Development and Characterization of Diamondback Moth Resistance to Transgenic Broccoli Expressing High Levels of Cry1C

Abstract

A field-collected colony of the diamondback moth, Plutella xylostella, had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis. After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400-and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous selection but decreased to 235-fold at G38 when selection ceased at G28. The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli. Saturable binding of 125 I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains. Significant differences in Cry1C binding to BBMV from the two strains were detected. BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain. These results suggest that reduced binding is not the major mechanism of resistance to Cry1C.

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

“…Along the same lines, changes in these toxin binding sites in the insect would not be reflected in a large change in binding parameters. Such a situation may have occurred in strains of Plutella xylostella and S. exigua that are highly resistant to Cry1Ca, for which binding studies reported just a small change in binding capacity [17,35]. This theory is supported by the observation that lack of a cadherin-like receptor in a Cry1A-resistant strain of Heliothis virescens (YHD2) [36] hardly affects the detectable binding of Cry1Ab or Cry1Ac in the resistant strain [37].…”

“…Toxin was eluted from the column by increasing the NaCl concentration. For iodine labelling of Cry1Ca, an additional purification step was performed, following the procedure described in Zhao et al [17], to remove adsorbed protoxin fragments that block iodination.…”

Inheritance of Resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

“…In fieldderived populations of the diamondback moth, resistance to Cry1Ac did not correspond to monogenic inheritance in a population from Malaysia (30), and two different genes that confer resistance to Cry1Ab were present in a population originating from the Philippines (10). Direct tests of monogenic inheritance of Cry1C resistance in a P. xylostella population originating from New York suggested that significant deviations between observed and expected mortalities were the result of nonadditive polygenic inheritance or experimental error (44), and further tests indicated that there might be polygenic inheritance (45).…”

Biological control of the diamondback moth,Plutella xylostella: A review

“…A field-collected population (Cry1C-Sel) from South Carolina displayed 31-fold resistance to Cry1C. Continuous laboratory selection, using first Cry1C protoxin, and in later generations transgenic broccoli expressing high levels of Cry1C, increased resistance to 12,400-fold (in neonates) and 63,100-fold (in second instars) after 26 generations (Zhao et al 2000). A Malaysian colony (SERD3) was reported to be resistant to both Btk and Bta .…”