Factors influencing the efficiency of an agroinjection-mediated SCMV-based systemic heterologous gene expression system in maize

Document Type : Research Paper


1 University of Zabol

2 Department of Biotechnology, Faculty of Agriculture, University of Zabol

3 Associate professor, Department of Biotechnology, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

4 Associate professor, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Zabol, Zabol, Iran.

5 Boyce Thompson Institute for Plant Research


Maize (Zea mays L.) is a key cereal crop throughout the world and important model for plant genetics and biology. Establishment of an efficient transient gene expression system in maize facilitates plant functional genomics projects using gene silencing or heterologous protein overexpression strategies. The present study was aimed at optimizing an Agroinjection-mediated SCMV-based systemic heterologous gene expression system in maize.
Materials and methods
Recombinant DNA encoding sugarcane mosaic virus (SCMV) containing the coding sequence of green fluorescent protein between the protein 1 (P1) and helper component‐proteinase (HC‐Pro) cistrons, in‐frame with the viral open reading frame, was introduced into the meristematic tissue, above the coleoptilar node, of maize seedlings via a direct Agroinoculation procedure. The efficiency of Agrobacterium tumefaciens strains EHA105 and GV3101 in delivering the recombinant vector into three and seven-day old seedlings of three maize varieties, including sweet corn (Iochief and Golden Bantam varieties) and dent corn (inbred line B73), was examined. Expression of GFP transgene in symptomatic Agroinoculated plants was assessed by confocal fluorescent microscopy and RT-PCR in comparison with controls.
Results of RT-PCR and confocal fluorescent microscopy revealed that: 1) A. tumefaciens GV3101 is significantly more successful in delivering the recombinant SCMV-based vector into maize plants than EHA105, 2) the percentage of GFP-expressing Golden Bantam plants is significantly higher than two other maize varieties, and 3) The effectiveness of growth stage of maize seedlings on the percentage of GFP expressing Agroinjected plants depends upon the interactions between Agrobacterium strain and maize genotype.
In conclusion, a combination of the Golden Bantam maize variety and A. tumefaciens strain GV3101 for direct Agroinjection of the SCMV-based vector into seedlings at the early two-leaf stage could be a fast and efficient system for investigating gene functions in maize.


Arafa RA, Rakha MT, Soliman NEK, Moussa OM et al. (2017) Rapid identification of candidate genes for resistance to tomato late blight disease using next-generation sequencing technologies. PloS one 12(12), e0189951.
Avesani L, Marconi G, Morandini F et al. (2007) Stability of Potato virus X expression vectors is related to insert size: implications for replication models and risk assessment. Transgenic Res 16(5), 587-597.
Beernink BM, Holan KL, Lappe RR, Whitham SA (2021) Direct agroinoculation of maize seedlings by injection with recombinant foxtail mosaic virus and sugarcane mosaic virus infectious clones. J Visualized Exp 168, e62277.
Bouton C, King RC, Chen H, Azhakanandam K et al. (2018) Foxtail mosaic virus: A viral vector for protein expression in cereals. Plant Physiol 177(4), 1352-1367.
Cao SL, Masilamany P, Li WB, Pauls KP (2014) Agrobacterium tumefaciens-mediated transformation of corn (Zea mays L.) multiple shoots. Biotechnol Biotechnol Equip 28(2), 208-216.
Chang AY, Chau VW, Landas JA, Pang Y (2017) Preparation of calcium competent Escherichia coli and heat-shock transformation. JEMI methods 1, 22-25.
Cheuk A, Houde M (2018) A new barley stripe mosaic virus allows large protein overexpression for rapid function analysis. Plant Physiol 176(3), 1919-1931.
Chung SH, Bigham M, Lappe RR et al. (2021) A sugarcane mosaic virus vector for rapid in planta screening of proteins that inhibit the growth of insect herbivores. Plant Biotechnol J. https://doi: 10.1111/pbi.13585 [Epub ahead of print] PMID: 33763921.
Cody WB, Scholthof HB, Mirkov TE (2017) Multiplexed gene editing and protein overexpression using a tobacco mosaic virus viral vector. Plant Physiol 175(1), 23-35.
Edwards D, Batley J (2010) Plant genome sequencing: applications for crop improvement. Plant biotechnol J 8(1), 2-9.
FAO (2019) World Food and Agriculture– Statistical pocketbook 2019. Rome.
Gao Q, Xu WY, Yan T et al. (2019) Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies. New Phytologist 223(4), 2120-2133.
Grimsley NH, Ramos C, Hein T, Hohn B (1988) Meristematic tissues of maize plants are most susceptible to agroinfection with maize streak virus. Biotechnol 6(2), 185-189.
Jones MW, Redinbaugh MG, Louie R (2007) The Mdm1 locus and maize resistance to Maize dwarf mosaic virus. Plant Dis 91(2), 185-190.
Karami O (2008) Factors affecting Agrobacterium-mediated transformation of plants. Transgenic Plant J 2(2), 127-137.
Kawazu K, Wasano N, Konno K et al. (2012) Evaluation of anti-herbivory genes using an Agrobacterium-mediated transient expression system. Plant Biotechnol 29 (5), 495-499.
King JL (2013) The development of an efficient method of Agrobacterium-mediated transient expression in soybean (Glycine max). PhD thesis, Ohio State University. pp. 19-21.
Kushwaha NK, Chakraborty S (2017) Chilli leaf curl virus-based vector for phloem-specific silencing of endogenous genes and overexpression of foreign genes. Appl Microbiol Biotechnol 101(5), 2121-2129.
Lawrence CJ, Harper LC, Schaeffer ML et al. (2008) MaizeGDB: the maize model organism database for basic, translational, and applied research. Int J Plant Genom 2008, 496957.
Lawrence SD, Novak NG (2001) A rapid method for the production and characterization of recombinant insecticidal proteins in plants. Mol Breed 8(2), 139-146.
Lee WS, Hammond-Kosack KE, Kanyuka K (2012) Barley stripe mosaic virus-mediated tools for investigating gene function in cereal plants and their pathogens: virus-induced gene silencing, host-mediated gene silencing, and virus-mediated overexpression of heterologous protein. Plant physiol 160(2), 582-590.
Lee WS, Hammond-Kosack KE, Kanyuka K (2015) In planta transient expression systems for monocots. In: Recent advancements in gene expression and enabling technologies in crop plants. Springer, New York, USA. pp. 391-422.
Li JF, Park E, von Arnim AG, Nebenführ 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(1), 1-15.
Lindbo J.A (2007) TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiol 145(4), 1232-1240.
Liu J, Fernie AR, Yan J (2020) The past, present, and future of maize improvement: domestication, genomics, and functional genomic routes toward crop enhancement. Plant Commun 1(1), 100010.
Mei Y, Beernink BM, Ellison EE et al. (2019a) Protein expression and gene editing in monocots using foxtail mosaic virus vectors. Plant Direct 3(11), e00181.
Mei Y, Liu G, Zhang C et al. (2019b) A sugarcane mosaic virus vector for gene expression in maize. Plant Direct 3(8), .e00158.
Mei Y, Zhang C, Kernodle BM et al. (2016) A Foxtail mosaic virus vector for virus-induced gene silencing in maize. Plant Physiol 171(2), 760-772.
Mohr I (2019) Examination of Cucumber Mosaic Virus and Sugarcane Mosaic Virus as VIGS and VOX vectors in Zea mays, MSc thesis, University of California, Davis. pp. 18-39.
Mor TS, Moon YS, Palmer KE, Mason HS (2003) Geminivirus vectors for high‐level expression of foreign proteins in plant cells. Biotechnol Bioeng 81(4), 430-437.
Nannas NJ, Dawe RK (2015) Genetic and genomic toolbox of Zea mays. Genet 199(3), 655-669.
Pirone T P (1972) Sugarcane mosaic virus. In: CMI/AAB descriptions of plant viruses. Gibbs AJ, Harrison BD, Murant AF (Eds), No. 88, Commonwealth Mycological Institute/Association of Applied Biologists, Kew, Surrey, England.
Redinbaugh MG, Louie R, Ngwira P et al. (2001) Transmission of viral RNA and DNA to maize kernels by vascular puncture inoculation. J Virol methods 98(2), 135-143.
Ritchie SW, Lui CN, Sellmer JC, Kononowicz H, Hodges TK, Gelvin SB (1993) Agrobacterium tumefaciens-mediated expression of gusA in maize tissues. Transgenic Res 2(5), 252-265.
Rosenkranz E (1978) Grasses native or adventives to the United States as new hosts of maize dwarf mosaic and sugarcane mosaic viruses. Phytopathol 68, 175-179.
Rosenkranz E (1983) Susceptibility of representative native Mississippi grasses in six subfamilies to maize dwarf mosaic virus strains A and B and sugarcane mosaic virus strain B. Phytopathol, 73(9), 1314-1321.
Schnable PS, Ware D, Fulton RS et al. (2009) The B73 maize genome: Complexity, diversity and dynamics. Sci 326, 1112–1115.
Seo JK, Lee HG, Kim KH (2009) Systemic gene delivery into soybean by simple rub-inoculation with plasmid DNA of a Soybean mosaic virus-based vector. Arch Virol 154(1), 87.
Shen WH, Hohn B (1994) Amplification and expression of the β‐glucuronidase gene in maize plants by vectors based on maize streak virus. The Plant J 5(2), 227-236.
Strable J, Scanlon MJ (2009) Maize (Zea mays): a model organism for basic and applied research in plant biology. Cold Spring Harb Protocol 2009(10), pdb-emo132.
Tao Y, Jiang L, Liu Q et al. (2013) Combined linkage and association mapping reveals candidates for Scmv1, a major locus involved in resistance to sugarcane mosaic virus (SCMV) in maize. BMC Plant Biol 13(1), 1-13.
Trzmiel K (2009) First report of Sugarcane mosaic virus infecting maize in Poland. Plant Dis 93(10), 1078-1078.
Unamba CI, Nag A, Sharma RK (2015) Next generation sequencing technologies: the doorway to the unexplored genomics of non-model plants. Front Plant Sci 6, 1074.
Vaghchhipawala Z, Rojas CM, Senthil-Kumar M, Mysore KS (2011) Agroinoculation and agroinfiltration: simple tools for complex gene function analyses. In: Plant Reverse Genetics. Humana Press, Totowa, NJ. pp. 65-76.
Wang Q, Zhang C, Wang C et al. (2017) Further characterization of Maize chlorotic mottle virus and its synergistic interaction with Sugarcane mosaic virus in maize. Sci Rep 7(1),1-10.
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(2), 259-273.
Wu H, Sparks C, Amoah B, Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21(7), 659-668.
Yang, A, He C, Zhang K (2006) Improvement of Agrobacterium-mediated transformation of embryogenic calluses from maize elite inbred lines. In Vitro Cell Dev Biol-Plant 42(3), 215-219.
Yu GR, Yan LIU, Du WP et al. (2013) Optimization of Agrobacterium tumefaciens-mediated immature embryo transformation system and transformation of glyphosate-resistant gene 2mG2-EPSPS in maize (Zea mays L.). J Integr Agric 12(12), 2134-2142.
Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Sci 296(5569), 913-916.
Zhang X, Ding X, Li Z, Wang, S (2020) Development of Tomato bushy stunt virus-based vectors for fusion and non-fusion expression of heterologous proteins in an alternative host Nicotiana excelsiana. Appl Microbiol Biotechnol 104(19), 8413-8425.