Carboxylesterases (CarEs) were considered as important phase-I detoxifying enzymes which participated in detoxification of different types of insecticides.  Up-regulation of CarE genes has been proved playing a major role in insecticide resistance in many pest insects, but its involvement in resistance to insecticides in Plutella xylostella has been rarely reported.  In this study, a CarE cDNA named PxαE8 was identified in P. xylostella, which has an open reading frame of 1599 nucleotides and putatively encodes 532 amino acids.  The investigation of spatial and temporal expression profiles of PxαE8 revealed that it was expressed in all developmental stages and body part/tissues, especially in larvae, adults as well as fat body, malpighian tubule and hemolymph of larvae.  Further, the relative expression of PxαE8 in two multi-resistant field populations, Hainan (HN) and Guangdong (GD) populations, was found 24.4- and 15.5-fold higher than that in susceptible population, respectively.  Knockdown of PxαE8 by RNAi dramatically increased the mortalities of larvae of HN population treated with LC₅₀ of beta-cypermethrin and phoxim by 25.3 and 18.3%, respectively.  These results suggested that up-regulation of PxαE8 was involved in resistance to both beta-cypermethrin and phoxim in P. xylostella, which shed light on further understanding of molecular mechanisms of multi-insecticide-resistance in P. xylostella and other pest insects.

Plants produce reactive oxygen species (ROS) to defend pathogens.  To counteract this attack, certain pathogens express superoxide dismutases (SODs) to scavenge host-derived ROS.  However, the roles of SODs in Verticillium dahliae, an important vascular pathogen, are not clear.  Our previous study has shown that a putative extracellular SOD (VdSOD5) of V. dahliae is significantly induced by culturing in cotton tissues, suggesting that VdSOD5 may play an important role in host–pathogen interactions and virulence.  Here, we showed that VdSOD5 encoded a superoxide dismutase with a co-factor copper-binding site and a functional signal peptide that can conduct protein secretion in an invertase-mutated yeast strain.  The mutations in VdSOD5 (ΔVdSOD5) did not change the normal vegetative growth and conidial production but reduced the virulence of V. dahliae on susceptible host cotton.  Further studies showed that the transcription of VdSOD5 was significantly up-regulated during the early stage of infection, and the loss-of-function of VdSOD5 decreased culture filtrate and fungal tissue SOD activities of V. dahliae by 74 and 28%, respectively.  Compared to the wild-type strain Vd991, the ΔVdSOD5 showed the same sensitivity to the intracellular ROS generator menadione.  Furthermore, nitroblue tetrazolium (NBT) staining demonstrated that VdSOD5 functioned in the detoxification of superoxides generated by host roots during infection.  These results suggest that VdSOD5 of V. dahliae is an important virulence factor, secreted out of cells to combat host-derived ROS.