Cecropins from Plutella xylostella and Their Interaction with Metarhizium anisopliae

Abstract

Cecropins are the most potent induced peptides to resist invading microorganisms. In the present study, two full length cDNA encoding cecropin2 (Px-cec2) and cecropin3 (Px-cec3) were obtained from P. xylostella by integrated analysis of genome and transcriptome data. qRT-PCR analysis revealed the high levels of transcripts of Px-cecs (Px-cec1, Px-cec2 and Px-cec3) in epidermis, fat body and hemocytes after 24, 30 and 36 h induction of Metarhizium anisopliae, respectively. Silencing of Spätzle and Dorsal separately caused the low expression of cecropins in the fat body, epidermis and hemocytes, and made the P.xylostella larvae more susceptible to M. anisopliae. Antimicrobial assays demonstrated that the purified recombinant cecropins, i.e., Px-cec1, Px-cec2 and Px-cec3, exerted a broad spectrum of antimicrobial activity against fungi, as well as Gram-positive and Gram-negative bacteria. Especially, Px-cecs showed higher activity against M. anisopliae than another selected fungi isolates. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that cecropins exerted the vital morphological alterations to the spores of M. anisopliae. Based on our results, cecropins played an imperative role in resisting infection of M. anisopliae, which will provide the foundation of biological control of insect pests by using cecorpins as a target in the future.

Fig 1. Nucleotide sequence of Px-cec2 and Px-cec3 cDNA from P. xylostella and their deduced amino acids.

The putative signal peptide is boxed, while the mature peptide is indicated in bold type, while the predicted transmembrane domain is shaded. The stop codon is marked as an asterisk.

Fig 2. qRT-PCR analysis of relative expression of cecropin genes from fat body, epidermis, hemocytes after inoculation of M. anisopliae.

Actin was used as an internal control. The mRNA levels of cecropins were highly expressed in 30 h after induction of M. anisopliae in fat body and 24 h in epidermis, Px-cec1, Px-cec2 and Px-cec3 showed maximum expression after 36 h, 30 h, 36 h in hemocytes, respectively. In addition, Px-cec3 depicted more sensitivity to M. anisopliae than Px-cec1 and Px-cec2. Relative expression levels of 6 h was arbitrarily set at 1. Three biological replications (n = 3) were conducted, and the 2^-ΔΔCt method was used to measure the relative transcription levels. Means with different number of asterisk are significantly different (P<0.05) (Duncan’s Multiple Range Test) among different time after treated with alive M. anisopliae. *: different with the lowest expression after treated with M. anisopliae; **: significant different with the lowest expression after treated with M. anisopliae; ***: highly significant different with the lowest expression after treated with M. anisopliae.

Fig 3. The knock-down of Spätzle and Dorsal independently by RNAi.

qRT-PCR analysis of cecropins on fat body, epidermis and hemocytes in P. xylostella after 36 h of RNAi. The relative expression levels of cecropins mRNA was different after treatments. The mRNA level of Px-cec1 was remarkably decreased in fat body, Px-cec2 in hemocytes, while Px-cec3 was dropped rapidly as compared to other tissues. Actin was used as an internal control. Each bar represents the mean ± S.E. (n = 3).

Fig 4. The analysis on susceptibility of P. xylostella larvae to alive M. anisopliae after knockdown of PxSpa and PxDor.

After dsRNA treatment, the larvae were treated with M. anisopliae spores (1×10⁹ conidia/ml). Mortality was recorded every 6h. Each treatment was replicated three times and each treatment consisted of 30 larvae.

Fig 5. SDS-PAGE analysis of recombinant protein from Drosophila S2 cells.

M: Kaleidoscope polypeptide weight protein marker; 1: recombinant protein of Px-cec1; 2: recombinant protein of Px-cec2; 3: recombinant protein of Px-cec3.The result showed that there was a single band corresponding to the expected size of 3.8 kDa.

Fig 6. Analysis of Pxcec2 and Pxcec3 using far-UV CD spectra under 10 mM phosphate buffer (pH 7.2) at room temperature.

Fig 7. SEM analysis of the spore of M. anisopliae interacted with cecropins.

A, C and E: CK (naive M. anisopliae spore); B, D and F: the spore of M. anisopliae interacted with Px-cec1, Px-cec2 and Px-cec3, respectively. The SEM analysis showed that M.anisopliae spore became short and wrinkled (B, D and F) after interacted with cecropins from P. xylostella as compared to the untreated spore (A, C and E), which had a bright and normal smooth surface.

Fig 8. TEM showing the effect of cecropins from P. xylostella on the spores of M. anisopliae.

A, C and E: CK (naive M. anisopliae spore); B, D and F: the spore of M. anisopliae interacted with Px-cec1, Px-cec2 and Px-cec3, respectively. The TEM images revealed that the cell membrane of the spore became wafery and the cellular cytoplasmic contents were dissolved and became vague (B, D and F). The entire cell membrane was disrupted, causing the cellular cytoplasmic contents to leak out after interaction with Px-cec1 and Px-cec2 (B and D), the untreated spore (A, C and E) had a bright and normal smooth surface and the cellular cytoplasmic content was clear.