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Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil ?-amylase.

ABSTRACT: BACKGROUND:Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An ?-amylase inhibitor (?-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis ?-amylase (AGA). RESULTS:We produced in a few days considerable amounts of ?-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high ?-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on ?-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active ?-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that ?-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. CONCLUSIONS:We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.

SUBMITTER: Prado GS

PROVIDER: S-EPMC6408794 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil α-amylase.

Prado Guilherme Souza GS Bamogo Pingdwende Kader Aziz PKA de Abreu Joel Antônio Cordeiro JAC Gillet François-Xavier FX Dos Santos Vanessa Olinto VO Silva Maria Cristina Mattar MCM Brizard Jean-Paul JP Bemquerer Marcelo Porto MP Bangratz Martine M Brugidou Christophe C Sérémé Drissa D Grossi-de-Sa Maria Fatima MF Lacombe Séverine S

BMC biotechnology 20190308 1

<h4>Background</h4>Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) i ...[more]

PMID: 30849970

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Project description:BACKGROUND: Cotton is a major fibre crop grown worldwide that suffers extensive damage from chewing insects, including the cotton boll weevil larvae (Anthonomus grandis). Transcriptome analysis was performed to understand the molecular interactions between Gossypium hirsutum L. and cotton boll weevil larvae. The Illumina HiSeq 2000 platform was used to sequence the transcriptome of cotton flower buds infested with boll weevil larvae. RESULTS: The analysis generated a total of 327,489,418 sequence reads that were aligned to the G. hirsutum reference transcriptome. The total number of expressed genes was over 21,697 per sample with an average length of 1,063 bp. The DEGseq analysis identified 443 differentially expressed genes (DEG) in cotton flower buds infected with boll weevil larvae. Among them, 402 (90.7%) were up-regulated, 41 (9.3%) were down-regulated and 432 (97.5%) were identified as orthologues of A. thaliana genes using Blastx. Mapman analysis of DEG indicated that many genes were involved in the biotic stress response spanning a range of functions, from a gene encoding a receptor-like kinase to genes involved in triggering defensive responses such as MAPK, transcription factors (WRKY and ERF) and signalling by ethylene (ET) and jasmonic acid (JA) hormones. Furthermore, the spatial expression pattern of 32 of the genes responsive to boll weevil larvae feeding was determined by "in situ" qPCR analysis from RNA isolated from two flower structures, the stamen and the carpel, by laser microdissection (LMD). CONCLUSION: A large number of cotton transcripts were significantly altered upon infestation by larvae. Among the changes in gene expression, we highlighted the transcription of receptors/sensors that recognise chitin or insect oral secretions; the altered regulation of transcripts encoding enzymes related to kinase cascades, transcription factors, Ca2+ influxes, and reactive oxygen species; and the modulation of transcripts encoding enzymes from phytohormone signalling pathways. These data will aid in the selection of target genes to genetically engineer cotton to control the cotton boll weevil.

Dataset Information

Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil ?-amylase.

Publications

Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil α-amylase.

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