Gall Wasp Transcriptomes Unravel Potential Effectors Involved in Molecular Dialogues With Oak and Rose

Abstract

To gain insight into wasp factors that might be involved in the initial induction of galls on woody plants, we performed high throughput (454) transcriptome analysis of ovaries and venom glands of two cynipid gall wasps, Biorhiza pallida and Diplolepis rosae, inducing galls on oak and rose, respectively. De novo assembled and annotated contigs were compared to sequences from phylogenetically related parasitoid wasps. The relative expression levels of contigs were estimated to identify the most expressed gene sequences in each tissue. We identify for the first time a set of maternally expressed gall wasp proteins potentially involved in the interaction with the plant. Some genes highly expressed in venom glands and ovaries may act to suppress early plant defense signaling. We also identify gall wasp cellulases that could be involved in observed local lysis of plant tissue following oviposition, and which may have been acquired from bacteria by horizontal gene transfer. We find no evidence of virus-related gene expression, in contrast to many non-cynipid parasitoid wasps. By exploring gall wasp effectors, this study is a first step toward understanding the molecular mechanisms underlying cynipid gall induction in woody plants, and the recent sequencing of oak and rose genomes will enable study of plant responses to these factors.

Keywords: gall induction; oak gall wasp; ovary secretions; rose gall wasp; venom.

FIGURE 1

Venom apparatus of B. pallida (A) and D. rosae (B) females. G, venom gland; R, venom reservoir. Scale bars = 0,6 mm.

FIGURE 2

The venom apparatus of B. pallida observed in fluorescence microscopy. (A) light microscopy, (B) FITC-conjugated phalloidin staining for actin detection, (C) DAPI staining for detection of nucleic acids, and (D) detailed view of (C). G, venom gland; R, venom reservoir; V, venom; arrow, secondary secretory zone; arrowhead, muscular fiber; boxes d1, d2, and d3 refer to detailed views shown in panel (D). Note the high density of the venom leaking from the ruptured reservoir in panel (A), and the different shapes of nuclei that allow to discriminate between the different tissues composing the organ. Scale bars = 0,3 mm for (A), (B), and (C) or 50 μm for (D).

FIGURE 3

View of the venom of B. pallida in light microscopy. At high magnification, numerous spherical particles of approximately 1 to 2 μm in diameter are visible in the venom of B. pallida. Scale bar = 50 μm.

FIGURE 4

The venom gland of D. rosae observed in confocal microscopy. Merge image of FITC-conjugated phalloidin staining for actin detection (green) and DAPI staining for detection of nucleic acids (blue). H, internal helix containing actin; arrow, surrounding mesenchymal tissue; arrowhead, muscular fiber. Note the round shape of the nuclei of glandular cells. Scale bars = 30 μm.

FIGURE 5

Contig expression levels in ovaries and venom glands and percentage of contigs encoding proteins with signal peptides in B. pallida (A) and D. rosae (B). In dark gray, contigs expressed at least 20 times higher in venom glands than in ovaries (v); in light gray, contigs expressed at least 20 times higher in ovaries than in venom glands (ov); in gray, contigs expressed in both ovaries and venom glands with relative expression levels differing by less than 20-fold. The distribution of contigs that had a blastx hit (black) or that are not assigned (NA; white) and the percentage of these contigs coding proteins with a signal peptide (SignalP4.0 prediction) are specified for each category of contigs.

FIGURE 6

Histogram presentation of Gene Ontology (GO) classification of B. pallida (A) and D. rosae (B) contigs. Results are given in the three GO categories at the level two of GO analysis for contigs highly expressed in venom gland (dark gray), those highly expressed in ovaries (light gray), and those equivalently expressed in the two tissues (gray).

FIGURE 7

Relative expression and annotation of B. pallida (A) and D. rosae (B) contigs differentially expressed in venom glands and potentially coding secreted proteins. Contigs considered here were only those returning a significant blastx hit (E-value < 10⁻⁴) and having a predicted signal peptide or homologous to a protein possessing a signal peptide (contigs indicated by Y in Tables 2, 3 from SignalP4.0 prediction). For each category, the sum of the reads (RPKM) in venom gland was calculated. For detailed description of contigs see Tables 2, 3.