Abstract
Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sunilkumar G, Campbell LM, Puckhaber L et al (2006) Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci USA 103:18054–18059
Yu J, Kohel RJ, Smith CW (2010) The construction of a tetraploid cotton genome wide comprehensive reference map. Genomics 95:230–240
Zhang HB, Li Y, Wang B, Chee PW (2008) Recent advances in cotton genomics. Int J Plant Genomics 2008:742304
http://www.fas.usda.gov/wap/circular/2010/10-05/productionfull05-10.pdf
Fradin EF, Thomma BP (2006) Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. alboatrum. Mol Plant Pathol 7:71–86
Bolek Y, El-Zik KM, Pepper AE et al (2005) Mapping of verticillium wilt resistance genes in cotton. Plant Sci 168:1581–1590
Yang C, Guo WZ, Li GY, Gao F, Lin SS, Zhang TZ (2008) QTLs mapping for Verticillium wilt resistance at seedling and maturity stages in Gossypium barbadense L. Plant Sci 174:290–298
Hashmi JA, Zafar Y, Arshad M et al (2011) Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences. Virus Genes 42:286–296
Chen ZJ, Scheffler BE, Dennis E et al (2007) Toward sequencing cotton (Gossypium) genomes. Plant Physiol 145:1303–1310
Burch-Smith TM, Anderson JC, Martin GB et al (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746
Dinesh-Kumar SP, Anandalakshmi R, Marathe R et al (2003) Virus-induced gene silencing. Methods Mol Biol 236:287–294
Becker A, Lange M (2010) VIGS–genomics goes functional. Trends Plant Sci 15:1–4
Hayward A, Padmanabhan M, Dinesh-Kumar SP (2011) Virus-induced gene silencing in Nicotiana benthamiana and other plant Âspecies. Plant Reverse Genetics: Methods and Protocols. Methods Mol Biol 678:55–63
Tuttle JR, Idris AM, Brown JK et al (2008) Geminivirus mediated gene silencing from Cotton leaf crumple virus is enhanced by low temperature in cotton. Plant Physiol 148:41–50
Kaloshian I (2007) Virus-Induced Gene silencing in plants roots. Plant-Pathogen Interactions: Methods and Protocols. Methods Mol Biol 354:173–181
Pflieger S, Blanchet S, Camborde L et al (2008) Efficient virus-induced gene silencing in Arabidopsis using a ‘one-step’ TYMV-derived vector. Plant J 56:678–690
Ding XS, Schneider WL, Chaluvadi SR et al (2006) Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts. Mol Plant Microbe Interact 19:1229–1239
Scofield SR, Huang L, Brandt AS et al (2005) Development of a virus induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol 138:2165–2173
Mei CS, Zhou X, Yang Y (2007) Use of RNA interference to dissect defense-signaling pathways in rice. Plant-Pathogen Interactions: Methods and Protocols. Methods Mol Biol 354:161–172
Gao X, Wheeler T, Li Z et al (2011) Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt. Plant J 66(2):293–305
Ellendorff U, Fradin EF, de Jonge R et al (2009) RNA silencing is required for Arabidopsis defence against Verticillium wilt disease. J Exp Bot 60:591–602
Parkhi V, Kumar V, Campbell LM et al (2010) Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis NPR1. Transgenic Res 19:959–975
Acknowledgments
We thank Dr. S. P. Dinesh-Kumar for pTRV-VIGS vectors and Dr. Terry Wheeler and Bayer CropScience (Lubbock, TX, USA) for cotton seeds. This work was supported by Texas AgriLife Research Cotton Improvement Program to L. S.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Gao, X., Shan, L. (2013). Functional Genomic Analysis of Cotton Genes with Agrobacterium-Mediated Virus-Induced Gene Silencing. In: Becker, A. (eds) Virus-Induced Gene Silencing. Methods in Molecular Biology, vol 975. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-278-0_12
Download citation
DOI: https://doi.org/10.1007/978-1-62703-278-0_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-277-3
Online ISBN: 978-1-62703-278-0
eBook Packages: Springer Protocols