Global Transcriptional Profile of Tranosema rostrale Ichnovirus Genes in Infected Lepidopteran Hosts and Wasp Ovaries

The ichnovirus TrIV, transmitted by the endoparasitic wasp Tranosema rostrale to its lepidopteran host during oviposition, replicates asymptomatically in wasp ovaries and causes physiological dysfunctions in parasitized caterpillars. The need to identify ichnoviral genes responsible for disturbances induced in lepidopteran hosts has provided the impetus for the sequencing and annotation of ichnovirus genomes, including that of TrIV. In the latter, 86 putative genes were identified, including 35 that could be assigned to recognized ichnoviral gene families. With the aim of assessing the relative importance of each TrIV gene, as inferred from its level of expression, and evaluating the accuracy of the gene predictions made during genome annotation, the present study builds on an earlier qPCR quantification of transcript abundance of TrIV rep ORFs, in both lepidopteran and wasp hosts, extending it to other gene families as well as to a sample of unassigned ORFs. We show that the majority (91%) of putative ORFs assigned to known gene families are expressed in infected larvae, while this proportion is lower (67%) for a sample taken among the remaining ORFs. Selected members of the TrV and rep gene families are shown to be transcribed in infected larvae at much higher levels than genes from any other TrIV gene family, pointing to their likely involvement in host subjugation. In wasp ovaries, the transcriptional profile is dominated by a rep gene and a member of a newly described gene family encoding secreted proteins displaying a novel cysteine motif, which we identified among previously unassigned ORFs.

Immunosuppression induced by expression of a viral RNase enhances susceptibility of Plutella xylostella to microbial pesticides

Polydnaviruses are a group of insect DNA viruses and are characterized in their segmented genome that is located in the chromosome（s） of host wasps. A polydnavirus, Cotesiaplutellae bracovirus （CpBV）, encodes a viral ribonuclease （RNase） T2 in a specific segment #3 （CpBV-S3）. This study tested its effect on gene expression associated with host immune responses in the diamondback moth, Plutella xylostella. Micro-inj ection of CpBV- $3 into nonparasitized larvae induced expression of its two encoded genes, CpBV-ORF301 （= CpBV-RNase T2） and CpBV-ORF302. In response to a bacterial challenge, four antimi- crobial peptide genes （hemolin, gloverin, cecropin and lysozyme） and six phenoloxidase （PO）-associated genes （proPO-activating proteinase, PO, serine proteinase homolog and serpins 1-3） were up-regulated in their expressions. However, the transient expression of CpBV-S3 suppressed the expressions of cecropin, PO and serpin 1. Double-stranded RNA specific to the viral RNase T2 could specifically knockdown the viral gene expression and restored the three gene expressions suppressed in the larvae injected with CpBV-S3. The inhibitory activity of the viral RNase T2 on the target genes was further proven by the suppression of PO activation in response to bacterial challenge in the larvae injected with CpBV-S3. This immunosuppression by the expression of the viral RNase T2 resulted in significant increase of pathogen susceptibility ofP. xylostella against Bacillus thuringiensis or baculovirus infection.

CLP gene family,a new gene family of Cotesia vestalis bracovirus inhibits melanization of Plutella xylostella hemolymph

Polydnaviruses(PDVs)are obligatory symbionts of parasitoid wasps and play an important role in suppressing host immune defenses.Although PDV genes that inhibit host melanization are known in Microplitis bracovirus,the functional homologs in Cotesia bracoviruses remain unknown.Here,we find that Cotesia vestalis bracovirus(CvBV)can inhibit hemolymph melanization of its host,Plutella xylostella larvae,during the early stages of parasitization,and that overexpression of highly expressed CvBV genes reduced host phenoloxidase activity.Furthermore,CvBV-7-I in particular reduced host phenolox-idase activity within 12 h,and the injection of anti-CvBV-7-1 antibody increased the melanization of parasitized host larvae.Further analyses showed that CvBV-7-1 and three homologs from other Cotesia bracoviruses possessed a C-terminal leucine/isoleucine-rich region and had a similar flinction in inhibiting melanization.Therefore,a new family of bracovirus genes was proposed and named as C-terminal Leucine/isoleucine-rich Protein(CLP).Ectopic expression of CvBV-7-1 in Drosophila hemocytes increased susceptibility to bacterial repression of melanization and reduced the melanotic encapsulation of par-asitized D.melanogaster by the parasitoid Leptopilina boulardi.The formation rate of wasp pupae and the eclosion rate of C.vestalis were affected when the function of CvBV-7-1 was blocked.Our findings suggest that CLP genes from Cotesia bracoviruses encoded proteins that contain a C-terminal leucine/isoleucine-rich region and function as melanization inhibitors during the early stage of parasitization,which is important for successful parasitization.

GENOMIC FEATURES OF COTESIA PLUTELLAE POLYDNAVIRUS

Polydnavirus was purified from the calyx fluid of Cotesia plutellae ovary. The genomic features of C. plutellae polydnavirus (CpPDV) were investigated. The viral genome consists of at least 12 different segments and the aggregate genome size is a lower estimate of 80kbp. By partial digestion of CpPDV DNA with BamHI and subsequent ligation with BamHI-cut plasmid Bluescript, a representative library of CpPDV genome was obtained.