Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants.

KEY MESSAGE: Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression. Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.

Hen Egg White Lysozyme (HEWL) Confers Resistance to Verticillium Wilt in Cotton by Inhibiting the Spread of Fungus and Generating ROS Burst.

Verticillium wilt is a soil-borne vascular disease caused by the fungal pathogen Verticillium dahliae. It causes great harm to upland cotton (Gossypium hirsutum) yield and quality. A previous study has shown that Hen egg white lysozyme (HEWL) exerts strong inhibitory activity against V. dahliae in vitro. In the current study, we introduced the HEWL gene into cotton through the Agrobacterium-mediated transformation, and the exogenous HEWL protein was successfully expressed in cotton. Our study revealed that HEWL was able to significantly inhibit the proliferation of V. dahlia in cotton. Consequently, the overexpression of HEWL effectively improved the resistance to Verticillium wilt in transgenic cotton. In addition, ROS accumulation and NO content increased rapidly after the V. dahliae inoculation of plant leaves overexpressing HEWL. In addition, the expression of the PR genes was significantly up-regulated. Taken together, our results suggest that HEWL significantly improves resistance to Verticillium wilt by inhibiting the growth of pathogenic fungus, triggering ROS burst, and activating PR genes expression in cotton.

A novel tasi RNA-based micro RNA-induced gene silencing strategy to tackle multiple pests and pathogens in cotton (Gossypium hirsutum L.).

MAIN CONCLUSION: This study demonstrates the combinatorial management of multiple pests through a trans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy. Transgenic cotton events demonstrated improved efficacy against cotton leaf curl disease, cotton leaf hopper and root-knot nematode. Cotton (Gossypium hirsutum L.), an important commercial crop grown worldwide is confronted by several pests and pathogens, thus reiterating interventions for their management. In this study, we report, the utility of a novel Arabidopsis miRNA173-directed trans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy for the simultaneous management of cotton leaf curl disease (CLCuD), cotton leaf hopper (CLH; Amrasca biguttula biguttula) and root-knot nematode (RKN, Meloidogyne incognita). Cotton transgenics were developed with the MIGS construct targeting a total of 7 genes by an apical meristem-targeted in planta transformation strategy. Stable transgenics were selected using stringent selection pressure, molecular characterization and stress-specific bio-efficacy studies. We identified 8 superior events with 50-100% resistance against CLCuD, while reduction in the root-knot nematode multiplication factor in the range of 35-75% confirmed resistance to RKN. These transgenic cotton events were also detrimental to the growth and development of CLH, as only 43.3-62.5% of nymphs could survive. Based on the corroborating evidences obtained by all the bioefficacy analyses, 3 events viz., L-75-1, E-27-11, E-27-7 were found to be consistent in tackling the target pests. To the best of our knowledge, this report is the first of its kind demonstrating the possibility of combinatorial management of pests/diseases in cotton using MIGS approach. These identified events demonstrate immense utility of the strategy towards combinatorial stress management in cotton improvement programs.

Silencing of a LIM gene in cotton exhibits enhanced resistance against Apolygus lucorum.

Plant bugs (Miridae species) have become major agricultural pests that cause increasing and severe economic damage. Plant-mediated RNA interference (RNAi) is emerging as an eco-friendly, efficient, and reliable strategy for pest management. In this study, we isolated and characterized a lethal gene of Apolygus lucorum and named it Apolygus lucorum LIM (AlLIM), which produced A. lucorum mortality rates ranging from 38% to 81%. Downregulation of the AlLIM gene expression in A. lucorum by injection of a double-stranded RNA (dsRNA) led to muscle structural disorganization that resulted in metamorphosis deficiency and increased mortality. Then we constructed a plant expression vector that enabled transgenic cotton to highly and stably express dsRNA of AlLIM (dsAlLIM) by Agrobacterium-mediated genetic transformation. In the field bioassay, dsAlLIM transgenic cotton was protected from A. lucorum damage with high efficiency, with almost no detectable yield loss. Therefore, our study successfully provides a promising genetically modified strategy to overpower A. lucorum attack.

Towards doubling fibre yield for cotton in the semiarid agricultural area by increasing tolerance to drought, heat and salinity simultaneously.

Abiotic stresses such as extreme temperatures, water-deficit and salinity negatively affect plant growth and development, and cause significant yield losses. It was previously shown that co-overexpression of the Arabidopsis vacuolar pyrophosphatase gene AVP1 and the rice SUMO E3 ligase gene OsSIZ1 in Arabidopsis significantly increased tolerance to multiple abiotic stresses and led to increased seed yield for plants grown under single or multiple abiotic stress conditions. It was hypothesized that there might be synergistic effects between AVP1 overexpression and OsSIZ1 overexpression, which could lead to substantially increased yields if these two genes are co-overexpressed in real crops. To test this hypothesis, AVP1 and OsSIZ1 were co-overexpressed in cotton, and the impact of OsSIZ1/AVP1 co-overexpression on cotton's performance under normal growth and multiple stress conditions were analysed. It was found that OsSIZ1/AVP1 co-overexpressing plants performed significantly better than AVP1-overexpressing, OsSIZ1-overexpressing and wild-type cotton plants under single, as well as under multiple stress conditions in laboratory and field conditions. Two field studies showed that OsSIZ1/AVP1 co-overexpressing plants produced 133% and 81% more fibre than wild-type cotton in the dryland conditions of West Texas. This research illustrates that co-overexpression of AVP1 and OsSIZ1 is a viable strategy for engineering abiotic stress-tolerant crops and could substantially improve crop yields in low input or marginal environments, providing a solution for food security for countries in arid and semiarid regions of the world.

The SikCuZnSOD3 gene improves abiotic stress resistance in transgenic cotton.

The expression of a gene encoding peroxisomal Cu-Zn superoxide dismutase from Saussurea involucrata Kar. et Kir. was induced by low temperature, PEG6000 treatment, and NaCl stress. To investigate the role of SikCuZnSOD3 in the mitigation of abiotic stress, we used Agrobacterium-mediated transformation to create transgenic cotton that overexpressed SikCuZnSOD3. Phenotypic analysis of T4 generation transgenic lines showed that they generally grew better than wild-type cotton under low temperature, PEG6000 treatment, and NaCl stress. Although there were no significant differences under control conditions, transgenic plants exhibited greater survival, fresh weight, and dry weight than wild-type plants under all three stress treatments. Additional physiological analyses demonstrated that the transgenic cotton had higher relative water content, proline and soluble sugar contents, and activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase), as well as lower relative conductivity, malondialdehyde content, and H2O2 and O2- accumulation. More importantly, overexpression of SikCuZnSOD3 increased the yield of cotton fiber. Our results confirm that the overexpression of SikCuZnSOD3 can improve the abiotic stress resistance of cotton by increasing the activity of antioxidant enzymes, maintaining ROS homeostasis, and reducing cell membrane damage. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01217-0.

A Modified Actin (Gly65Val Substitution) Expressed in Cotton Disrupts Polymerization of Actin Filaments Leading to the Phenotype of Ligon Lintless-1 (Li1) Mutant.

Dynamic remodeling of the actin cytoskeleton plays a central role in the elongation of cotton fibers, which are the most important natural fibers in the global textile industry. Here, a high-resolution mapping approach combined with comparative sequencing and a transgenic method revealed that a G65V substitution in the cotton actin Gh_D04G0865 (GhACT17D in the wild-type) is responsible for the Gossypium hirsutum Ligon lintless-1 (Li1) mutant (GhACT17DM). In the mutant GhACT17DM from Li1 plant, Gly65 is substituted with valine on the lip of the nucleotide-binding domain of GhACT17D, which probably affects the polymerization of F-actin. Over-expression of GhACT17DM, but not GhACT17D, driven by either a CaMV35 promoter or a fiber-specific promoter in cotton produced a Li1-like phenotype. Compared with the wild-type control, actin filaments in Li1 fibers showed higher growth and shrinkage rates, decreased filament skewness and parallelness, and increased filament density. Taken together, our results indicate that the incorporation of GhACT17DM during actin polymerization disrupts the establishment and dynamics of the actin cytoskeleton, resulting in defective fiber elongation and the overall dwarf and twisted phenotype of the Li1 mutant.

A Na+/H+ antiporter, K2-NhaD, improves salt and drought tolerance in cotton (Gossypium hirsutum L.).

KEY MESSAGE: Overexpression of K2-NhaD in transgenic cotton resulted in phenotypes with strong salinity and drought tolerance in greenhouse and field experiments, increased expression of stress-related genes, and improved regulation of metabolic pathways, such as the SOS pathway. Drought and salinity are major abiotic stressors which negatively impact cotton yield under field conditions. Here, a plasma membrane Na+/H+ antiporter gene, K2-NhaD, was introduced into upland cotton R15 using an Agrobacterium tumefaciens-mediated transformation system. Homozygous transgenic lines K9, K17, and K22 were identified by PCR and glyphosate-resistance. TAIL-PCR confirmed that T-DNA carrying the K2-NhaD gene in transgenic lines K9, K17 and K22 was inserted into chromosome 3, 19 and 12 of the cotton genome, respectively. Overexpression of K2-NhaD in transgenic cotton plants grown in greenhouse conditions and subjected to drought and salinity stress resulted in significantly higher relative water content, chlorophyll, soluble sugar, proline levels, and SOD, CAT, and POD activity, relative to non-transgenic plants. The expression of stress-related genes was significantly upregulated, and this resulted in improved regulation of metabolic pathways, such as the salt overly sensitive pathway. K2-NhaD transgenic plants growing under field conditions displayed strong salinity and drought tolerance, especially at high levels of soil salinity and drought. Seed cotton yields in transgenic line were significantly higher than in wild-type plants. In conclusion, the data indicate that K2-NhaD transgenic lines have great potential for the production of stress-tolerant cotton under field conditions.

Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker.

Cotton (Gossypium hirsutum L.), a mercantile crop plant, is grown worldwide for fiber and seed oil. As with other economically important crops, cotton is bogged down with many biotic and abiotic stress factors. Towards this, genetic engineering offers numerous protocols to engineer plants for better resilience. However, recalcitrance of cotton to plant tissue culture has been the major constraint for successful in vitro regeneration. Hence, alternate methods that evade tissue culture regeneration have been envisaged. Non tissue culture-based in planta transformation strategies are in vogue due to amenability and ease in the generation of transgenic plants. In the present study, we demonstrate the utility of an in planta transformation protocol and establishment of a stringent selection agent-based screening for the identification of transgenics. The genotype independent nature of the protocol was validated in cotton cv. Pusa 8-6 using GFP. Preliminary transformation efficiency of 28% was achieved with a screening efficiency of 20% in the presence of hygromycin. The proof of T-DNA integration by various molecular and expression analysis in T1 and T2 generations proved that this technique can be employed to generate transgenic cotton.

Neighbouring crop diversity mediates the effect of Bt cotton on insect community and leaf damage in fields.

Effects of large-scale cultivation of transgenic crops on agricultural biodiversity remain unclear, particularly in the context of complex ecological interactions between transgenic crops and other organisms. Here we conducted a comprehensive survey to investigate the number of species, population abundance, community evenness and dominance of insects and weeds as well as leaf damage to weeds in Bt and non-Bt cotton fields at 27 sites across northern China. The role of neighbouring crop diversity around cotton fields in controlling insects and weeds in the cotton fields was also assessed. In addition, we conducted a 3-year field experiment to verify the results of the survey. Weed diversity in Bt and non-Bt cotton fields was similar, but the species number and diversity indices of insects are significantly decreased in Bt fields aligning with reduced leaf damage to broadleaf plant species including cotton as well as crops in neighbouring plots. The leaf damage to Bt and non-Bt cotton negatively associates with the diversity of neighbouring crops in cotton fields. Our study demonstrates the neighbouring crop diversity mediates the effects of Bt crops on agricultural diversity in complex interactions among transgenic crops, in-field weed and insect communities, and neighbouring crops.

Resistance status of Helicoverpa armigera against Bt cotton in Pakistan.

Transgenic cotton expressing the toxin Cry1Ac from Bacillus thuringiensis L. (Bt) is widely cultivated in Pakistan after its formal approval in 2010. The exposure of the local target pests to the Cry1Ac endotoxin for this duration might have changed the baseline susceptibility. To probe the status of resistance in one of the main target pests, Helicoverpa armigera, field-collected larvae were reared in the lab for conducting leaf fed bioassays. Twenty-six cotton accessions collected from farmers, including 25 Bt-cotton and one non-Bt, were tested to quantify the level of Cry1Ac, an insecticidal crystalline protein (ICP), in leaves of lower, middle and upper canopies of plants. The concentration of ICP was tested through Enzyme-linked Immunosorbent Assay and found significantly variable (P < 0.01) between and within accessions. The highest mean expression was observed in Accession-2 and Accession-4, while the lowest in Accession-21 and Accession-19. Among fresh leaf tissues from different parts of the plant, the highest mean expression was recorded at 60 days after sowing in upper canopy leaves of cotton accessions, which decreased in lower parts of the plant with the lowest mean expression in lower canopy leaves. Laboratory bioassays, to calculate lethal dose, for H. armigera showed that LD50 and LD95 were 0.62 µg/g and 1.59 µg/g of fresh tissue weight, respectively. A strong positive correlation also exists between the levels of Cry1Ac protein and insect mortality (r = 0.84). These findings suggested the future risk of cultivation of Bt cotton, carrying single Cry1Ac gene, in Pakistan, as resistance surging in H. armigera against Cry protein. These results may also have significant implications for the resistance management in Bt crops, especially cotton, in future.

Up-regulation of GhTT2-3A in cotton fibres during secondary wall thickening results in brown fibres with improved quality.

Brown cotton fibres are the most widely used naturally coloured raw materials for the eco-friendly textile industry. Previous studies have indicated that brown fibre pigments belong to proanthocyanidins (PAs) or their derivatives, and fibre coloration is negatively associated with cotton productivity and fibre quality. To date, the molecular basis controlling the biosynthesis and accumulation of brown pigments in cotton fibres is largely unknown. In this study, based on expressional and transgenic analyses of cotton homologs of ArabidopsisPA regulator TRANSPARENT TESTA 2 (TT2) and fine-mapping of the cotton dark-brown fibre gene (Lc1), we show that a TT2 homolog, GhTT2-3A, controls PA biosynthesis and brown pigmentation in cotton fibres. We observed that GhTT2-3A activated GhbHLH130D, a homolog of ArabidopsisTT8, which in turn synergistically acted with GhTT2-3A to activate downstream PA structural genes and PA synthesis and accumulation in cotton fibres. Furthermore, the up-regulation of GhTT2-3A in fibres at the secondary wall-thickening stage resulted in brown mature fibres, and fibre quality and lint percentage were comparable to that of the white-fibre control. The findings of this study reveal the regulatory mechanism controlling brown pigmentation in cotton fibres and demonstrate a promising biotechnological strategy to break the negative linkage between coloration and fibre quality and/or productivity.

Bacterial communities under long-term conventional and transgenic cotton farming systems using V3-V5 and V5-V9 of 16s rDNA.

Understanding the community structure of soil microbes is required to evaluate the potential effects of genetically modified (GM) plants on ecological environments. Bacterial communities in soil planted with conventional cotton (CC) and transgenic cultivar (TC) in a natural ecosystem for three years were characterized by 454 pyrosequencing of the V3-V5 and V5-V9 regions of 16S rDNA from June to September 2013. V3-V5 and V5-V9 regions yielded a total of 12,848 and 10,541 OTUs, respectively. The V5-V9 amplicon was additionally used to detect phyla that were poorly sequenced by V3-V5 (such as Chlamydiae, Crenarchaeota and Archaea). Among the species detected by each primer pair, 46% of the species identified from V3-V5 and 60% of those identified from V5-V9 were detected by both primer pairs. Although distinct bacterial compositions existed between the two amplified regions, statistical analysis revealed no significant difference in the diversity indexes or phylogenetic patterns in TC versus compared to those in the CC control. Further, clustering analysis in both regions indicated that there was no unambiguous aggregation in TC compared to that in CC control. Of all 26 phyla detected by both regions, each region detected 2 distinct phyla exhibiting significant variations in abundance. The species unique to each treatment field accounted for less than 27% of all species and were rare taxa (abundance < 0.15%). However, a small fraction of diagnostic taxa with specific ecological functions differed significantly between TC and CC. These differences were not driven by any obvious environmental factors. The results established a comprehensive inventory of the bacterial communities associated with GM plants and indicated that transgenic cotton may not significantly affect soil microorganisms compared with conventional cotton over a three-year period. Furthermore, diagnostic taxa were provided for monitoring the perturbation in soil, but further verification in future studies is required.

Overexpression of the Rice SUMO E3 Ligase Gene OsSIZ1 in Cotton Enhances Drought and Heat Tolerance, and Substantially Improves Fiber Yields in the Field under Reduced Irrigation and Rainfed Conditions.

The Arabidopsis SUMO E3 ligase gene AtSIZ1 plays important roles in plant response to abiotic stresses as loss of function in AtSIZ1 leads to increased sensitivity to drought, heat and salt stresses. Overexpression of the AtSIZ1 rice homolog, OsSIZ1, leads to increased heat and drought tolerance in bentgrass, suggesting that the function of the E3 ligase SIZ1 is highly conserved in plants and it plays a critical role in abiotic stress responses. To test the possibility that the SUMO E3 ligase could be used to engineer drought- and heat-tolerant crops, the rice gene OsSIZ1 was overexpressed in cotton. We report here that overexpression of OsSIZ1 in cotton results in higher net photosynthesis and better growth than wild-type cotton under drought and thermal stresses in growth chamber and greenhouse conditions. Additionally, this tolerance to abiotic stresses was correlated with higher fiber yield in both controlled-environment and field trials carried out under reduced irrigation and rainfed conditions. These results suggest that OsSIZ1 is a viable candidate gene to improve crop yields under water-limited and rainfed agricultural production systems.

ABP9, a maize bZIP transcription factor, enhances tolerance to salt and drought in transgenic cotton.

MAIN CONCLUSION: ABP9 , encoding a bZIP transcription factor from maize, enhances tolerance to multiple stresses and may participate in the ABA signaling pathway in transgenic cotton by altering physiological and biochemical processes and stress-related gene expression. Abiotic stresses, such as soil salinity and drought, negatively affect growth, development, and yield in cotton. Gene ABP9, which encodes a bZIP transcription factor, binds to the abscisic acid (ABA)-responsive-element (ABRE2) motif of the maize catalase1 gene. Its expression significantly improves tolerance in Arabidopsis to multiple abiotic stresses, but little is known about its role in cotton. In the present study, the ABP9 gene was introduced into upland cotton (Gossypium hirsutum L.) cultivar R15 by Agrobacterium tumefaciens-mediated transformation, and 12 independent transgenic cotton lines were obtained. Cotton plants over-expressing ABP9 have enhanced tolerance to salt and osmotic stress. Under stress, they developed better root systems in a greenhouse and higher germination, reduced stomatal aperture, and stomatal density in a growth chamber. Under drought conditions, survival rate and relative water content (RWC) of transgenic cotton were higher than those of R15 plants. Under salt and osmotic stresses, chlorophyll, proline, and soluble sugar contents significantly increased in transgenic cotton leaves and the malondialdehyde (MDA) content was lower than in R15. Overexpression of ABP9 also enhanced oxidative stress tolerance, reduced cellular levels of reactive oxygen species (ROS) through increased activities of antioxidative enzymes, and alleviated oxidative damage to cell. Interestingly, ABP9 over-expressing cotton was more sensitive to exogenous ABA than R15 at seed germination, root growth, stomatal aperture, and stomatal density. Moreover, ABP9 overexpression upregulated significantly the transcription levels of stress-related genes such as GhDBP2, GhNCED2, GhZFP1, GhERF1, GhHB1, and GhSAP1 under salt treatment. Conjointly, these results showed that overexpression of ABP9 conferred enhanced tolerance to multiple abiotic stresses in cotton. The stress-tolerant transgenic lines provide valuable resources for cotton breeding.

A transgenic strategy for controlling plant bugs (Adelphocoris suturalis) through expression of double-stranded RNA homologous to fatty acyl-coenzyme A reductase in cotton.

Plant bugs (Miridae species), which are sap-sucking insects, have emerged as major pests of cotton in China. Most Miridae species are not sensitive to commercial Bacillus thuringiensis (Bt) cotton, resulting in significant economic losses and an increased application of insecticide, which eventually may compromise the future of Bt cotton. We demonstrate that FATTY ACYL-COA REDUCTASE (AsFAR) plays an essential role in the reproduction of the bug Adelphocoris suturalis. Down-regulation of AsFAR expression by injection of double-stranded RNA suppresses ovarian development and female fertility, resulting in females producing few viable offspring. To determine the viability of an RNA interference approach to limit FAR expression and reproductive ability in A. suturalis, a dsRNA targeting the AsFAR gene (dsAsFAR) of A. suturalis was expressed in transgenic cotton plants. AsFAR transcription levels were significantly downregulated in A. suturalis feeding on the transgenic plants. In contained field trials, the transgenic cotton lines significantly suppressed the development of A. suturalis populations and were resistant to damage caused by plant bug infestation. These results suggest a new strategy for the management of plant bug pests of cotton.

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm.

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for pest resistance management in China.

Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests.

Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for integrated pest management in China.

Transgenic Cotton Plants Expressing the HaHR3 Gene Conferred Enhanced Resistance to Helicoverpa armigera and Improved Cotton Yield.

RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3, a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA-HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing dsHaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera. Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls.

Response of AtNPR1-expressing cotton plants to Fusarium oxysporum f. sp. vasinfectum isolates.

In our earlier investigation, we had demonstrated that transgenic cotton plants expressing AtNPR1 showed significant tolerance to Fusarium oxysporum f. sp. vasinfectum, isolate 11 (Fov11) and several other pathogens. The current study was designed to further characterize the nature of the protection provided by AtNPR1 expression and its limitations. Green Fluorescent Protein-expressing Fov11 was generated and used to study the progression of the disease within the plant. The results show that the spread of the pathogen was slower in the AtNPR1-transformants compared to the wild type plants. Transcript analysis in the seedling root and hypocotyl showed that the transgenic lines are capable of launching a stronger defense response when infected with Fov11. We further confirmed that AtNPR1 transformants showed greater degree of tolerance to Fov11. However, little or no protection was observed against a related, but more virulent isolate, Fov43, and a highly virulent isolate, CA9.