Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin

Abstract

The bacterium Bacillus thuringiensis produces ICPs (insecticidal crystal proteins) that are deposited in their spore mother cells. When susceptible lepidopteran larvae ingest these spore mother cells, the ICPs get solubilized in the alkaline gut environment. Of approx. 140 insecticidal proteins described thus far, insecticidal protein Cry1Ac has been applied extensively as the main ingredient of spray formulation as well as the principal ICP introduced into crops as transgene for agricultural crop protection. The 135 kDa Cry1Ac protein, upon ingestion by the insect, is processed successively at the N- and C-terminus by the insect midgut proteases to generate a 65 kDa bioactive core protein. The activated core protein interacts with specific receptors located at the midgut epithilium resulting in the lysis of cells and eventual death of the larvae. A laboratory-reared population of Helicoverpa armigera displayed 72-fold resistance to the B. thuringiensis insecticidal protein Cry1Ac. A careful zymogram analysis of Cry1Ac-resistant insects revealed an altered proteolytic profile. The altered protease profile resulted in improper processing of the insecticidal protein and as a consequence increased the LC50 concentrations of Cry1Ac. The 135 kDa protoxin-susceptible insect larval population processed the protein to the biologically active 65 kDa core protein, while the resistant insect larval population yielded a mixture of 95 kDa and 68 kDa Cry1Ac polypeptides. N-terminal sequencing of these 95 and 68 kDa polypeptides produced by gut juices of resistant insects revealed an intact N-terminus. Protease gene transcription profiling by semi-quantitative RT (reverse transcription)-PCR led to the identification of a down-regulated HaSP2 (H. armigera serine protease 2) in the Cry1Ac-resistant population. Protease HaSP2 was cloned, expressed and demonstrated to be responsible for proper processing of insecticidal protoxin. The larval population displaying resistance to Cry1Ac do not show an altered sensitivity against another insecticidal protein, Cry2Ab. The implications of these observations in the context of the possibility of development of resistance and its management in H. armigera to Cry1Ac through transgenic crop cultivation are discussed.

Cis-mediated down-regulation of a trypsin gene associated with Bt resistance in cotton bollworm

“…Various mechanisms of resistance to Cry1Ac have been identified previously in H. armigera , including qualitative changes in or reduced levels of the confirmed and putative midgut receptors cadherin, aminopeptidase, alkaline phosphatase, and ABCC2 proteins14151617. In addition, a serine protease from the midgut of a Cry1Ac-resistant strain of H. armigera from India had significantly reduced expression, resulting in improper processing of the protoxin18. Our previous work with ten laboratory-selected strains of H. armigera from China showed that total protease activity in the larval gut was negatively associated with resistance to Cry1Ac19, which suggests that reduced protease activity may contribute to resistance in these strains.…”

Cotton bollworm resistance to Bt transgenic cotton: A case analysis

“…A similar pro- teinase-related Bt resistance mechanism was reported for Bt-resistant Ostrinia nubilalis and Heliothis virescens [34,35]. In addition, resistant larvae of H. armigera showed significantly decreased expression of a specific protease, HaSP2 (H. armigera serine protease 2), which is one of the six groups of serine proteases from H. armigera midgut that can process and activate the Cry1Ac polypeptide [36].…”

Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentidae)

“…Sequencing of the N-terminal end of the purified trypsin-activated Cry7Aa fragments revealed that the 70-kDa fragment starts at Glu 47 , whereas the 65-kDa fragment is produced after the removal of the N-terminal sequence up to Ile 88 . The cleavage of the N terminus has been described as a critical step for some Cry proteins to be considered active proteins (48)(49)(50)(51)(52)(53). Indirect evidence based on the structure of the unprocessed Cry2Aa protein revealed that the N-terminal region masks a region of the protein that could be involved in the interaction between the protein and the membrane of the target insect (54).…”