast, overexpression of Rpf84 promoted nodulation, and the overexpression nodules maintained a larger infection/differentiation region and had more infected cells filled with bacteroids than the control at 45 days post inoculation, suggesting a retarded ageing process in nodules. These results indicate for the first time that RP regulates the symbiotic nodulation of legumes and that RPL22 may function in initiating the invasion of rhizobia and preventing bacteroids from degradation in R. pseudoacacia.


Black locust Infection process Nodule symbiosis Ribosomal protein Subcellular location Symbiosome 



Days post-inoculation


Green fluorescent protein




Infection thread




RNA interference


Ribosomal protein


Ribosomal large subunit protein


Transmission electron microscopy



This work was financially supported by the National Key Research and Development Program of China (Grant no. 2016YFD0800706), the National Natural Science Foundation of China (Grant no. 31172252) and the Shaanxi Province Natural Science Foundation of China (Grant no. 2016JM3004).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

425_2019_3267_MOESM1_ESM.pdf (182 kb)
Supplementary material 1 (PDF 181 kb)


  1. Antolin-Llovera M, Petutsching EK, Ried MK, Lipka V, Nurnberger T, Robatzek S, Parniske M (2014) Knowing your friends and foes–plant receptor-like kinases as initiators of symbiosis or defence. New Phytol 204:791–802PubMedPubMedCentralGoogle Scholar
  2. Barakat A, Szick-Miranda K, Chang IF, Guyot R, Blanc G, Cooke R, Delseny M, Bailey-Serres J (2001) The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome. Plant Physiol 127:398–415PubMedPubMedCentralGoogle Scholar
  3. Bloemberg GV, Wijfjes AHM, Lamers GEM, Stuurman N, Lugtenberg BJJ (2000) Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities. Mol Plant Microbe Interact 13:1170–1176PubMedPubMedCentralGoogle Scholar
  4. Boisson-Dernier A, Chabaud M, Garcia F, Becard G, Rosenberg C, Barker DG (2001) Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol Plant Microbe Interact 14:695–700PubMedPubMedCentralGoogle Scholar
  5. Breakspear A, Liu CW, Roy S et al (2014) The root hair “infectome” of Medicago truncatula uncovers changes in cell cycle genes and reveals a requirement for auxin signaling in rhizobial infection. Plant Cell 26:4680–4701PubMedPubMedCentralGoogle Scholar
  6. Byrne ME (2009) A role for the ribosome in development. Trends Plant Sci 14:512–519PubMedPubMedCentralGoogle Scholar
  7. Carroll AJ, Heazlewood JL, Ito J, Millar AH (2008) Analysis of the Arabidopsis cytosolic ribosome proteome provides detailed insights into its components and their post-translational modification. Mol Cell Proteom 7:347–369Google Scholar
  8. Carvalho CM, Santos AA, Pires SR, Rocha CS, Saraiva DI, Machado JPB, Mattos EC, Fietto LG, Fontes EPB (2008) Regulated nuclear trafficking of rpL10A mediated by NIK1 represents a defense strategy of plant cells against virus. PLoS Pathog 4:e1000247PubMedPubMedCentralGoogle Scholar
  9. Casati P, Walbot V (2003) Gene expression profiling in response to ultraviolet radiation in maize genotypes with varying flavonoid content. Plant Physiol 132:1739–1754PubMedPubMedCentralGoogle Scholar
  10. Chang IF, Szick-Miranda K, Pan SQ, Bailey-Serres J (2005) Proteomic characterization of evolutionarily conserved and variable proteins of arabidopsis cytosolic ribosomes. Plant Physiol 137:848–862PubMedPubMedCentralGoogle Scholar
  11. Chen HY, Chou MX, Wang XY, Liu SS, Zhang FL, Wei GH (2013) Profiling of differentially expressed genes in roots of Robinia pseudoacacia during nodule development using suppressive subtractive hybridization. PLoS One 8:e63930PubMedPubMedCentralGoogle Scholar
  12. Choi YL, Tsukasaki K, O’Neill MC et al (2007) A genomic analysis of adult T-cell leukemia. Oncogene 26:1245–1255Google Scholar
  13. Dobbelstein M, Shenk T (1995) In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein–Barr virus-expressed RNA by using randomized and cDNA-derived RNA libraries. J Virol 69:8027–8034PubMedPubMedCentralGoogle Scholar
  14. Fahl SP, Wang MS, Zhang Y, Duc ACE, Wiest DL (2015) Regulatory roles of rpl22 in hematopoiesis: an old dog with new tricks. Crit Rev Immunol 35:379–400PubMedPubMedCentralGoogle Scholar
  15. Fåhraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16:374–381Google Scholar
  16. Falcone Ferreyra ML, Pezza A, Biarc J, Burlingame AL, Casati P (2010) Plant L10 ribosomal proteins have different roles during development and translation under ultraviolet-B stress. Plant Physiol 153:1878–1894Google Scholar
  17. Falcone Ferreyra ML, Casadevall R, Luciani MD, Pezza A, Casati P (2013) New evidence for differential roles of L10 ribosomal proteins from Arabidopsis. Plant Physiol 163:378–391PubMedPubMedCentralGoogle Scholar
  18. Fournier J, Teillet A, Chabaud M, Ivanov S, Genre A, Limpens E, de Carvalho-Niebel F, Barker DG (2015) Remodeling of the infection chamber before infection thread formation reveals a two-step mechanism for rhizobial entry into the host legume root hair. Plant Physiol 167:1233–1242PubMedPubMedCentralGoogle Scholar
  19. Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13:89–102Google Scholar
  20. Horiguchi G, Molla-Morales A, Perez-Perez JM, Kojima K, Robles P, Ponce MR, Micol JL, Tsukaya H (2011) Differential contributions of ribosomal protein genes to Arabidopsis thaliana leaf development. Plant J 65:724–736PubMedPubMedCentralGoogle Scholar
  21. Houmani JL, Davis CI, Ruf IK (2009) Growth-promoting properties of Epstein–Barr virus EBER-1 RNA correlate with ribosomal protein L22 binding. J Virol 83:9844–9853PubMedPubMedCentralGoogle Scholar
  22. Kearse MG, Ireland JA, Prem SM, Chen AS, Ware VC (2013) RpL22e, but not RpL22e-like-PA, is SUMOylated and localizes to the nucleoplasm of Drosophila meiotic spermatocytes. Nucleus 4:241–258PubMedPubMedCentralGoogle Scholar
  23. Kim SJ, Strich R (2016) Rpl22 is required for IME1 mRNA translation and meiotic induction in S. cerevisiae. Cell Div 11:10PubMedPubMedCentralGoogle Scholar
  24. Kim KY, Park SW, Chung YS, Chung CH, Kim JI, Lee JH (2004) Molecular cloning of low-temperature-inducible ribosomal proteins from soybean. J Exp Bot 55:1153–1155Google Scholar
  25. Le SY, Sternglanz R, Greider CW (2000) Identification of two RNA-binding proteins associated with human telomerase RNA. Mol Biol Cell 11:999–1010PubMedPubMedCentralGoogle Scholar
  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408PubMedPubMedCentralGoogle Scholar
  27. Moin M, Bakshi A, Madhav MS, Kirti PB (2017) Expression profiling of ribosomal protein gene family in dehydration stress responses and characterization of transgenic rice plants overexpressing RPL23A for water-use efficiency and tolerance to drought and salt stresses. Front Chem 5:97PubMedPubMedCentralGoogle Scholar
  28. Nagaraj S, Senthil-Kumar M, Ramu VS, Wang KR, Mysore KS (2016) Plant ribosomal proteins, RPL12 and RPL19, play a role in nonhost disease resistance against bacterial pathogens. Front Plant Sci 6:1192PubMedPubMedCentralGoogle Scholar
  29. Ni JQ, Liu LP, Hess D, Rietdorf J, Sun FL (2006) Drosophila ribosomal proteins are associated with linker histone H1 and suppress gene transcription. Genes Dev 20:1959–1973PubMedPubMedCentralGoogle Scholar
  30. Nishimura R, Hayashi M, Wu GJ, Kouchi H, Imaizumi-Anraku H, Murakami Y, Kawasaki S, Akao S, Ohmori M, Nagasawa M, Harada K, Kawaguchi M (2002) HAR1 mediates systemic regulation of symbiotic organ development. Nature 420:426–429PubMedPubMedCentralGoogle Scholar
  31. Nishimura T, Wada T, Yamamoto KT, Okada K (2005) The Arabidopsis STV1 protein, responsible for translation reinitiation, is required for auxin-mediated gynoecium patterning. Plant Cell 17:2940–2953PubMedPubMedCentralGoogle Scholar
  32. Oldroyd GED (2013) Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 11:252–263Google Scholar
  33. O’Leary MN, Schreiber KH, Zhang Y et al (2013) The ribosomal protein Rpl22 controls ribosome composition by directly repressing expression of its own paralog, Rpl22l1. PLoS Genet 9:e1003708PubMedPubMedCentralGoogle Scholar
  34. Ouyang S, Zhu W, Hamilton J et al (2007) The TIGR rice genome annotation resource: improvements and new features. Nucleic Acids Res 35:D883–D887PubMedPubMedCentralGoogle Scholar
  35. Rao SY, Lee SY, Gutierrez A et al (2012) Inactivation of ribosomal protein L22 promotes transformation by induction of the stemness factor, Lin28B. Blood 120:3764–3773PubMedPubMedCentralGoogle Scholar
  36. Rosado A, Li RX, van de Ven W, Hsu E, Raikhel NV (2012) Arabidopsis ribosomal proteins control developmental programs through translational regulation of auxin response factors. Proc Natl Acad Sci USA 109:19537–19544PubMedPubMedCentralGoogle Scholar
  37. Roux B, Rodde N, Jardinaud MF et al (2014) An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing. Plant J 77:817–837PubMedPubMedCentralGoogle Scholar
  38. Roy S, Robson F, Lilley J et al (2017) MtLAX2, a functional homologue of the Arabidopsis auxin influx transporter AUX1, is required for nodule organogenesis. Plant Physiol 174:326–338PubMedPubMedCentralGoogle Scholar
  39. Rutkowski R, Hofmann K, Gartner A (2010) Phylogeny and function of the invertebrate p53 superfamily. Cold Spring Harb Perspect Biol 2:a001131PubMedPubMedCentralGoogle Scholar
  40. Savada RP, Bonham-Smith PC (2014) Differential transcript accumulation and subcellular localization of Arabidopsis ribosomal proteins. Plant Sci 223:134–145Google Scholar
  41. Solanki NR, Stadanlick JE, Zhang Y, Duc AC, Lee SY, Lauritsen JPH, Zhang ZQ, Wiest DL (2016) Rp122 loss selectively impairs αβ T cell development by dysregulating endoplasmic reticulum stress signaling. J Immunol 197:2280–2289PubMedPubMedCentralGoogle Scholar
  42. Sormani R, Masclaux-Daubresse C, Daniele-Vedele F, Chardon F (2011) Transcriptional regulation of ribosome components are determined by stress according to cellular compartments in Arabidopsis thaliana. PLoS One 6:e28070PubMedPubMedCentralGoogle Scholar
  43. Soyano T, Hirakawa H, Sato S, Hayashi M, Kawaguchi M (2014) NODULE INCEPTION creates a long-distance negative feedback loop involved in homeostatic regulation of nodule organ production. Proc Natl Acad Sci USA 111:14607–14612PubMedPubMedCentralGoogle Scholar
  44. Steffen KK, MacKay VL, Kerr EO et al (2008) Yeast life span extension by depletion of 60S ribosomal subunits is mediated by Gcn4. Cell 133:292–302PubMedPubMedCentralGoogle Scholar
  45. Steffen KK, McCormick MA, Pham KM, MacKay VL, Delaney JR, Murakami CJ, Kaeberlein M, Kennedy BK (2012) Ribosome deficiency protects against ER stress in Saccharomyces cerevisiae. Genetics 191:107–118PubMedPubMedCentralGoogle Scholar
  46. Suzaki T, Yano K, Ito M, Umehara Y, Suganuma N, Kawaguchi M (2012) Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus is accompanied by auxin response. Development 139:3997–4006PubMedPubMedCentralGoogle Scholar
  47. Szakonyi D, Byrne ME (2011) Ribosomal protein L27a is required for growth and patterning in Arabidopsis thaliana. Plant J 65:269–281PubMedPubMedCentralGoogle Scholar
  48. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729PubMedPubMedCentralGoogle Scholar
  49. Toczyski DP, Matera AG, Ward DC, Steitz JA (1994) The Epstein–Barr-Virus (EBV) small RNA EBER1 binds and relocalizes ribosomal rrotein L22 in EBV-infected human B-lymphocytes. Proc Natl Acad Sci USA 91:3463–3467PubMedPubMedCentralGoogle Scholar
  50. Vasse J, De Billy F, Camut S, Truchet G (1990) Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules. J Bacteriol 172:4295–4306PubMedPubMedCentralGoogle Scholar
  51. Voisin AS, Salon C, Jeudy C, Warembourg FR (2003) Symbiotic N2 fixation activity in relation to C economy of Pisum sativum L. as a function of plant phenology. J Exp Bot 54(393):2733–2744Google Scholar
  52. Wei GH, Chen WM, Zhu WF, Chen C, Young JPW, Bontemps C (2009) Invasive Robinia pseudoacacia in China is nodulated by Mesorhizobium and Sinorhizobium species that share similar nodulation genes with native American symbionts. FEMS Microbiol Ecol 68:320–328Google Scholar
  53. Xiao TT, Schilderink S, Moling S, Deinum EE, Kondorosi E, Franssen H, Kulikova O, Niebel A, Bisseling T (2014) Fate map of Medicago truncatula root nodules. Development 141:3517–3528Google Scholar
  54. Yang M, Sun H, He J, Wang H, Yu X, Ma L, Zhu C (2014) Interaction of ribosomal protein L22 with casein kinase 2alpha: a novel mechanism for understanding the biology of non-small cell lung cancer. Oncol Rep 32:139–144Google Scholar
  55. Zhang Y, Duc AC, Rao S, Sun XL, Bilbee AN, Rhodes M, Li Q, Kappes DJ, Rhodes J, Wiest DL (2013) Control of hematopoietic stem cell emergence by antagonistic functions of ribosomal protein paralogs. Dev Cell 24:411–425PubMedPubMedCentralGoogle Scholar
  56. Zhang Y, O’Leary MN, Peri S et al (2017) Ribosomal proteins Rpl22 and Rpl22l1 control morphogenesis by regulating pre-mRNA splicing. Cell Rep 18:545–556PubMedPubMedCentralGoogle Scholar
  57. Zheng M, Wang YH, Liu X et al (2016) The RICE MINUTE-LIKE1 (RML1) gene, encoding a ribosomal large subunit protein L3B, regulates leaf morphology and plant architecture in rice. J Exp Bot 67:3457–3469PubMedPubMedCentralGoogle Scholar
  58. Zhou F, Roy B, von Arnim AG (2010) Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3 h and the ribosomal protein RPL24. BMC Plant Biol 10:193PubMedPubMedCentralGoogle Scholar
  59. Zhou X, Liao WJ, Liao JM, Liao P, Lu H (2015) Ribosomal proteins: functions beyond the ribosome. J Mol Cell Biol 7:92–104PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Stress Biology in Arid Areas, Shaanxi Key Laboratory of Agricultural and Environmental Microbiology, College of Life SciencesNorthwest A&F UniversityYanglingChina
  2. 2.College of Medical TechnologyShaanxi University of Chinese MedicineXianyangChina

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