Skip to main content
Log in

Transgenic Tea Over-expressing Solanum tuberosum Endo-1,3-beta-d-glucanase Gene Conferred Resistance Against Blister Blight Disease

  • Original Paper
  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript

Abstract

Tea (Camellia sinensis [L.] O. Kuntze) plant, one of the most important plantation crops in the world, is infected by a fungus called Exobasidium vexans leading to dreaded blister blight disease. The disease may result in crop losses up to 35% which directly affect the tea industry. Solanum tuberosum endo-1,3-beta-d-glucanase was cloned into tea genome via Agrobacterium-mediated transformation. The transformation event initially gave 32 kanamycin-resistant plantlets, out of which PCR analysis confirmed only 10 plantlets about the integration of transgene in the plant genome. Real-time PCR study detected transgene expression in six transgenic plantlets. Upregulation of endogenous C. sinensis pathogenesis-related (PR) genes like PR3 (chitinase I) gene and PR5 (thaumatin-like protein) gene also occurred in transgenic plantlets. Detached leaf infection assay showed resistance to E. vexans in greenhouse-acclimated transgenic plantlets. An inhibitory activity against E. vexans was noticed on the detached leaves of transgenic plantlets compared to control. Transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area unlike the formation of fungal lesion on control plantlet. Thus, it can be inferred that constitutive expression of the potato endo-1,3-beta-d-glucanase gene can be a strategy to produce blister blight-resistant tea.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Anand A, Zhou T, Trick HN, Gill BZ, Bockus WW, Muthukrishnan S (2003) Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin like protein, chitinase and glucanase against Fusarium graminearum. J Exp Bot 54(384):1101–1111. https://doi.org/10.1093/jxb/erg110

    Article  CAS  PubMed  Google Scholar 

  • Atkinson NJ, Urwin PE (2012) The interaction of plant biotic and abiotic stresses: from genes to the field. J Exp Bot 63(10:3523–3543

    Article  Google Scholar 

  • Balasubramanian V, Vashisht D, Cletus J, Sakthivel N (2012) Plant β-1,3-glucanases: their biological functions and transgenic expression against phytopathogenic fungi. Biotechnol Lett 34(11):1983–1990. https://doi.org/10.1007/s10529-012-1012-6

    Article  CAS  PubMed  Google Scholar 

  • Beffa RS, Neuhaus JM, Meins F Jr (1993) Physiological compensation in antisense transformants: specific induction of an “ersatz” glucan endo-1, 3-beta-glucosidase in plants infected with necrotizing viruses. Proc Natl Acad Sci USA 90(19):8792–8796. https://doi.org/10.1073/pnas.90.19.8792

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Begam J, Sharma GD (2015) Comparative impact of physico- chemical and nutritional parameters on some phytopathogenic fungi isolated from the phyllosphere of diseased tea leaf (Camellia sinensis L. O. Kuntze). Indian J Appl Res 5(1):235–238

    Google Scholar 

  • Berrocal-Lobo M, Molina A, Solano R (2002) Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi. Plant J 29(1):23–32. https://doi.org/10.1046/j.1365-313x.2002.01191.x

    Article  CAS  PubMed  Google Scholar 

  • Borkowoska M, Krzymowska M, Talarczyk A, Awan MF, Yakovleva L, Kleczkowski K, Wielgat B (1998) Transgenic potato plants expressing soybean beta-1, 3-endoglucanase gene exhibit an increased resistance to Phytophthora infestans. Z Naturforsch 53:1012–1016

    Google Scholar 

  • Bradeen JM, Iorizzo M, Mollov DS, Raasch J, Colton Kramer L, Millett BP, Austin-Phillips S, Jiang J, Carputo D (2009) Higher copy numbers of the potato RB transgene correspond to enhanced transcript and late blight resistance levels. Mol Plant Microb Interact 22(4):437–446. https://doi.org/10.1094/MPMI-22-4-0437

    Article  CAS  Google Scholar 

  • Ceasar SA, Ignacimuthu S (2012) Genetic engineering of crop plants for fungal resistance: role of antifungal genes. Biotechnol Lett 34(6):995–1002. https://doi.org/10.1007/s10529-012-0871-1

    Article  PubMed  Google Scholar 

  • Chen L, Zhang ZY, Liang HX, Liu HX, LP D, Xu H, Xin Z (2008) Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat. J Exp Bot 59(15):4195–4204. https://doi.org/10.1093/jxb/ern259

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Christensen AB, Cho BH, Naesby M, Gregersen PL, Brandt J, Madriz-Ordenana K, Collinge DB, Thordal-Christensen H (2002) The molecular characterization of two barley proteins establishes the novel PR-17 family of pathogenesis-related proteins. Mol Plant Pathol 3(3):135–144. https://doi.org/10.1046/j.1364-3703.2002.00105.x

    Article  CAS  PubMed  Google Scholar 

  • Dandekar AM, Fisk HJ (2005) Plant transformation: Agrobacterium-mediated gene transfer. Methods Mol Biol 286:35–46

    CAS  PubMed  Google Scholar 

  • Donzelli B, Lorito M, Scala F, Harman G (2001) Cloning, sequence and structure of a gene encoding an antifungal glucan 1, 3-beta-glucosidase from Trichoderma atroviride (T. harzianum). Gene 277(1-2):199–208. https://doi.org/10.1016/S0378-1119(01)00681-3

    Article  CAS  PubMed  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Felcher KJ, Douches DS (2003) Expression of a fungal glucose oxidase gene in three potato cultivars with different susceptibility to late blight (Phytophthora infestans Mont. deBary). J Am Soc Hortic Sci 128(2):238–245

    CAS  Google Scholar 

  • Gohain B, Borchetia S, Bhuyan LP, Rahman A, Sakata K, Mizutani M, Shimizu B, Gurusubramaniam G, Ravindranath R, Hazarika M, Das S (2012) Understanding Darjeeling tea flavour on a molecular basis. Plant Mol Biol 78(6):577–597. https://doi.org/10.1007/s11103-012-9887-0

    Article  CAS  PubMed  Google Scholar 

  • Gulati A, Gulati A, Ravindranath SD, Chakrabarty DN (1993) Indian. Phytopathology 46:155–159

    Google Scholar 

  • Gupta P, Ravi I, Sharma V (2013) Induction of β-1, 3-glucanase and chitinase activity in the defense response of Eruca sativa plants against the fungal pathogen Alternaria brassicicola. J Plant Interact 8(2):155–161. https://doi.org/10.1080/17429145.2012.679705

    Article  CAS  Google Scholar 

  • Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2(4):208–218. https://doi.org/10.1007/BF01977351

    Article  CAS  Google Scholar 

  • Jach G, Görnhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R, Schell J, Mass C (1995) Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 8(1):97–109. https://doi.org/10.1046/j.1365-313X.1995.08010097.x

    Article  CAS  PubMed  Google Scholar 

  • Jayaswall K, Mahajan P, Singh G, Parmar R, Seth R, Raina A, Swarnkar MK, Singh AK, Shankar R, Sharma RK (2016) Transcriptome analysis reveals candidate genes involved in blister blight defense in tea (Camellia sinensis (L) Kuntze). Sci Rep 6 (30412). https://doi.org/10.1038/srep30412

  • Jongedijk E, Tigelaar H, van Roekel JSC, Bres-Vloemans SA, Dekker I, van den Elzen PJM, Cornelissen BJC, Melchers LS (1995) Synergistic activity of chitinases and β-1, 3-glucanases enhances fungal resistance in transgenic tomato plants. Euphytica 85(1-3):173–180. https://doi.org/10.1007/BF00023946

    Article  CAS  Google Scholar 

  • Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382. https://doi.org/10.1016/0076-6879(87)48036-1

    Article  CAS  Google Scholar 

  • Lin B, Xiao Y (1995) Sources of resistance to Verticillium wilt in Solanum melongena and its affinities identified by improved root dip method. Capsicum Eggplant Newsl 14:81–84

    Google Scholar 

  • Lin WC, Lu CF, Wu JW, Cheng ML, Lin YM, Yang NS, Black L, Green SK, Wang JF, Cheng CP (2004) Transgenic tomato plants expressing the Arabidopsis AtNPR1 gene display enhanced resistance to a spectrum of fungal and bacterial diseases. Transgenic Res 13(6):567–581. https://doi.org/10.1007/s11248-004-2375-9

    Article  CAS  PubMed  Google Scholar 

  • Malik EP, Singh MB (1980) Plant enzymology and hittoenzymology, 1st edn. Kalyani Publishers, New Delhi, p 286

    Google Scholar 

  • Mao B, Liu X, Hu D, Li D (2013) Co-expression of RCH10 and AGLU1 confers rice resistance to fungal sheath blight Rhizoctonia solani and blast Magnaporthe oryzae and reveals impact on seed germination. World J Microbiol Biotechnol 30(4):1229–1238. https://doi.org/10.1007/s11274-013-1546-3

    Article  PubMed  Google Scholar 

  • Masoud SA, Zhu Q, Lamb C, Dixon RA (1996) Constitutive expression of an inducible β-1, 3-glucanase in alfalfa reduces disease severity caused by the oomycete pathogen Phytophthora megasperma f. spmedicaginis, but does not reduce disease severity of chitin-containing fungi. Transgenic Res 5(5):313–323. https://doi.org/10.1007/BF01968941

    Article  CAS  Google Scholar 

  • Maziah M, Saraih M, Sreeramanan S (2007) Transgenic banana Rastali (AAB) with β-1, 3-glucanase gene for tolerance to Fusarium wilt race 1 disease via Agrobacterium-mediated ransformation system. Plant Pathol J 6:271–282

    Article  CAS  Google Scholar 

  • Mondal T K, Bhattacharya A, Ahuja P S and Chand P K (2001) Transgenic tea (Camellia sinensis (L.) O. Kuntze cv. Kangra Jat) plants obtained by Agrobacterium mediated transformation of somatic embryos; Plant Cell Rep. 20 712–720

  • Moustafa SA, Ismail IM, Metry EA, Ghazal S, Ibrahim MA (2007) Cloning and characterization of β-1, 3- glucanase from blight resistant and susceptible potato (Solanum tuberosum L.) cultivars. J Appl Sci Res 3(12):1960–1968

    CAS  Google Scholar 

  • Nakamura Y, Sawada H, Kobayashi S, Nakajima I, Yoshikawa M (1999) Expression of soybean β-1, 3-endoglucanase cDNA and effect on disease tolerance in kiwifruit plants. Plant Cell Rep 18(7-8):527–532. https://doi.org/10.1007/s002990050616

    Article  CAS  Google Scholar 

  • O’Kennedy MM, Crampton BG, Lorito M, Chakauya E, Breese WA, Burger JT, Botha FC (2011) Expression of a β-1, 3-glucanase from a biocontrol fungus in transgenic pearl millet. S Afr J Bot 77(2):335–345. https://doi.org/10.1016/j.sajb.2010.09.016

    Article  Google Scholar 

  • Prabhavathi V, Rajam MV (2007) Mannitol-accumulating transgenic eggplants exhibit enhanced resistance to fungal wilts. Plant Sci 173(1):50–54. https://doi.org/10.1016/j.plantsci.2007.04.004

    Article  CAS  Google Scholar 

  • Premkumar R, Ponmurugan P, Manian S (2008) Growth and photosynthetic and biochemical responses of tea cultivars to blister blight infection. Photosynthetica 46(1):135–138. https://doi.org/10.1007/s11099-008-0021-0

    Article  CAS  Google Scholar 

  • Punja ZK (2001) Genetic engineering of plants to enhance resistance to fungal pathogens—a review of progress and future prospects. Can J Plant Pathol 23(3):216–235. https://doi.org/10.1080/07060660109506935

    Article  CAS  Google Scholar 

  • Qiu DY, Xiao J, Ding XH, Xiong M, Cai M, Cao YL, Li XH, CG X, Wang SP (2007) OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling. Mol Plant-Microbe Interact 20(5):492–499. https://doi.org/10.1094/MPMI-20-5-0492

    Article  CAS  PubMed  Google Scholar 

  • Radhakrishnan B, Baby UI (2004) Economic threshold level for blister blight of tea. Indian Phytopathol 57:195–196

    Google Scholar 

  • Rahman M, Rao AQ, Batool F, Azam S, Shahid AA, Husnain T (2012) Transgene copy number and phenotypic variations in transgenic basmati rice. J Anim Plant Sci 22(4):1004–1013

    Google Scholar 

  • Rejeb IN, Pastor V, Mauch-Mani B (2014) Plant responses to simultaneous biotic and abiotic stress: molecular mechanisms. Plants 3(4):458–475. https://doi.org/10.3390/plants3040458

    Article  PubMed  PubMed Central  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Singh HR, Bhattacharyya N, Agarwala N, Bhagawati P, Deka M, Das S (2014) Exogenous gene transfer in Assam tea [Camellia assamica (Masters)] by Agrobacterium-mediated transformation using somatic embryo. Eur J Exp Biol 4(3):166–175

    CAS  Google Scholar 

  • Singh HR, Deka M, Das S (2015) Enhanced resistance to blister blight in transgenic tea (Camellia sinensis [L.] O. Kuntze) by overexpression of class I chitinase gene from potato (Solanum tuberosum). Funct Integr Genomics 15(4):461–480

    Article  CAS  PubMed  Google Scholar 

  • Sundaresha S, Manoj Kumar A, Rohini S, Math S, Keshamma E, Chandrashekar S, Udayakumar M (2010) Enhanced protection against two major fungal pathogens of groundnut, Cercospora arachidicola and Aspergillus flavus in transgenic groundnut over-expressing a tobacco β-1, 3-glucanase. Eur J Plant Pathol 126(4):497–508. https://doi.org/10.1007/s10658-009-9556-6

    Article  CAS  Google Scholar 

  • Thordal-Christensen H, Gregersen PL, Collinge DB (2000) The barley/Blumeria (syn. Erysiphe) graminis interaction. In: Slusarenko A, Fraser RSS and Van Loon LC (eds) Mechanisms of Resistance to Plant Diseases, The Netherlands. Kluwer Academic Publishers, Dordrecht, pp 77–100

  • Van der Krol AR, Mur LA, Beld M, Mol JNM, Stuitje AR (1990) Flavonoid genes in petunia: addition of a limited number of genes copies may lead to a suppression of gene expression. Plant Cell 2(4):291–299. https://doi.org/10.1105/tpc.2.4.291

    Article  PubMed  PubMed Central  Google Scholar 

  • Wally O, Punja ZK (2010) Genetic engineering for increasing fungal and bacterial disease resistance in crop plants. GM Crops 1(4):199–206. https://doi.org/10.4161/gmcr.1.4.13225

    Article  PubMed  Google Scholar 

  • Wróbel-Kwiatkowskaa M, Lorenc-Kukulaa K, Starzyckib M, Oszmiańskic J, Kepczyńskad E, Szopaa J (2004) Expression of β-1, 3-glucanase in flax causes increased resistance to fungi. Physiol Mol Plant Pathol 65(5):245–256. https://doi.org/10.1016/j.pmpp.2005.02.008

    Article  Google Scholar 

  • Wu HY, Liu KH, Wang YC, Wu JF, Chiu WL, Chen CY, Wu SH, Sheen J, Lai EM (2014) AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings. Plant Methods 10(1):19. https://doi.org/10.1186/1746-4811-10-19

    Article  PubMed  PubMed Central  Google Scholar 

  • Xu ZZ, Zhou GS, Li H (2004) Responses of chlorophyll fluorescence and nitrogen level of Leymus chinensis seedling to changes of soil moisture and temperature. J Environ Sci 16:666–669

    CAS  Google Scholar 

  • Yang L, Hu C, Li N, Zhang J, Yan J, Deng Z (2011) Transformation of sweet orange [Citrus sinensis (L.) Osbeck] with pthA-nls for acquiring resistance to citrus canker disease. Plant Mol Biol 75(1-2):11–23. https://doi.org/10.1007/s11103-010-9699-z

    Article  CAS  PubMed  Google Scholar 

  • Yin Z, Malepszy S (2003) The transgenes are expressed with different level in plants. Biotechnologia 2(61):236–260

    Google Scholar 

  • Yoshikawa M, Tsuda M, Takeuchi Y (1993) Resistance to fungal disease in transgenic tobacco plants expressing the phytoalexin elicitor-releasing factor, β-1, 3-glucanase from soybean. Naturwissenschaften 80(9):417–420. https://doi.org/10.1007/BF01168337

    Article  CAS  Google Scholar 

  • Zhu Q, Maher EA, Masoud S, Dixon RA, Lamb CJ (1994) Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Nat Biotechnol 12(8):807–812. https://doi.org/10.1038/nbt0894-807

    Article  CAS  Google Scholar 

Download references

Acknowledgements

HRS was a senior research fellow supported by the Council of Scientific and Industrial Research, Govt. of India. The authors also acknowledge the support of the director, Tocklai Tea Research Institute, Jorhat, Assam, India.

Funding

This study was supported by generous funding from Department of Biotechnology, Govt. of India.

Author information

Authors and Affiliations

Authors

Contributions

S Das and HR Singh planned the research objective. HR Singh was awarded senior research fellowship by CSIR (Govt. of India) to take up the research work. HR Singh performed the experiments and wrote the manuscript. P Hazarika helped with tissue culture and genomic DNA isolation. N Agarwala helped in the preparation of plant transformation vector. B Gohain helped in doing real-time PCR experiments. N Bhattacharyya, P Bhagawati, T Bandyopadhyay, R Bharalee, S Gupta, and M Deka helped during literature survey and planning of experiments.

Corresponding author

Correspondence to H. Ranjit Singh.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

ESM 1

(DOC 289 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Singh, H.R., Hazarika, P., Agarwala, N. et al. Transgenic Tea Over-expressing Solanum tuberosum Endo-1,3-beta-d-glucanase Gene Conferred Resistance Against Blister Blight Disease. Plant Mol Biol Rep 36, 107–122 (2018). https://doi.org/10.1007/s11105-017-1063-x

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11105-017-1063-x

Keywords

Navigation