Transmission of a Protease-Secreting Bacterial Symbiont Among Pea Aphids via Host Plants

Abstract

Aphids are economically important pest insects that damage plants by phloem feeding and the transmission of plant viruses. Their ability to feed exclusively on nutritionally poor phloem sap is dependent on the obligatory symbiotic bacterium Buchnera aphidicola, but additional facultative symbionts may also be present, a common example of which is Serratia symbiotica. Many Serratia species secrete extracellular enzymes, so we hypothesised that S. symbiotica may produce proteases that help aphids to feed on plants. Molecular analysis, including fluorescence in situ hybridization (FISH), revealed that S. symbiotica colonises the gut, salivary glands and mouthparts (including the stylet) of the pea aphid Acyrthosiphon pisum, providing a mechanism to transfer the symbiont into host plants. S. symbiotica was also detected in plant tissues wounded by the penetrating stylet and was transferred to naïve aphids feeding on plants containing this symbiont. The maintenance of S. symbiotica by repeated transmission via plants may explain the high frequency of this symbiont in aphid populations. Proteomic analysis of the supernatant from a related but cultivable S. symbiotica strain cultured in liquid medium revealed the presence of known and novel proteases including metalloproteases. The corresponding transcripts encoding these S. symbiotica enzymes were detected in A. pisum and in plants carrying the symbiont, although the mRNA was much more abundant in the aphids. Our data suggest that enzymes from S. symbiotica may facilitate the digestion of plant proteins, thereby helping to suppress plant defense, and that the symbionts are important mediators of aphid-plant interactions.

Keywords: Serratia symbiotica; Vicia faba; extracellular proteases; phloem sap; symbiosis.

FIGURE 1

The detection of S. symbiotica genomic DNA by PCR. M, DNA marker (size in base pairs); PC, positive control (pGEM T-Easy vector with S. symbiotica 16S rDNA); NC, negative control (distilled water); lane 1, Serratia-positive aphids; lane 2, Serratia-free aphids; lane 3, Serratia-reinfected aphids (2 months after infection event); lane 4, V. faba plant infested with Serratia-positive aphids; lane 5, V. faba plant infested with Serratia-free aphids. The Serratia specific primers used for PCR are listed in Supplementary Table S1. Amplicon size ∼480 bp.

FIGURE 2

Localization of S. symbiotica by fluorescence in situ hybridization (FISH) in A. pisum mouthparts and V. faba tissues. Detection of S. symbiotica (red) in the head (mouthparts, salivary glands and gut) of a 10-day-old adult aphids (A–D) and V. faba longitudinal stem sections under dark field (E) and bright field (F) imaging. Nuclei were counterstained with DAPI (dark blue). Abbreviations: MD, mandible; SG, salivary gland; St, stylet; LR, labrum; FSC, food and salivary canal; G, gut.

FIGURE 3

Quantitative PCR analysis of S. symbiotica in A. pisum and V. faba. Data show the relative abundance of the S. symbiotica dnaK gene compared to the rpl32 reference gene in aphids and the actin reference gene in plants. This was used to determine the abundance of S. symbiotica in the Serratia-positive and Serratia-free aphid lines (A), and in V. faba leaves after exposure to each aphid line, after retention times of 2, 5, and 10 days (B). Statistical significance is indicated as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 4

Quantitative RT-PCR analysis showing the relative expression of five S. symbiotica genes (DegQ, HtpX, YfgC, SohB, and PepA) encoding proteolytic enzymes associated with the host plant (Table 1) in Serratia-positive and Serratia-free aphids. The expression data were normalised to the aphid reference gene rpl32. Statistical significance is indicated as follows: ^∗p < 0.05, ^∗∗p < 0.01.