Transcript and protein profiling analysis of the Destruxin A-induced response in larvae of Plutella xylostella

Abstract

Background: Destruxins (dtxs) are the mycotoxin produced by certain entomopathogenic fungi, such as Metarhizium anisopliae, Aschersonia sp, Alternaria brassicae and Ophiosphaerella herpotrichae. It can affect a wide variety of biological processes in insects, including innate immune, Ca(2+) channel in cells, and apoptosis in a dose-dependent manner. Dtxs have been used as biological control agent for a long time, however, their molecular mechanism of action is still unknown.

Principal findings: In this study, both digital gene expression (DGE) and two-dimensional electrophoresis (2-DE) approaches were adopted to examine the effects of dtx A on Plutella xyllostella (L.) larvae. By using DGE and 2-DE analyses, 1584 genes and 42 protein points were identified as being up- or down regulated at least 2-fold in response to dtx A. Firstly, injection of dtx A to larvae accelerated the increase of peptidoglycan recognition protein (PGRP), which could activate the Toll signal pathway inducing production of antibacterial substances such as cecropin and gloverin. Dtx A also stimulated prophenoloxidase (proPO) system which plays an important role in innate immunity and leads to melanization of external organisms. Secondly, dtx A suppressed the expression of genes related to the Toll pathway, and induced expression of serine proteinase inhibitors (serpins), especially the serpin 2 that blocked process of the proPO system. Finally, other physiological process like xenobiotics detoxification, apoptosis, calcium signaling pathway and insect hormone biosynthesis, were also mediated in response to dtx A toxicity.

Conclusions: Transcript and protein profiling analyses will provide an insight into the potential molecular mechanism of action in P. xylostella larvae in response to dtx A.

Figure 1. Distribution of genes’ coverage in each library.

Figure 2. qRT-PCR validation of DGE result.

Note: The left y-axis indicates the relative expression level by qRT-PCR, and the right y-axis indicates the log₂Ratio of 4H/CK by DGE.

Figure 3. Two-dimensional electrophoresis map of hemolymph proteins.

Note: 4^th instar larvae of Plutella xylostella were treated with destruxin A for 4 hour. Hemolymph proteins (1 mg) were separated on 2D gels (pH 4–7) and stained with Coomassie Brilliant Blue R-250. The differential expression and successfully identified protein spots are number, corresponding to the number in Table S2.

Figure 4. The MALDI-TOF/TOF-MS/MS analysis of protein spot 22.

Note: The MALDI-TOF-MS peptide mass fingerprint spectrum of trypsin-digested protein (a) and its MS/MS peptide mass fingerprint spectrum of ionic peak 2565.23 (b).

Figure 5. Western blot analysis of expression of PxSerpin 2.

Note: Those were visualized by DAB. Actin was used as an internal control.