, Volume 28, Issue 1, pp 69–80 | Cite as

Inducible expression of magnesium protoporphyrin chelatase subunit I (CHLI)-amiRNA provides insights into cucumber mosaic virus Y satellite RNA-induced chlorosis symptoms

  • Sachin Ashok Bhor
  • Chika Tateda
  • Tomofumi Mochizuki
  • Ken-Taro Sekine
  • Takashi Yaeno
  • Naoto Yamaoka
  • Masamichi Nishiguchi
  • Kappei KobayashiEmail author
Original Article


Recent studies with Y satellite RNA (Y-sat) of cucumber mosaic virus have demonstrated that Y-sat modifies the disease symptoms in specific host plants through the silencing of the magnesium protoporphyrin chelatase I subunit (CHLI), which is directed by the Y-sat derived siRNA. Along with the development of peculiar yellow phenotypes, a drastic decrease in CHLI-transcripts and a higher accumulation of Y-sat derived siRNA were observed. To investigate the molecular mechanisms underlying the Y-sat—induced chlorosis, especially whether or not the reduced expression of CHLI causes the chlorosis simply through the reduced production of chlorophyll or it triggers some other mechanisms leading to the chlorosis, we have established a new experimental system with an inducible silencing mechanism. This system involves the expression of artificial microRNAs targeting of Nicotiana tabacum CHLI gene under the control of chemically inducible promoter. The CHLI mRNA levels and total chlorophyll content decreased significantly in 2 days, enabling us to analyze early events in induced chlorosis and temporary changes therein. This study revealed that the silencing of CHLI did not only result in the decreased chlorophyll content but also lead to the downregulation of chloroplast and photosynthesis-related genes expression and the upregulation of defense-related genes. Based on these results, we propose that the reduced expression of CHLI could activate unidentified signaling pathways that lead plants to chlorosis.


Chlorosis Artificial microRNA CHLI Inducible silencing Satellite RNA 



Authors thank Kazue Obara for technical assistance. This study was supported in part by The United Graduate School of Agricultural Sciences, Ehime University, and JSPS KAKENHI Grants 26292026 and 15K14664 to Kobayashi. Bhor has been supported by Rotary Yoneyama Memorial Foundation for doctoral studies.

Supplementary material

13337_2017_360_MOESM1_ESM.docx (214 kb)
Supplementary material 1 (DOCX 213 kb)


  1. 1.
    Almon E, Horowitz M, Wang HL, Lucas WJ, Zamski E, Wolf S. Phloem-specific expression of the Tobacco mosaic virus movement protein alters carbon metabolism and partitioning in transgenic potato plants. Plant Physiol. 1997;115:1599–607.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Aoyama T, Chua NH. A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 1997;11:605–12.CrossRefPubMedGoogle Scholar
  3. 3.
    Balachandran S, Osmond CB, Daley PF. Diagnosis of the earliest strain-specific interactions between Tobacco mosaic virus and chloroplasts of tobacco leaves in vivo by means of chlorophyll fluorescence imaging. Plant Physiol. 1994;104:1059–65.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Bhat S, Folimonova SY, Cole AB, Ballard KD, Lei Z, Watson BS, Sumner LW, Nelson RS. Influence of host chloroplast proteins on Tobacco mosaic virus accumulation and intercellular movement. Plant Physiol. 2012;161:134–47.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bhor SA, Tateda C, Mochizuki T, Sekine KT, Takashi Y, Yamaoka Y, Nishiguchi M, Kobayashi K. Inducible transgenic tobacco system to study the mechanisms underlying chlorosis mediated by the silencing of chloroplast heat shock protein 90. VirusDis. 2017. doi: 10.1007/s13337-017-0361-0.
  6. 6.
    Biondi S, Scaramagli S, Capitani F, Altamura MM, Torrigiani P. Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers. J Exp Bot. 2001;52:231–42.CrossRefPubMedGoogle Scholar
  7. 7.
    Christov I, Stefanov D, Velinov T, Goltsev V, Georgieva K, Abracheva P, Genova Y, Christov N. The symptomless leaf infection with Grapevine leaf roll associated virus 3 in grown in vitro plants as a simple model system for investigation of viral effects on photosynthesis. J Plant Physiol. 2007;164:1124–33.CrossRefPubMedGoogle Scholar
  8. 8.
    Dardick C. Comparative expression profiling of Nicotiana benthamiana leaves systemically infected with three fruit tree viruses. Mol Plant Microbe Interact. 2007;20:1004–17.CrossRefPubMedGoogle Scholar
  9. 9.
    Guo DP, Guo YP, Zhao JP, Liu H, Peng Y, Wang QM, Chen JS, Rao GZ. Photosynthetic rate and chlorophyll fluorescence in leaves of stem mustard (Brassica juncea var. tsatsai) after turnip mosaic virus infection. Plant Sci. 2005;168:57–63.CrossRefGoogle Scholar
  10. 10.
    Herbers K, Takahata Y, Melzer M, Mock HP, Hajirezaei M, Sonnewald U. Regulation of carbohydrate partitioning during the interaction of Potato virus Y with tobacco. Mol Plant Pathol. 2000;1:51–9.CrossRefPubMedGoogle Scholar
  11. 11.
    Herrera-Vasquez A, Salinas P, Holuigue L. Salicylic acid and reactive oxygen species interplay in the transcriptional control of defense genes expression. Front Plant Sci. 2015;. doi: 10.3389/fpls.2015.00171.PubMedPubMedCentralGoogle Scholar
  12. 12.
    Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eichholtz D, Rogers SG. A simple method for transferring genes into plants. Science. 1985;227:1229–31.CrossRefGoogle Scholar
  13. 13.
    Hu CC, Hsu YH, Lin NS. Satellite RNAs and satellite viruses of plants. Viruses. 2009;1:1325–50.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Inoue H, Li M, Schnell DJ. An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts. Proc Natl Acad Sci USA. 2013;110(8):3173–8.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Johanningmeier U, Howell SH. Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi. J Biol Chem. 1984;259(21):13541–9.PubMedGoogle Scholar
  16. 16.
    Kyseláková H, Prokopová J, Nauš J, Novák O, Navrátil M, Šafářová D, Špundová M, Ilik P. Photosynthetic alterations of pea leaves infected systemically by Pea enation mosaic virus: a coordinated decrease in efficiencies of CO2 assimilation and photosystem II photochemistry. Plant Physiol Biochem. 2011;49:1279–89.CrossRefPubMedGoogle Scholar
  17. 17.
    Lehto K, Tikkanen M, Hiriart J-B, Paakkarinen V, Aro E-M. Depletion of the photosystem II core complex in mature tobacco leaves infected by the flavum strain of Tobacco mosaic virus. Mol Plant Microbe Interact. 2003;16:1135–44.CrossRefPubMedGoogle Scholar
  18. 18.
    Luo T, Fan T, Liu Y, Rothbart M, Yu J, Zhou S, Grimm B, Luo M. Thioredoxin redox regulates ATPase activity of magnesium chelatase CHLI subunit and modulates redox-mediated signaling in tetrapyrrole biosynthesis and homeostasis of reactive oxygen species in pea plants. Plant Physiol. 2012;159:118–30.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Manfre A, Glenn M, Nuñez A, Moreau RA, Dardick C. Light quantity and photosystem function mediate host susceptibility to Turnip mosaic virus via a salicylic acid-independent mechanism. Mol Plant Microbe Interact. 2011;24:315–27.CrossRefPubMedGoogle Scholar
  20. 20.
    Mochizuki N, Brusslan JA, Larkin R, Nagatani A, Chory J. Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction. Proc Natl Acad Sci USA. 2001;98:2053–8.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Mochizuki T, Ogata Y, Hirata Y, Ohki ST. Quantitative transcriptional changes associated with chlorosis severity in mosaic leaves of tobacco plants infected with Cucumber mosaic virus. Mol Plant Pathol. 2014;15:242–54.CrossRefPubMedGoogle Scholar
  22. 22.
    Navarro B, Gisel A, Rodio ME, Delgado S, Flores R, Di Serio F. Small RNAs containing the pathogenic determinant of a chloroplast- replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing. Plant J. 2012;70:991–1003.CrossRefPubMedGoogle Scholar
  23. 23.
    Olesinski AA, Almon E, Navot N, Perl A, Galun E, Lucas WJ, Wolf S. Tissue-specific expression of the Tobacco mosaic virus movement protein in transgenic potato plants alters plasmodesmal function and carbohydrate partitioning. Plant Physiol. 1996;111:541–50.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ossowski S, Schwab R, Weigel D. Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J. 2008;53:674–90.CrossRefPubMedGoogle Scholar
  25. 25.
    Otulak K, Chouda M, Bujarski J, Garbaczewska G. The evidence of Tobacco rattle virus impact on host plant organelles ultrastructure. Micron. 2015;70:7–20.CrossRefPubMedGoogle Scholar
  26. 26.
    Papenbrock J, Pfündel E, Mock HP, Grimm B. Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants. Plant J. 2000;22:155–64.CrossRefPubMedGoogle Scholar
  27. 27.
    Porra RJ, Thompson WA, Kriedemann PE. Determination of accurate extinction coefficients and simultaneous-equations for assaying chlorophyll a and chlorophyll b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic-absorption spectroscopy. Biochim Biophys Acta. 1989;975:384–94.CrossRefGoogle Scholar
  28. 28.
    Qin J, Zuo K, Zhao J, Ling H, Cao Y, Qiu C, Li F, Sun X, Tang K. Overexpression of GbERF confers alteration of ethylene-responsive gene expression and enhanced resistance to Pseudomonas syringae in transgenic tobacco. J Biosci. 2006;31:255–63.CrossRefPubMedGoogle Scholar
  29. 29.
    Rahoutei J, García-Luque I, Barón M. Inhibition of photosynthesis by viral infection: effect on PSII structure and function. Physiol Plant. 2000;110:286–92.CrossRefGoogle Scholar
  30. 30.
    Rissler HM, Collakova E, DellaPenna D, Whelan J, Pogson BJ. Chlorophyll biosynthesis. Expression of a second Chl I gene of magnesium chelatase in Arabidopsis supports only limited chlorophyll synthesis. Plant Physiol. 2002;128:770–9.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Sawers RJH, Viney J, Farmer PR, Bussey RR, Olsefski G, Anufrikova K, Hunter N, Brutnell TP. The maize Oil Yellow1 (Oy1) gene encodes the I subunit of magnesium chelatase. Plant Mol Biol. 2006;60:95–106.CrossRefPubMedGoogle Scholar
  32. 32.
    Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell. 2006;18:1121–33.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Shi Y, Chen J, Hong X, Chen J, Adams MJ. A potyvirus P1 protein interacts with the Rieske Fe/S protein of its host. Mol Plant Pathol. 2007;8:785–90.CrossRefPubMedGoogle Scholar
  34. 34.
    Shimura H, Pantaleo V, Ishihara T, Myojo N, Inaba J-I, Sueda K, Burgyán J, Chikara M. A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery. PLoS Pathog. 2011;7:1–12.CrossRefGoogle Scholar
  35. 35.
    Smith NA, Eamens AL, Wang MB. Viral small interfering RNAs target host genes to mediate disease symptoms in plants. PLoS Pathog. 2011;7:1–9.Google Scholar
  36. 36.
    Strand Å, Asami T, Alonso J, Ecker JR, Chory J. Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrin IX. Nature. 2003;5:79–83.CrossRefGoogle Scholar
  37. 37.
    Strand Å. Plastid-to-nucleus signalling. Curr Opin Plant Biol. 2004;7:621–5.CrossRefPubMedGoogle Scholar
  38. 38.
    Surpin M, Larkin RM, Chory J. Signal transduction between the chloroplast and the nucleus. Plant Cell. 2002;14:S327–38.PubMedPubMedCentralGoogle Scholar
  39. 39.
    Susek RE, Ausubel FM, Chory J. Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development. Cell. 1993;74:787–99.CrossRefPubMedGoogle Scholar
  40. 40.
    Tripathy BC, Oelmüller R. Reactive oxygen species generation and signaling in plants. Plant Signal Behav. 2012;7:1621–33.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Waliullah S, Kosaka N, Yaeno T, Ali ME, Sekine K-T, Atsumi G, Yamaoka N, Nishiguchi M, Takahashi H, Kobayashi K. Cauliflower mosaic virus Tav protein induces leaf chlorosis in transgenic tobacco through a host response to virulence function of Tav. J Gen Plant Pathol. 2015;81:261–70.CrossRefGoogle Scholar
  42. 42.
    Waliullah S, Mochizuki T, Sekine K-T, Atsumi G, Ali ME, Yaeno T, Yamaoka N, Nishiguchi M, Kobayashi K. Artificial induction of a plant virus protein in transgenic tobacco provides a synchronous system for analyzing the process of leaf chlorosis. Physiol Mol Plant Pathol. 2014;88:43–51.CrossRefGoogle Scholar
  43. 43.
    Yang J, Zhang F, Li J, Chen JP, Zhang HM. Integrative analysis of the microRNAome and transcriptome illuminates the response of susceptible rice plants to Rice stripe virus. PLoS ONE. 2016;11:1–21.Google Scholar
  44. 44.
    Zhao J, Zhang X, Hong Y, Liu Y. Chloroplast in plant-virus interaction. Front Microbiol. 2016;7:1–20.Google Scholar

Copyright information

© Indian Virological Society 2017

Authors and Affiliations

  1. 1.The United Graduate School of Agricultural SciencesEhime UniversityMatsuyamaJapan
  2. 2.Iwate Biotechnology Research CenterKitakamiJapan
  3. 3.Graduate School of Life and Environmental SciencesOsaka Prefecture UniversitySakaiJapan
  4. 4.Faculty of AgricultureUniversity of the RyukyusNakagamiJapan
  5. 5.Faculty of AgricultureEhime UniversityMatsuyamaJapan
  6. 6.Research Unit for CitromicsEhime UniversityMatsuyamaJapan

Personalised recommendations