Figure 1.
Symptoms of maize rough dwarf disease (MRDD) caused by Rice black-streaked dwarf virus (RBSDV) in the field.
(A) MRDD symptoms on maize heavily infected with RBSDV under natural field conditions. (B and C) Lower surfaces of a RBSDV infected leaf showing dark leaf greening and vein clearing with small white enations and plant dwarfing. Note: Plants were photographed at 4 weeks after feeding access by viruliferous planthoppers. (D) Effects of different planting dates on MRDD incidence. The center plot shows MRDD in heavily infected RBSDV maize that had been planted in northern China at the end of May, 2011 before harvest of surrounding wheat fields. Under these conditions, high concentrations of planthoppers migrate from maturing wheat to emerging maize seedlings and transmit RBSDV. The vigorous plants on each side of the RBSDV-infected plants were planted on June 18, 2011 after wheat in surrounding was harvested and most of the planthoppers had dispersed. The infected plants failed to develop tassels and had very small cobs with greatly reduced seed set, and often died. Plants were photographed on Sep. 7, 2011. Note: This area of China is in a wheat-maize rotation region and maize planted before wheat harvesting generally develops very high levels of MRDD because planthoppers preferentially move from mature wheat to new maize seedlings.
Figure 2.
Construction of plant expression vectors and molecular detection of transgenes.
(A) Schematic diagram of the plant expression vectors pAMM2024 and pAMM2025, which are derivatives of vector pCB301 [68]. The bacterial RNase III rnc (pAMM2024) and the E117K (rnc70) mutant (pAMM2025) genes were inserted between the rice Actin promoter and the nopaline synthase 3′ terminator (Tnos). Enh-35S, enhanced 35S promoter with a duplicated enhancer; NPT II, neomycin phosphotransferase II gene; RB, right border; LB, left border. (B and C) PCR and PCR-southern blot detection of transgenes. T0 maize plants were detected by PCR first (B), and the 592 bp PCR products from positive plants were confirmed by southern blot using the rnc-specific probe (C). P: The pAMM2025 plasmid was used as a positive control; M: λDNA/EcoRΙ+Hind III markers; NT: non-transgenic plants.
Table 1.
PCR detection of regenerated maize carrying the rnc and mutant (rnc70) transgenes.
Figure 3.
Screening fields with an unusually high disease incidence in the area.
The screening field depicted above had serious infections of RBSDV in the surrounding wheat crops and in weeds. The screening fields were chosen because they provided wheat and weed habitats sufficient to permit growth of large populations of the small brown planthopper before transgenic maize was planted in the spring (A). Consequently, high densities RBSDV-infected planthoppers migrated into the fields after planting of the test plants in the summer (B). After maize was planted, high levels of infection were observed in the control non-transgenic plants and in the highly susceptible maize variety, and high densities of RBSDV-infected planthoppers were detected in all plants. To mediate bio-safety protection, an isolation belt consisting of surrounding pear trees was considered in the initial design to provide protection against pollen flow and spread of transgenic maize [74]. The photographs were taken on May 15 (A) and June 12 (B), 2010 respectively.
Figure 4.
MRDD symptoms scored on a 0 to 4 disease scale.
Disease scores were assessed as follows: 0 (no visible symptoms), 1 (4/5 of normal healthy plant height, with white streaks throughout the veins of upper leaves), 2 (2/3 of healthy plant height, with visible dwarfing, combined with dark greening and white streak symptoms throughout the plant), 3 (more extreme dwarfing; ∼1/2 of healthy plant height, delayed tassels that failed to develop pollen, and very small cobs; and 4 (1/3 of healthy plant height, with no cobs or dwarf cobs containing only a few seed, or plant death). The inset shows cobs taken from plants with scores of 0 to 4, respectively.
Table 2.
Numbers of MRDD resistant and susceptible transgenic progeny in the field.
Table 3.
Field evaluations of ND60 and ND67 transgenic rnc70 and control lines for MRDD resistance in 2008, 2009 and 2010.
Figure 5.
Field showing MRDD in transgenic maize plants with (rnc70) resistance to RBSDV infection.
(A) Comparison of immature transgenic plants (ND67-1-3-5-5) that are highly resistant to RBSDV compared to non-transgenic Z31 plants that are susceptible to RBSDV. The plants were photographed at 9 weeks after sowing. Note: The plants in the background are part of a protective buffer designed to ensure the biosafety of transgenic plants. (B and C) Appearance of mature transgenic plants (ND67-1-3-5-5) and non-transgenic Z31 maize near harvest time. Note the differences in sizes and appearances of the transgenic and non-transgenic plants. ND67-1-3-5-5 leaves maintained a normal green color, but leaves of the non-transgenic Z31 stunted plants developed strong yellow chlorotic regions.
Table 4.
MRDD symptoms on ND60-2-5-4-1 and ND67-1-3-5-5 T12 transgenic lines at 2 weeks after access feeding by viruliferous planthoppers.
Figure 6.
ELISA and western blotting to assess the resistance of transgenic maize under artificial inoculation conditions.
(A) ELISA of three independent lines derived from ND67-1-3-5-5 and ND60-2-5-4-1 transformations, compared with healthy and infected non-transgenic Z31 controls. The OD450 values illustrate the accumulation of RBSDV and the data presented are the means from three independent samples of each line with the standard deviation shown as error bars. The transgenic lines were compared with infected non-transgenic lines, and differences within each line from infected non-transgenic lines were tested for significance by Student’s t-tests (*P<0.05). (B) Western blot analysis of the same samples as those used for the ELISA. Protein was isolated from the infected transgenic and non-transgenic maize leaves. Antibodies specific for the RBSDV P10 protein (anti-P10) [73] was used for RBSDV detection, and healthy Z31 was used as the negative control. The bottom panel shows a protein loading control with Coomassie blue staining.
Figure 7.
Molecular detection of transgene expression in T12 transgenic maize and assays for RNC70 binding to RBSDV dsRNA.
(A and B) The rnc70 gene was stably integrated into the genomes of transgenic maize, as shown by southern blot analysis with an rnc70 probe, excised by EcoR I and Hind III respectively (A). Expression of the RNC70 protein in the transgenic maize was confirmed by western blot analysis using anti-RNC70 antibodies (B). (C and D) In the binding assays, the RNC70 and RNC proteins were expressed and purified in E. coli (C). RBSDV dsRNA (75 ng) was incubated with RNC (0.06 µg) and RNC70 (0.11, 0.22 or 0.33 µg) for 30 min at 37°C and separated on a 5% native polyacrylamide gel. In contrast to RNC, in which RBSDV dsRNA was completely cleaved, the rates of RBSDV dsRNA migration were progressively reduced after incubation with increasing amounts of the RNC70 protein (D).