Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

Thuanne Pires Ribeiro,Leonardo Lima Pepino Macedo,Isabela Tristan Lourenço‐Tessutti,Maria Fatima Grossi‐De‐Sa,Maria Eugênia Lisei‐De‐Sá,Wagner Alexandre Lucena,Fabricio Barbosa Monteiro Arraes,Maria Cristina Mattar Silva,Márcio Alves‐Ferreira,Sinara Artico,Marilia Santos Silva,Regina Maria Santos Amorim,Janaina Nascimento Lima

doi:10.1111/pbi.12694

Abstract

SummaryGenetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination‐related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR‐based 2−ΔΔCt analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g−1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g−1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

Highlights

Cotton (Gossypium hirsutum) production is highly influenced by a large number of insect pests, and a major pest in the Americas is the cotton boll weevil (CBW) Anthonomus grandis (Coleoptera: Curculionidae), which causes significant losses to cotton production and impacts fiber quality (Azambuja and Degrande, 2014; Bastos et al, 2005; De Lima et al, 2013; Gallo et al, 2002; Habib and Fernandes, 1983; Instituto Mato-grossense do Algod~ao, 2015; Ribeiro et al, 2010; Soria et al, 2013)
Concerning hemipteran insect control, a recent study showed that a Bacillus thuringiensis (Bt) toxin variant (Cry51Aa2.834_16) could reduce populations of Lygus spp. in whole-genetically modified (GM) cotton plants evaluated in caged-field trials (Gowda et al, 2016)
These preliminary results reveal that Cry10Aa toxin has a crystal proteins (Cry) typical 3D-deltaendotoxin conformation, which is typical of pore-forming toxins, with seven helixes in domain I, three beta sheets in domain II and a beta sandwich in domain III (Figures S1, S2), and presents LC50 6.35 lg mLÀ1 against the CBW, which indicates a similar potency of Cry10Aa to the same insect found by Aguiar et al (2012) (Table S1)

Summary

Introduction

Cotton (Gossypium hirsutum) production is highly influenced by a large number of insect pests, and a major pest in the Americas is the cotton boll weevil (CBW) Anthonomus grandis (Coleoptera: Curculionidae), which causes significant losses to cotton production and impacts fiber quality (Azambuja and Degrande, 2014; Bastos et al, 2005; De Lima et al, 2013; Gallo et al, 2002; Habib and Fernandes, 1983; Instituto Mato-grossense do Algod~ao, 2015; Ribeiro et al, 2010; Soria et al, 2013). The endophytic habit of CBW larvae into cotton reproductive structures can result in crop losses of up to 100%, especially because chemical control is only applicable during the adult weevil stage, when it feeds on immature cotton bolls (Busoli and Michelotto, 2005; Ribeiro et al, 2015). There are currently more than 750 characterized Bt-encoded entomotoxic crystal proteins (Cry), which are grouped into at least 74 different classes and are collectively active against insects, nematodes, mites and protozoans (Crickmore et al, 1998). Substantial knowledge on the direct use of Bt for the biological control of insects has accumulated over the last decades, its commercial application is limited due to high production costs and instability of the Cry proteins under field conditions (Navon, 2000, 2013).

Objectives

Results

Conclusion