Analysis of reporter proteins GUS and DsRed driven under the control of CaMV35S promoter in syncytia induced by beet cyst nematode Heterodera schachtii in Arabidopsis roots

Muhammad Amjad Ali, Amjad Abbas

Abstract


Background: Cyst nematodes induce specialized feeding structures called syncytia in the plant roots. The expression of CaMV promoter in syncytia has remained topic of debate. The objective of this research was to study the activity of CaMV promoter by using reporter proteins like GUSand DsRed under the control of CaMV35S promoter in syncytia induced by H. schachtii in Arabidopsis roots.

Methods: pMAA-Red and pPZP3425 plasmids were used to study expression of GUS and DsRedin syncytia.  The plants were grown in 2% Knop medium under sterile conditions in growth chambers at 25°C in long day conditions. GUS activity in syncytia was studied through staining of syncytia using X-gluc solution. Ds-Red fluorescence in syncytia was detected by using an inverse microscope equipped with UV filter.

Results: The expression analysis of DsRed protein driven by CaMV promoter demonstrated that this promoter is active in syncytia at all the time points. All the syncytia showed DsRed expression at 5 dpi. At 7 dpi, 10 dpi and 15 dpi over 90%, 80% and 50% of the syncytia showed DsRedfluorescence respectively. There was very high fluorescence in the syncytia as compared to the uninfected root segments due to high expression. CaMV::GUS lines showed GUS expression in 80% of 5dpi syncytia. However, unlike expression of DsRed, the number of GUS stained syncytia decreased quickly to around 50% at 7 dpi and to about 5% in the 15 dpi syncytia.

Conclusions: The results conclude that CaMV promoter is more active in younger syncytia as compared to older syncytia but can be used for expression in syncytia. Moreover, DsRed protein could be used as better reporter for evaluation of gene expression in syncytia as compared to GUS.


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References


Abad P, Gouzy J, Aury JM, Castagnone-Sereno P, Danchin EGJ, et al. Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita. Nature Biotechnology, (2008); 26(8): 909-915.

Jones MGK. Host cell responses to endoparasitic nematode attack: structure and function of giant cells and syncytia. Annals of Applied Biology (1981); 97(3): 353–372.

Golinowski W, Grundler FMW, Sobczak M. Changes in the structure of Arabidopsis thaliana during female development of the plant-parasitic nematode Heterodera schachtii. Protoplasma, (1996); 194(1-2): 103-116.

Endo B. Ultrastructure of initial responses of susceptible and resistant soybean roots to infection by Heterodera glycines. Revue de Nematologie (1991); 14(1): 73–94.

Grundler FMW, Sobczak M, Lange S. Defence responses of Arabidopsis thaliana during invasion and feeding site induction by the plant-parasitic nematode Heterodera glycines. Physiological and Molecular Plant Pathology, (1997); 50(6): 419-429.

de Almeida Engler J, Favery B, Engler G, Abad P. Loss of susceptibility as an alternative for nematode resistance. Current Opinion in Biotechnology, (2005); 16(2): 112-117.

Siddique S, Sobczak M, Tenhaken R, Grundler FM, Bohlmann H. Cell Wall Ingrowths in Nematode Induced Syncytia Require UGD2 and UGD3. Plos One, (2012); 7(7): e41515.

Hussey RS, Grundler FM. Nematode parasitism of plants: The Physiology and Biochemistry of Free-living and Plant-parasitic nematodes. (1998), pp. 213-243. CAB International.

Williamson VM, Hussey RS. Nematode pathogenesis and resistance in plants. Plant Cell, (1996); 8(10): 1735-1745.

Benfey PN, Ren L, Chua NH. The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO Journal, (1989); 8(8): 2195-2202.

Odell JT, Nagy F, Chua NH. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature, (1985); 313(6005): 810-812.

Gallie DR, Walbot V. Identification of the motifs within the tobacco mosaic virus 5'-leader responsible for enhancing translation. Nucleic Acids Research, (1992); 20(17): 4631-4638.

Holtorf S, Apel K, Bohlmann H. Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana. Plant Molecular Biology, (1995); 29(4): 637-646.

Jefferson RA. The GUS reporter gene system. Nature, (1989); 342(6251): 837-838.

Ow DW, JR DEW, Helinski DR, Howell SH, Wood KV, et al. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science, (1986); 234(4778): 856-859.

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science, (1994); 263(5148): 802-805.

Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnology, (1999); 17(10): 969-973.

Baird GS, Zacharias DA, Tsien RY. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proceedings of National Academy of Sciences (USA), (2000); 97(22): 11984-11989.

Jach G, Binot E, Frings S, Luxa K, Schell J. Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression. Plant Journal, (2001); 28(4): 483-491.

Ali MA, Shah KH, Bohlmann H. pMAA-Red: a new pPZP-derived vector for fast visual screening of transgenic Arabidopsis plants at the seed stage. BMC Biotechnology, (2012); 12(1): 37.

Urwin PE, Lilley CJ, McPherson MJ, Atkinson HJ. Resistance to both cyst and root-knot nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin. Plant Journal, (1997); 12(2): 455-461.

Liu B, Hibbard JK, Urwin PE, Atkinson HJ. The production of synthetic chemodisruptive peptides in planta disrupts the establishment of cyst nematodes. Plant Biotechnology Journal, (2005); 3(5): 487-496.

Goddijn OJ, Lindsey K, van der Lee FM, Klap JC, Sijmons PC. Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter-gusA fusion constructs. Plant Journal, (1993); 4(5): 863-873.

Van Poucke K, Karimi M, Gheysen G. Analysis of nematode-responsive promoters in sugar beet hairy roots. Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, (2001); 66(2b): 591-598.

Urwin PE, Moller SG, Lilley CJ, McPherson MJ, Atkinson HJ. Continual green-fluorescent protein monitoring of cauliflower mosaic virus 35S promoter activity in nematode-induced feeding cells in Arabidopsis thaliana. Molecular Plant-Microbe Interactions, (1997); 10(3): 394-400.

Goverse A, Biesheuvel J, Wijers GJ, Gommers FJ, Bakker J, et al. In planta monitoring of the activity of two constitutive promoters, CaMV 35S and TR2 ', in developing feeding cells induced by Globodera rostochiensis using green fluorescent protein in combination with confocal laser scanning microscopy. Physiological and Molecular Plant Pathology, (1998); 52(4): 275-284.

Fuller VL, Lilley CJ, Urwin PE. Nematode resistance. New Phytologist, (2008); 180(1): 27-44.

Lilley CJ, Wang D, Atkinson HJ, Urwin PE. Effective delivery of a nematode-repellent peptide using a root-cap-specific promoter. Plant Biotechnology Journal, (2011); 9(2): 151-161.

Szakasits D, Siddique S, Bohlmann H. An improved pPZP vector for Agrobacterium-mediated plant transformation. Plant Molecular Biology Reporter, (2007); 25(3-4): 115-120.

Logemann E, Birkenbihl RP, Ulker B, Somssich IE. An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods, (2006); 2.

Sijmons PC, Grundler FMW, Vonmende N, Burrows PR, Wyss U. Arabidopsis-Thaliana as a New Model Host for Plant-Parasitic Nematodes. Plant Journal, (1991); 1(2): 245-254.

Epple P, Apel K, Bohlmann H. Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporum. Plant Cell, (1997); 9(4): 509-520.

Ali MA, Abbas A, Kreil DP, Bohlmann H. Overexpression of the transcription factor RAP2.6 leads to enhanced callose deposition in syncytia and enhanced resistance against the beet cyst nematode Heterodera schachtii in Arabidopsis roots. BMC Plant Biology, (2013); 1347.

Ali MA, Wieczorek K, Kreil DP, Bohlmann H. The beet cyst nematode Heterodera schachtii modulates the expression of WRKY transcription factors in syncytia to favour its development in Arabidopsis roots. Plos One, (2014); 9(7): e102360.

Bakhetia M, Charlton WL, Urwin PE, McPherson MJ, Atkinson HJ. RNA interference and plant parasitic nematodes. Trends in Plant Science, (2005); 10(8): 362-367.

Ali MA, Abbas A, Azeem F, Javed N, Bohlmann H. Plant-nematode Interactions: From Genomics to Metabolomics. International Journal of Agriculture and Biology, (2015); 17(06): 1071-1082.




DOI: http://dx.doi.org/10.62940/als.v3i3.180

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