Cry78Aa, a novel Bacillus thuringiensis insecticidal protein with activity against Laodelphax striatellus and Nilaparvata lugens
Graphical abstract
Introduction
Rice is one of the most important food crops globally and most people living in Asia depend on this staple food. Rice planthoppers, which have piercing-sucking mouthparts, not only feed on the phloem sap of rice plants but also serve as vectors of virus infections that can cause serious yield losses (Heong and Hardy, 2009). The brown planthopper (Nilaparvata lugens), small brown planthopper (Laodelphax striatellus) and white back planthopper (Sogatella furcifera) are three primary hemipteran pests of rice and seriously threaten rice production. Currently, planthopper control methods rely mainly on the application of chemical insecticides. These chemicals can induce resistance in the pest and also produce unintended mortality of non-target organisms.
Bacillus thuringenesis (Bt) and plants expressing Bt insecticidal proteins have been successfully applied in insect control (Palma et al., 2014a), and efforts have been made to develop rice planthopper specific Bt insecticidal proteins. Shao et al. used protein engineering to modify a lepidopteran-specific Cry1Ab toxin with known gut binding peptides to create a hybrid protein with limited activity against the brown planthopper N. lugens (Shao et al., 2016). Using a membrane feeding protocol (Wang et al., 2014) we previously identified a number of Bt strains demonstrating some level of activity against L. striatellus. One of these strains (1012) encoded two toxins, Cry64Ba and Cry64Ca, that were confirmed to have high toxicity against rice planthoppers (Liu et al., 2018). Another strain identified in that screen (C9F1) was phenotypically distinct from the above strain and is the subject of this investigation.
Section snippets
Strains, plasmid and growth conditions
The C9F1 (CGMCC10782) strain was isolated from soil collected from the BaiWangShan Forest Park in Beijing and preserved at Institute of Plant Protection, Chinese Academy of Agricultural Sciences (IPPCAAS), Beijing. Scanning electron microscopy and SDS-PAGE analysis of the spore-crystal mixture of C9F1 were conducted following the methods described by Shu et al. (2007). For Q-Exactive Mass Spectrometry analysis crystals solubilized in sodium carbonate buffer were subjected to SDS-PAGE, bands
Initial characterization of the C9F1 strain
The spore and crystal mixture of C9F1 were examined under a scanning electron microscope and small spherical crystals were observed (Fig. 1A). Total protein of sporulated C9F1 was analyzed by SDS-PAGE and revealed one major protein of around 140 kDa as well as other minor proteins (Fig. 1B).
Draft genome sequence and gene annotation of C9F1 putative pesticidal proteins
A total of 6,422,579 nucleotide base pairs were generated using an Illumina sequencing platform and were assembled to 610 scaffolds with a genome size 6.21 Mb. The number of predicted protein coding
Discussion
Only Cry64Ba, Cry64Ca (Liu et al., 2018) and modified Cry1Ab (Shao et al., 2016) were previously confirmed as having high toxicity against rice planthoppers. The discovery of another toxin will hopefully increase the potential for control of these economically important pests. The SDS-PAGE profile of C9F1 indicates that the main protein(s) expressed by this strain are around 140 kDa in size and, based on the genome sequence, are most likely Cry8 toxins. These toxins are typically reported to be
Acknowledgments
This study was supported by National Key R&D Program of China (Grant 2017YFD0200400) and the National Science and Technology Major Project of China (Grant 2014ZX0800912B).
Compliance with ethical standards
The manuscript does not contain experiments using mammals and does not contain studies on humans.
Conflict of interest
The authors declare no competing interests.
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