RNA Interference-Based Transgenic Maize Resistant to Maize Dwarf Mosaic Virus

Abstract

Maize dwarf mosaic virus (MDMV) is a widespread pathogenic virus that causes serious loss of yield in maize (Zea mays). RNA interference (RNAi) triggered by hairpin RNA (hpRNA) transcribed from a transgenic inverted-repeat sequence is an effective way to defend against viruses in plants. In this study, an hpRNA expression vector containing a sense arm and an antisense arm of 150 bp separated by an intron of the maize actin gene was constructed to target the P1 protein (protease) gene of MDMV and used to transform Agrobacterium tumefaciens strain EHA105. The transformed Agrobacterium strain was used to transform maize embryonic calli isolated from immature embryos by an improved culture technique. In all, 46 plants were regenerated after stringent hygromycin B selection, and 18 of them were certified to be positive by PCR amplification. Of these positive plants, 13 were grown to produce offspring, and nine were identified by Southern blotting to have the transgene integrated with one or two copies. The resistance of three T 2 lines was evaluated in a field trial of dual MDMV inoculation in two environments and was found to be improved compared with the non-transformed control. The disease indexes of the transgenic plant lines h2, 13, and h1 were not significantly different from the highly resistant control line H9-21. The viral titers of the inoculated plants were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and the result was in accord with the resistance evaluated in the field trial. The addition of uniconazole S3307 (0.25 mgl −1 ) and ABT rootpromoting powder (0.5 mg l −1 ) showed a significant improvement of hardening in regenerated plantlets, which were stronger and generated a better fibrous root system than the control. This improvement could facilitate the transgenic operation of maize.

Control of Virus Diseases in Maize

“…Pathogen-derived resistance, achieved by expressing viral proteins or RNAs in transgenic plants, is another way to incorporate resistance into crop plants (Prins et al, 2008). For maize, plants transformed with MCDV coat protein(s), the MDMV P1, or the MSV replication associated protein genes were resistant to MCDV, MDMV, or MSV, respectively, under controlled conditions (Liu, Tan, Li, Zhang, & He, 2009;McMullen, Roth, & Townsend, 1996;Shepherd et al, 2007;Zhang, Fu, Gou, Wang, & Li, 2010). Interestingly, transgenic plants expressing SCMV (MDMV-B) were resistant to that virus, but also had pleiotropic resistance to MCMV (Murry et al, 1993).…”

Improvement of resistance to maize dwarf mosaic virus mediated by transgenic RNA interference

“…According to Zhang et al (2010), embryonic calli were produced from immature embryos of inbred line "18-599" on the N6 medium, improved by increasing the Ca 2+ content from 1.13 to 5 mmol/L and the supplement of 0.25 mg/L uniconazole S3307 (Fu et al, 2005), and transformed by co-cultivation with the Agrobacterium strain EH␣105 harboring the expression vectors pASC404 and pASC100, respectively. After being screened on the selection medium containing hygromycin B at the gradient concentrations 5, 10 and 15 mg/L for 20 days each, the positive calli were differentiated on the regeneration medium [N6 salt mixture (Chu et al, 1975), B5 vitamins (Gamborg et al, 1968), 30 g/L sucrose, 100 mg/L casein hydrolysate, 1 mg/L kinetin, 6 g/L agar, pH 5.8] with supplement of 0.25 mg/L uniconazole S3307 and 0.5 mg/L ABT root-promoting powder (Zhonglin Jiacheng Sci-Tech Co., Ltd., Beijing, China) at 27 • C under illumination of 1000 E/m 2 s for a photoperiod of 12 h light/12 h dark.…”

“…In recent years, RNA interference (RNAi) triggered by selfcomplementary hairpin RNA (hpRNA) transcribed from the transgenic inverted-repeat sequence has been proved to be more efficient to generate MDMV resistance for its straight-forward natural protection mechanism Gan et al, 2010;Zhang et al, 2010). However, different transgenic lines still showed a wide range of phenotypes from extreme resistance to susceptibility, even they were isolated from the same transformation event.…”

Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

“…A few attempts have been made in animal and plant models where the antiviral trait was installed by transgenesis to confer stable protection to the transformed individuals and to their progeny. Successes have been reached in plants ( Bucher et al 2006;Bonfim et al 2007;Zhang 2010) but, to our knowledge, no case has yet been reported in animals showing a stable and robust protection against a virus after a RNAi-aided antiviral trait was introduced through germline transformation.The baculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV), is a major pathogen that affects silkworm rearings and hampers silk cocoon production in Asia. In India alone .50% of silk cocoon crop losses are attributed to baculovirus infection (Khurad et al 2006).…”

“…A few attempts have been made in animal and plant models where the antiviral trait was installed by transgenesis to confer stable protection to the transformed individuals and to their progeny. Successes have been reached in plants ( Bucher et al 2006;Bonfim et al 2007;Zhang 2010) but, to our knowledge, no case has yet been reported in animals showing a stable and robust protection against a virus after a RNAi-aided antiviral trait was introduced through germline transformation.…”