The microRNA-7322-5p/p38/Hsp19 axis modulates Chilo suppressalis cell-defences against Cry1Ca: an effective target for a stacked transgenic rice approach.

Bacillus thuringiensis (Bt)-secreted crystal (Cry) toxins form oligomeric pores in host cell membranes and are a common element in generating insect-resistant transgenic crops. Although Cry toxin function has been well documented, cellular defences against pore-formation have not been as well developed. Elucidation of the processes underlying this defence, however, could contribute to the development of enhanced Bt crops. Here, we demonstrate that Cry1Ca-mediated downregulation of microRNA-7322-5p (miR-7322-5p), which binds to the 3' untranslated region of p38, negatively regulates the susceptibility of Chilo suppressalis to Cry1Ca. Moreover, Cry1Ca exposure enhanced phosphorylation of Hsp19, and hsp19 downregulation increased susceptibility to Cry1Ca. Further, Hsp19 phosphorylation occurs downstream of p38, and pull-down assays confirmed the interactions between Hsp19 and Cry1Ca, suggesting that activation of Hsp19 by the miR-7322-5p/p38/Hsp19 pathway promotes Cry1Ca sequestration. To assess the efficacy of targeting this pathway in planta, double-stranded RNA (dsRNA) targeting C. suppressalis p38 (dsp38) was introduced into a previously generated cry1Ca-expressing rice line (1CH1-2) to yield a single-copy cry1Ca/dsp38 rice line (p38-rice). Feeding on this rice line triggered a significant reduction in C. suppressalis p38 expression and the line was more resistant to C. suppressalis than 1CH1-2 in both short term (7-day) and continuous feeding bioassays as well as field trials. These findings provide new insights into invertebrate epithelium cellular defences and demonstrate a potential new pyramiding strategy for Bt crops.