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A protocol for functional study of genes in Brassica juncea by Agrobacterium-mediated transient expression: applicability in other Brassicaceae

  • Madhumanti Das
  • Haraprasad Naiya
  • Ananya Marik
  • Gairik Mukherjee
  • Anindita SealEmail author
Original Article
  • 18 Downloads

Abstract

Agrobacterium-mediated transient expression in plant organs is a quick and reliable method for studying gene functions. Due to the significance of transient transformation, substantial efforts have been dedicated to developing such protocols in various plants including the model Arabidopsis thaliana. Despite the importance, a reliable protocol is still lacking in Brassicaceae due to their recalcitrance towards Agrobacterium-mediated transient transformation. We have developed protocols for transient expression in Brassica juncea (PI 211000) and tested three other Brassica sp. for the suitability of the protocol. Co-infiltration of a bacteria-derived avirulence protein AvrPto1 significantly improved expression in B. juncea cotyledonary leaves. The protocol was used successfully in studying protein localization, protein–protein interaction by co-immunoprecipitation assay and transient silencing in B. juncea indicating it to be an excellent model system for transient expression. The efficiency of the protocol varied between Brassica sp. and depended highly on the Agrobacterium strain used. The protocol would be useful in designing functional analyses of genes using transient expression in Brassicaceae, including Arabidopsis and enable inclusion of mutant lines for such studies.

Keywords

Agrobacterium Brassica juncea Transient expression 

Abbreviations

Avr

Avirulence

GUS

β-Glucuronidase

GFP

Green fluorescent protein

CFP

Cyan fluorescent protein

BjTTP

Brassica juncea tetratricopeptide

BjHCF

Brassica juncea high-chlorophyll fluorescence

BjNRAMP

Brassica juncea natural resistance associated macrophage protein

Notes

Acknowledgements

We thank Prof. Fumiaki Katagiri, University of Minnesota Twin Cities, for providing us the AvrPto1 clone. The pMDC vectors were obtained from ABRC. We also thank Dr. Naveen Bisht, NIPGR, India for providing us the B. nigra, B. rapa and B. napus seeds. We thank Dr. Ronita Nag for her assistance with A. thaliana work. We acknowledge the Department of Biotechnology—Interdisciplinary Program in Life Sciences, the University of Calcutta for the confocal microscopy facility and Mr. Arijit Pal and Mr. Souvik Roy jointly for their technical assistance. The organellar markers in pCMU vectors were gifts from Prof. M. J. Harrison and obtained from Dr. Senjuti Sinha Roy, NIPGR, India. We thank Prof. Daisuke Miki and Prof. Ko Shimamoto for providing us with the pANDA35HK vector. Madhumanti Das acknowledges Council of Scientific and Industrial Research (CSIR) and Haraprasad Naiya acknowledges University Grants Commission (UGC) for their fellowships. The work was funded by Council of Scientific and Industrial Research (CSIR) [Project No. 38(1276)/10/EMR-II] and Department of Science and Technology (DST) [SERB/SR/SO/PS/19/2012], Govt. of India.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13562_2019_543_MOESM1_ESM.tif (6.9 mb)
Figure S1Expression of pCAMBIA1305.1-GUS containing catalase intron in different time point. GUS-plus gene was expressed transiently in B. juncea leaves 48hpi and 72hpi. n=15 (2 leaves/plant) (TIFF 7105 kb)
13562_2019_543_MOESM2_ESM.tif (67 kb)
Figure S2A scheme showing agroinfiltration of B. juncea leaves. B. juncea (or other Brassica sp) leaves were pricked at the abaxial side and infiltrated with desired constructs mixed with AvrPto1 containing Agrobacterium culture. Gene/protein expression was checked 72 hours post infiltration (hpi) (TIFF 66 kb)
13562_2019_543_MOESM3_ESM.tif (841 kb)
Figure S3Localization study with negative controls. (a) The untransformed leaf in the wavelength range of mCherry (upper panel) and CFP (middle panel). Leaf transformed with empty GFP (pMDC45) vector (lower panel). (b) The untransformed root in the wavelength range of mCherry (upper panel) and CFP (lower panel). B.F. bright field (Scale 20 µm), (each in 3 experimental replicates) (TIFF 841 kb)
13562_2019_543_MOESM4_ESM.docx (12 kb)
Supplementary material 4 (DOCX 11 kb)

References

  1. Abramovitch RB, Anderson JC, Martin GB (2006) Bacterial elicitation and evasion of plant innate immunity. Nat Rev Mol Cell Biol 7:601–611PubMedPubMedCentralCrossRefGoogle Scholar
  2. Barampuram S, Zhang ZJ (2011) Recent advances in plant transformation. Methods Mol Biol 701:1–35PubMedCrossRefPubMedCentralGoogle Scholar
  3. Berger B, Stracke R, Yatusevich R, Weisshaar B, Flugge UI, Gigolashvili T (2007) A simplified method for the analysis of transcription factor-promoter interactions that allows high-throughput data generation. Plant J 50:911–916PubMedCrossRefPubMedCentralGoogle Scholar
  4. Campanoni P, Sutter JU, Davis CS, Littlejohn GR, Blatt MR (2007) A generalized method for transfecting root epidermis uncovers endosomal dynamics in Arabidopsis root hairs. Plant J 51:322–330PubMedCrossRefPubMedCentralGoogle Scholar
  5. Cardillo AB, Rodriguez Talou J, Giulietti AM (2016) Establishment, culture, and scale-up of Brugmansia candida hairy roots for the production of tropane alkaloids. Methods Mol Biol 1391:173–186PubMedCrossRefPubMedCentralGoogle Scholar
  6. Chilton MD, Drummond MH, Merio DJ, Sciaky D, Montoya AL, Gordon MP, Nester EW (1977) Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11:263–271PubMedCrossRefPubMedCentralGoogle Scholar
  7. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469PubMedPubMedCentralCrossRefGoogle Scholar
  8. Das S, Sen M, Saha C, Chakraborty D, Das A, Banerjee M, Seal A (2011) Isolation and expression analysis of partial sequences of heavy metal transporters from Brassica juncea by coupling high throughput cloning with a molecular fingerprinting technique. Planta 234:139–156PubMedCrossRefPubMedCentralGoogle Scholar
  9. Escobar MA, Dandekar AM (2003) Agrobacterium tumefaciens as an agent of disease. Trends Plant Sci 8:380–386PubMedCrossRefPubMedCentralGoogle Scholar
  10. Escudero J, Hohn B (1997) Transfer and Integration of T-DNA without Cell Injury in the Host Plant. The Plant Cell 9(12):2135PubMedPubMedCentralCrossRefGoogle Scholar
  11. Goossens J, De Geyter N, Walton A, Eeckhout D, Mertens J, Pollier J, Fiallos-Jurado J, De Keyser A, De Clercq R, Van Leene J, Gevaert K, De Jaeger G, Goormachtig S, Goossens A (2016) Isolation of protein complexes from the model legume Medicago truncatula by tandem affinity purification in hairy root cultures. Plant J 88:476–489PubMedCrossRefPubMedCentralGoogle Scholar
  12. Gordon JE, Christie PJ (2014) The Agrobacterium Ti plasmids. Microbiol Spectr 2:1–18CrossRefGoogle Scholar
  13. Gutièrrez-Pesce P, Taylor K, Muleo R, Rugini E (1998) Somatic embryogenesis and shoot regeneration from transgenic roots of the cherry rootstock Colt (Prunus avium × P. pseudocerasus) mediated by pRi 1855 T-DNA of Agrobacterium rhizogenes. Plant Cell Rep 17:574–580PubMedCrossRefPubMedCentralGoogle Scholar
  14. Harvey JJ, Lincoln JE, Gilchrist DG (2008) Programmed cell death suppression in transformed plant tissue by tomato cDNAs identified from an Agrobacterium rhizogenes-based functional screen. Mol Genet Genom MGG 279:509–521CrossRefGoogle Scholar
  15. Holsters M, de Waele D, Depicker A, Messens E, van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187PubMedCrossRefPubMedCentralGoogle Scholar
  16. Horsch RB, Klee HJ, Stachel S, Winans SC, Nester EW, Rogers SG, Fraley RT (1986) Analysis of Agrobacterium tumefaciens virulence mutants in leaf discs. Proc Natl Acad Sci USA 83:2571–2575PubMedCrossRefPubMedCentralGoogle Scholar
  17. Hwang HH, Yu M, Lai EM (2017) Agrobacterium-mediated plant transformation: biology and applications. Arabidopsis Book 15:e0186PubMedPubMedCentralCrossRefGoogle Scholar
  18. Ivanov S, Harrison MJ (2014) A set of fluorescent protein-based markers expressed from constitutive and arbuscular mycorrhiza-inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula. Plant J 80:1151–1163PubMedCrossRefPubMedCentralGoogle Scholar
  19. Ivanov S, Fedorova EE, Limpens E, De Mita S, Genre A, Bonfante P, Bisseling T (2012) Rhizobium-legume symbiosis shares an exocytotic pathway required for arbuscule formation. Proc Natl Acad Sci USA 109:8316–8321PubMedCrossRefPubMedCentralGoogle Scholar
  20. Jen GC, Chilton MD (1986) Activity of T-DNA borders in plant cell transformation by mini-T plasmids. J Bacteriol 166:491–499PubMedPubMedCentralCrossRefGoogle Scholar
  21. Koroleva OA, Tomlinson ML, Leader D, Shaw P, Doonan JH (2005) High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. Plant J 41:162–174PubMedCrossRefPubMedCentralGoogle Scholar
  22. Krenek P, Samajova O, Luptovciak I, Doskocilova A, Komis G, Samaj J (2015) Transient plant transformation mediated by Agrobacterium tumefaciens: principles, methods and applications. Biotechnol Adv 33:1024–1042CrossRefGoogle Scholar
  23. Li JF, Park E, von Arnim AG, Nebenfuhr A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5:6PubMedPubMedCentralCrossRefGoogle Scholar
  24. Li GYL, Li F, Zhang S, Zhang H, Qian W, Fang Z, Wu J, Wang X, Zhang S, Sun R (2018) Research progress on Agrobacterium tumefaciens-based transgenic technology in Brassica rapa. Horticult Plant J 4(3):126–132CrossRefGoogle Scholar
  25. Lioshina LG, Bulko OV (2014) Plant regeneration from hairy roots and calluses of periwinkle Vinca minor L. and foxglove purple Digitalis purpurea L. Cytol Genet 48:302–307CrossRefGoogle Scholar
  26. Ma L, Lukasik E, Gawehns F, Takken FL (2012) The use of agroinfiltration for transient expression of plant resistance and fungal effector proteins in Nicotiana benthamiana leaves. Methods Mol Biol 835:61–74PubMedCrossRefPubMedCentralGoogle Scholar
  27. Marik A, Naiya H, Das M, Mukherjee G, Basu S, Saha C, Chowdhury R, Bhattacharyya K, Seal A (2016) Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea. Plant Mol Biol 92:519–537PubMedCrossRefPubMedCentralGoogle Scholar
  28. Marion J, Bach L, Bellec Y, Meyer C, Gissot L, Faure JD (2008) Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J 56:169–179PubMedCrossRefPubMedCentralGoogle Scholar
  29. Miki D, Shimamoto K (2004) Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol 45:490–495PubMedCrossRefPubMedCentralGoogle Scholar
  30. Moloney MM, Walker JM, Sharma KK (1989) High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep 8:238–242PubMedCrossRefPubMedCentralGoogle Scholar
  31. Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136PubMedCrossRefPubMedCentralGoogle Scholar
  32. Noel LD, Cagna G, Stuttmann J, Wirthmuller L, Betsuyaku S, Witte CP, Bhat R, Pochon N, Colby T, Parker JE (2007) Interaction between SGT1 and cytosolic/nuclear HSC70 chaperones regulates Arabidopsis immune responses. Plant Cell 19:4061–4076PubMedPubMedCentralCrossRefGoogle Scholar
  33. Porter JR, Flores H (1991) Host range and implications of plant infection by Agrobacterium rhizogenes. Crit Rev Plant Sci 10:387–421CrossRefGoogle Scholar
  34. Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K, Garcha J, Winte S, Masson H, Inagaki S, Federici F, Sinha N, Deal RB, Bailey-Serres J, Brady SM (2014) Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 166:455–469PubMedPubMedCentralCrossRefGoogle Scholar
  35. Rosas-Diaz T, Cana-Quijada P, Amorim-Silva V, Botella MA, Lozano-Duran R, Bejarano ER (2017) Arabidopsis NahG plants as a suitable and efficient system for transient expression using Agrobacterium tumefaciens. Mol Plant 10:353–356PubMedCrossRefPubMedCentralGoogle Scholar
  36. Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475PubMedPubMedCentralCrossRefGoogle Scholar
  37. Stachel SE, Nester EW (1986) The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J 5(7):1445–1454PubMedPubMedCentralCrossRefGoogle Scholar
  38. Tai TH, Dahlbeck D, Clark ET, Gajiwala P, Pasion R, Whalen MC, Stall RE, Staskawicz BJ (1999) Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato. Proc Natl Acad Sci USA 96:14153–14158PubMedCrossRefPubMedCentralGoogle Scholar
  39. Toro N, Datta A, Yanofsky M, Nester E (1988) Role of the overdrive sequence in T-DNA border cleavage in Agrobacterium. Proc Natl Acad Sci USA 85:8558–8562PubMedCrossRefPubMedCentralGoogle Scholar
  40. Tsuda K, Qi Y, le Nguyen V, Bethke G, Tsuda Y, Glazebrook J, Katagiri F (2012) An efficient Agrobacterium-mediated transient transformation of Arabidopsis. Plant J 69:713–719PubMedCrossRefPubMedCentralGoogle Scholar
  41. Tzfira T, Vaidya M, Citovsky V (2004) Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature 431:87–92PubMedCrossRefPubMedCentralGoogle Scholar
  42. Van den Ackerveken G, Marois E, Bonas U (1996) Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell 87:1307–1316PubMedCrossRefPubMedCentralGoogle Scholar
  43. Van der Hoorn RA, Laurent F, Roth R, De Wit PJ (2000) Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis. Molecul Plant Microbe Interact 13:439–446CrossRefGoogle Scholar
  44. Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol J 3:259–273PubMedCrossRefPubMedCentralGoogle Scholar
  45. 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 10:19PubMedPubMedCentralCrossRefGoogle Scholar
  46. Yamazaki Y, Kitajima M, Arita M, Takayama H, Sudo H, Yamazaki M, Aimi N, Saito K (2004) Biosynthesis of camptothecin. In silico and in vivo tracer study from [1-13C]glucose. Plant Physiol 134:161–170PubMedPubMedCentralCrossRefGoogle Scholar
  47. Yang L, Wang H, Liu J, Li L, Fan Y, Wang X, Song Y, Sun S, Wang L, Zhu X (2008) A simple and effective system for foreign gene expression in plants via root absorption of agrobacterial suspension. J Biotechnol 134:320–324PubMedCrossRefPubMedCentralGoogle Scholar
  48. Yasmin A, Debener T (2010) Transient gene expression in rose petals via Agrobacterium infiltration. Plant Cell Tissue Organ Cult 102:245–250CrossRefGoogle Scholar
  49. Young JM, Kuykendall LD, Martinez-Romero E, Kerr A, Sawada H (2001) A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. Int J Syst Evol Microbiol 51:89–103PubMedPubMedCentralCrossRefGoogle Scholar
  50. Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D, Wang J, Wang H (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7:30PubMedPubMedCentralCrossRefGoogle Scholar
  51. Zhang Z-Y, Li G-Y, Wang J-L, Guo X-J, Wang Z, Tan X-L (2017) Establishment of rapeseed (Brassica Napus L.) cotyledon transient transformation system for gene function analysis. Pak J Bot 49:2227–2233Google Scholar
  52. Zottini M, Barizza E, Costa A, Formentin E, Ruberti C, Carimi F, Lo Schiavo F (2008) Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells. Plant Cell Rep 27:845–853PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyUniversity of CalcuttaKolkataIndia
  2. 2.ICAR-Indian Institute of Natural Resins and GumsNamkum, RanchiIndia

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