Prophenoloxidase from Pieris rapae: gene cloning, activity, and transcription in response to venom/calyx fluid from the endoparasitoid wasp Cotesia glomerata.
Prophenoloxidase (PPO) plays an important role in melanization, necessary for defense against intruding parasitoids. Parasitoids have evolved to inject maternal virulence factors into the host hemocoel to suppress hemolymph melanization for the successful development of their progeny. In this study, the full-length complementary DNA (cDNA) of a Pieris rapae PPO was cloned. Its cDNA contained a 2 076-base pair (bp) open reading frame (ORF) encoding 691 amino acids (aa). Two putative copper-binding sites, a proteolytic activation site, three conserved hemocyanin domains, and a thiol ester motif were found in the deduced amino acid sequence. According to both multiple alignment and phylogenetic analysis, P. rapae PPO gene cloned here is a member of the lepidopteran PPO-2 family. Injection of Cotesia glomerata venom or calyx fluid resulted in reduction of P. rapae hemolymph phenoloxidase activity, demonstrating the ability to inhibit the host's melanization. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of P. rapae PPO-2 in the haemocytes from larvae had not significantly changed following venom injection, suggesting that the regulation of PPO messenger RNA (mRNA) expression by venom was not employed by C. glomerata to cause failure of melanization in parasitized host. While decreased P. rapae PPO-2 gene expression was observed in the haemocytes after calyx fluid injection, no detectable transcriptional change was induced by parasitization, indicating that transcriptional down-regulation of PPO by calyx fluid might play a minor role involved in inhibiting the host's melanization.
Key Laboratory of Forest Disaster Warning and Control of Yunnan Province and Key Laboratory of Southwest Mountain Forest Resources Conservation and Utilization of Ministry of Education, Southwest Forestry University, Kunming 650224, China. jiayingzhu001@yahoo.com.cn
Jia-ying
JY
Zhu
Pu
P
Yang
Guo-xing
GX
Wu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004156:Catechol Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D017344:Genes, Insect; D006458:Hemolymph / Q000201:enzymology; D006790:Host-Parasite Interactions / Q000235:genetics / Q000276:immunology / Q000502:physiology; D007113:Immunity, Innate; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007915:Lepidoptera / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology / Q000469:parasitology; D008543:Melanins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014862:Wasp Venoms / Q000633:toxicity; D014863:Wasps / Q000469:parasitology*
D017931:DNA Primers; D018076:DNA, Complementary; D004792:Enzyme Precursors; D019476:Insect Proteins; D008543:Melanins; D012333:RNA, Messenger; D014862:Wasp Venoms; C038451:pro-phenoloxidase; D004156:Catechol Oxidase
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