| Summary: | Many insects, including the <i>Plutella xylostella</i> (L.), have developed varying degrees of resistance to many insecticides, including <i>Bacillus thuringiensis</i> (<i>Bt</i>) toxins, the bioinsecticides derived from <i>Bt</i>. The polycalin protein is one of the potential receptors for <i>Bt</i> toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of <i>P. xylostella</i>, but whether polycalin is associated with the resistance of <i>Bt</i> toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the <i>Pxpolycalin</i> gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of <i>Pxpolycalin</i> showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the <i>Pxpolycalin</i> gene and its transcript level were not linked to Cry1Ac resistance, whereas both the <i>PxABCC2</i> gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the <i>Pxpolycalin</i> gene in a short term. Furthermore, the knockout of <i>polycalin</i> and ATP-binding cassette transporter subfamily C2 <i>(ABCC2)</i> genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to <i>Bt</i> toxins.
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