Insect resistance management in Bacillus thuringiensis cotton by MGPS(multiple genes pyramiding and silencing)

The introduction of Bacillus thuringiensis(Bt)cotton has reduced the burden of pests without harming the environment and human health.However,the efficacy of Bt cotton has decreased due to field-evolved resistance in insect pests over time.In this review,we have discussed various factors that facilitate the evolution of resistance in cotton pests.Currently,different strategies like pyramided cotton expressing two or more distinct Bt toxin genes,refuge strategy,releasing of sterile insects,and gene silencing by RNAi are being used to control insect pests.Pyramided cotton has shown resistance against different cotton pests.The multiple genes pyramiding and silencing(MGPS)approach has been proposed for the management of cotton pests.The genome information of cotton pests is necessary for the development of MGPS-based cotton.The expression cassettes against various essential genes involved in defense,detoxification,digestion,and development of cotton pests will successfully obtain favorable agronomic characters for crop protection and production.The MGPS involves the construction of transformable artificial chromosomes,that can express multiple distinct Bt toxins and RNAi to knockdown various essential target genes to control pests.The evolution of resistance in cotton pests will be delayed or blocked by the synergistic action of high dose of Bt toxins and RNAi as well as compliance of refuge requirement.

Comparative Analysis of Cry1Ac Toxin Oligomerization and Pore Formation Between Bt-Susceptible and Bt-Resistant Helicoverpa armigera Larvae

With the long-term use of Bacillus thuringiensis （Bt） insecticide and expansion of CrylA-expressing transgenic plants, some insect pests have developed resistance to Bt in open fields, greenhouses, and in the laboratory. Bt resistance is complex and there appear to be different ways for resistance development. Understanding the Bt resistance mechanisms is critical to prolong its usefulness. In this article, Bt receptors, the cadherin and aminopeptidase N （APN）, in brush border membrane vesicles （/3BMV） of Helicoverpa armigera were examined in both CrylAc-susceptible （96S） and CrylAc- resistant （LF120） strains, to compare CrylAc toxin oligomerization and pore formation in these two strains. CrylAc toxin oligomerization and pore formation in these two strains were compared. Results showed that cadherin and aminopeptidase N proteins could express normally in both susceptible and resistant 11. armigera strains. The ability to form CrylAc oligomers and ion channels on BBMVs was also not significantly different between these two strains.

Selection,effective dominance,and completeness of Cry1A.105/Cry2Ab2 dual-protein resistance in Helicoverpa zea(Boddie)(Lepidoptera:Noctuidae)

In the U.S.,Helicoverpa zea(Boddie)is a major pest targeted by both transgenic maize and cotton expressing Bacillus thuringiensis(Bt)proteins.Resistance of insect to Bt maize and cotton containing cry1A and cry2A genes has widely occurred in the U.S.In this study,two trials were performed to investigate larval survival and development of a Cry1A.105/Cry2Ab2 dual-protein resistant(VT2P-RR),a susceptible,and an F1 heterozygous(VT2P-RS)populations of H.zea on ears of nine Bt and three non-Bt maize hybrids.The Bt maize hybrids evaluated represent five common pyramided traits expressing two or three of the Cry1A.105,Cry1Ab,Cry1F,Cry2Ab2,and Vip3Aa20 proteins.In the laboratory,neonates of the three H.zea populations were inoculated on silks of ears collected from maize at R1-R2 plant stages;and larval survivorship was checked 10 d after neonate release.All three insect populations survived normally on non-Bt maize ears.Varied numbers of VT2P-RR and VT2P-RS survived on ears of Cry1A.105/Cry2Ab2 maize,while all larvae of the three populations died or could not develop on ears of Vip3Aa20-expressing maize.The results demonstrated that the dual-protein resistant H.zea was not cross-resistant to Vip3Aa20-expressing maize,and thus traits with vip3Aa20 gene should be effective to manage Cry1A.105/Cry2Ab2-resistant H.zea.The resistance in VT2P-RR was determined to be incomplete on Cry1A.105/Cry2Ab2 maize.The effective dominance levels varied greatly,from recessive to incompletely dominant,depending on maize hybrids and trials,suggesting that proper selection of maize hybrids could be important for mitigating the Cry1A.105/Cry2Ab2 resistance.The data generated should aid in modeling multiple-protein Bt resistance in H.zea.

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda

The fall armyworm(FAW),Spodoptera frugiperda,is a major pest native to the Americas that has recently invaded the Old World.Point mutations in the target-site proteins acetylcholinesterase-1(ace-1),voltage-gated sodium channel(VGSC)and ryanodine receptor(RyR)have been identified in S.frugiperda as major resistance mechanisms to organophosphate,pyrethroid and diamide insecticides respectively.Mutations in the adenosine triphosphate-binding cassette transporter C2 gene(ABCC2)have also been identified to confer resistance to Cry IF protein.In this study,we applied a whole-genome sequencing(WGS)approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China,Malawi,Uganda and Brazil.This approach revealed three amino acid substitutions(A201S,G227A and F290V)of S.frugiperda ace-1,which are known to be associated with organophosphate resistance.The Brazilian population had all three ace-1 point mutations and the 227A allele(mean frequency=0.54)was the most common.Populations from China,Malawi and Uganda harbored two of the three ace-1 point mutations(A201S and F290V)with the 290V allele(0.47-0.58)as the dominant allele.Point mutations in VGSC(T929I,L932F and L1014F)and RyR(I4790M and G4946E)were not detected in any of the 150 individuals.A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations.Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations,but also provide insights for improvement of resistance management tactics in S.frugiperda.

Detection and monitoring of insect resistance to transgenic Bt crops

Transgenic crops expressing Bacillus thuringiensis （Bt） endotoxins havebecome one of the most important tools for managing corn and cotton insect pests in the US and other countries. The widespread adoption of transgenic Bt crops could place a high degree of selection pressure on the target insect populations and accelerate development of resistance, raising concerns about the long-term durability of Bt plants as an effective pest management tool. Conservation of Bt susceptibility in insects has become one of the most active research areas in modern agriculture. One of the key factors for a successful Bt resistance management plan is to have a cost-effective monitoring system that can provide information on. （i） the initial Bt resistance allele frequencies at low levels in field insect populations; and （ii） early shifts in Bt resistance allele frequencies so that proactive measures for managing resistance can be deployed well before field control failures. Developing such a monitoring program has been difficult because： （i） resistance traits that occur at very low frequencies are hard to detect; （ii） many factors affect the sensitivity and accuracy of a Bt resistance monitoring program; and （iii） monitoring resistance is costly. Several novel methods for detecting Bt resistance alleles developed during the last decade have made a cost-effective monitoring system possible. Future studies should focus on how to improve and standardize the methodologies for insect sampling and Bt resistance detection.

Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene

Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis （Bt） insecticidal genes, CrylAc and Cryllg, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） gene （GIO） were combined into a single transferred DNA （T-DNA） fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice.