A virus-induced gene silencing (VIGS) system for functional genomics in the parasitic plant Striga hermonthica

Plant Methods

Amos Alakonya²  Steven Runo¹  Dinah Kirigia² 

[1] Department of Biochemistry and Biotechnology, Kenyatta University, P.O. Box 43844, 00100 Nairobi Kenya;Jomo Kenyatta University of Agriculture and Technology, Institute of Biotechnology Research, P. O. Box 62000-00200, Nairobi, Kenya

关键词: Phytoene desaturase;    Tobacco rattle virus;    Agro-infiltration;    Agro-drench;    Viral induced gene silencing;    Striga hermonthica;   

Others  :  802536
DOI  :  10.1186/1746-4811-10-16

 received in 2014-01-24, accepted in 2014-05-22,  发布年份 2014

【 摘 要 】

Background

Striga hermonthica is a hemiparasitic weed that infects cereals in Sub Sahara Africa (SSA) resulting in up to 100% grain yield loss. This significant loss in grain yields is a major contributor to food insecurity and poverty in the region. Current strategies to control the parasite are costly, unavailable and remain unpracticed by small-scale farmers, underscoring the need for more economical and sustainable control strategies. Development of resistant germplasm is the most sustainable strategy in the control of S. hermonthica, but is constrained by paucity of resistance genes for introduction into crop germplasm. RNA interference (RNAi) has potential for developing host-derived resistance against S. hermonthica by transformation of host crops with RNAi sequences targeted at critical Striga genes. The application of RNAi in management of S. hermonthica is however constrained by lack of efficient high throughput screening protocols for the candidate genes for silencing, as well as sub optimal delivery of siRNAs into the parasite. In comparison to stable transformation, viral induced gene silencing (VIGS) is a rapid and powerful tool for plant functional genomics and provides an easy and effective strategy in screening for putative candidate genes to target through RNAi. In addition, VIGS allows for a secondary amplification of the RNAi signal increasing the siRNA threshold and facilitates siRNA transport through viral movement proteins. We tested the efficiency of the Tobacco rattle virus (TRV1 and TRV2) VIGS vectors in silencing S. hermonthica phytoene desaturase (PDS) gene through agrodrench and agro-infiltration.

Results

We report the validation of VIGS in S. hermonthica using a silencing cassette generated from TRV with a PDS gene insert. Agro-infiltrated and agro-drenched S. hermonthica leaves showed photo-bleaching phenotypes typical for PDS silencing within 7 and 14 days post infection respectively. In both cases S. hermonthica plants recovered from photo-bleaching effects within 28 days post inoculation. The transformation efficiency of the VIGS protocol in S. hermonthica was (60 ± 2.9)%.

Conclusion

These results demonstrate that the TRV-VIGS system work in S. hermonthica and can be used for candidate gene validation for their role in the parasite development and parasitism, with the ultimate goal of developing resistant transgenic maize.

【 授权许可】

   
2014 Kirigia et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

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【 参考文献 】

• [1]Food and Agriculture Organization: The state of food insecurity in the world: addressing food insecurity in protracted crises. 2010. [https://www.fao.org/docrep/013/i1683e/i1683e webcite]
• [2]Erenstein O, Kassie G-T, Langyintuo A, Mwangi W: Characterization of Maize Producing Household in Drought Prone Regions of Eastern Africa. CIMMYT; 2011. [http://repository.cimmyt.org/xmlui/bitstream/handle/10883/1306/95930.pdf?sequence=3 webcite]
• [3]AATF: Empowering African Farmers to Eradicate S. Hermonthica from Maize Crop Lands. Nairobi, Kenya: The African Agricultural Technology Foundation; 2006. [http://www.aatf-africa.org/userfiles/Empowering_African_farmers webcite]
• [4]Ejeta G: Breeding for S. hermonthica resistance in Sorghum: exploitation of intricate host parasite biology. Crop Sci 2007, 47:216-227.
• [5]M’mboyi F, Mugo S, Murenga M, Ambani L: Maize Production and Improvement in Sub-Saharan Africa. Nairobi, Kenya: African Biotechnology Stakeholders Forum(ABSF); 2010.
• [6]Rich PJ, Ejeta G: Towards effective resistance to S. hermonthica in African maize. Plant Signal Behav 2008, 3:618-621.
• [7]Parker C: Observations on the current status of Orobanche and Striga problems worldwide. Pest Manag Sci 2009, 65:453-459.
• [8]Yoder J: Parasitic plant responses to host plant signals: a model for subterranean plant-plant interactions. Curr Opin Plant Biol 1999, 2:65-70.
• [9]Westwood JH, Yoder JI, Timko MP, DePamphilis CW: The evolution of parasitism in plants. Trends Plant Sci 2010, 15:227-235.
• [10]Khan Z, Pickett J, Wadhams L, Hassanali A, Midega C: Combined control of S. hermonthica and stemborers by maize-Desmodium spp intercrops. Crop Prot 2006, 25:989-995.
• [11]De Groote H, Wangare L, Kanampiu F, Odendo M, Diallo A, Karaya H, Friesen D: The potential of herbicide Tolerant maize technology for S. hermonthica control in Africa. Agr Syst 2008, 97:83-94.
• [12]Tomilov A, Tomilova N, Wroblewski T, Michelmore R, Yoder J: Trans-specific gene silencing between host and parasitic plants. Plant J 2008, 56:389-397.
• [13]Yoder J, Gunathilake P, Wu B, Tomilova N, Tomilov AA: Engineering host resistance against parasitic weeds with RNA interferance. Pest Manag Sci 2009, 65:460-466.
• [14]Aly R, Cholakh H, Joel DM, Leibman D, Steinitz B, Zelcer A, Naglis A, Yarden O, Gal-On A: Gene silencing of mannose 6-phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant. Plant Biotechnol 2009, 7:487-498.
• [15]Runo S, Alakonya A, Machuka J, Sinha N: RNA interference as a resistance mechanism against crop parasites in Africa: a ‘Trojan horse’ approach. Pest Manag Sci 2011, 67:129-136.
• [16]Runo S: Engineering host-derived resistance against plant parasites through RNA interferance: challanges and opportunities. Bioeng Bugs 2011, 2:208-213.
• [17]Yoder J, Scholes JD: Host plant resistance to parasitic weeds;recent progress and bottlenecks. Curr Opin Plant Biol 2010, 13:478-484.
• [18]Alakonya A, Kumar R, Koenig D, Kimura S, Townsley B, Runo S, Garces HM, Kang J, Yanez A, David-Schwartz R, Machuka J, Sinha N, Rakefet: Interspecific RNA interference of SHOOT MERISTEMLESS-like disrupts Cuscuta pentagona plant parasitism. Plant Cell 2012, 24:3153-3166.
• [19]Bandaranayake P, Yoder J: Trans-specific gene silencing of acetyl-CoA carboxylase in a root-parasitic plant. Mol Plant Microbe Interact 2013, 26:575-584.
• [20]De Framond A, Rich PJ, McMillan J, Ejeta G: Effects of Striga Parasitism of Transgenic Maize Armed With RNAi Constructs tar-Geting Essential S. Asiatica Genes. In Integrating New Technologies for Striga Control. Edited by Ejeta G, Gressel J. Singapore: World Scientific Publishing Co; 2007:185-196.
• [21]Yoshida S, Maruyama S, Nozaki H, Shirasu K: Horizontal gene transfer by the parasitic plant Striga hermonthica. Science 2010, 328(5982):1128.
• [22]Hiei Y, Ohta S, Komari T, Kumashiro T: Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequences-analysis of the boundaries of the T-DNA. Plant J 1994, 6:271-282.
• [23]Ishida Y, Saito S, Ohta S, Hiei Y, Komari T, Kumashiro T: High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 1996, 14:745-750.
• [24]Zhao ZY, Gu WN, Cai TS, Tagliani L, Hondrd D, Bond D, Schroeder S, Rudert M, Pierce D: High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 2002, 8:323-333.
• [25]Cheng M, Hu T, Layton J, Liu CN, Fry JE: Desiccation of plant tissue post-agrobacterium infection enhances T-DNA delivery and increase stable transformation efficiency in wheat. In Vitro Cell Dev Biol Plant 2003, 39:595-604.
• [26]Zhao ZY, Cai TS, Tagliani L, Miller M, Wang N, Pang H: Agrobacterium-mediated sorghum transformation. Plant Mol Biol 2000, 44:789-798.
• [27]Voinnet O: RNAi silencing as a plant immune system against viruses. Trends Genet Engin 2001, 17:449-459.
• [28]Lu R, Martin-Hernandez AM, Peart JR, Malcuit I, Baulcombe DC: Virus- induced gene silencing. Plants Methods 2003, 30:296-303.
• [29]Robertson D: VIGS vectors for gene silencing: many targets, many tools. Ann Rev Plant Biol 2004, 55:495-519.
• [30]Donaire L, Wang Y, Gonzalez-Ibeas D, Mayer KF, Aranda MA, Lave CL: Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology 2009, 392:203-214.
• [31]Waterhouse PM, Fusaro AF: Plant science. Viruses face a double defense by plant small RNAs. Science 2006, 313:54-55.
• [32]Ding SW, Voinnet O: Antiviral immunity directed by small RNAs. Cell 2007, 130:413-426.
• [33]Kalantidis K, Schumacher HT, Alexiadis T, Helm JM: RNA silencing movement in plants. Biol Cell 2008, 100:13-26.
• [34]Baulcombe DC: RNA silencing. Curr Biol 2002, 21:82-84.
• [35]Himber C, Dunoyer P, Moissiard G, Ritzenthaler C, Voinnet O: Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. EMBO J 2003, 22:4523-4533.
• [36]Voinnet O, Baulcombe DC: Systemic signaling in gene silencing. Nature 1997, 389:553-555.
• [37]Dunoyer P, Himber C, Voinnet O: DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. Nat Genet 2005, 37:1356-1360.
• [38]Becker A, Lange l: VIGS -genomics goes functiona. Trends Plant Sci 2009, 15:1-4.
• [39]Choong-Min R, Anand A, Kang L, Mysore K: Agrodrench: a novel and effective agroinoculation method forvirus-induced gene silencing in roots and diverseSolanaceous species. Plant J 2004, 40:322-331.
• [40]Verchot-Lubicz: Soil borne viruses: advances in cell-to-cell movement, gene silencing, and pathogen-derived resistance. J Plant Pathol 2002, 62:55-63.
• [41]Arabidopsis biological resource center data base http://abrc.osu.edu webcite