Horizontal transmission of the insect symbiont Rickettsia is plant-mediated

Abstract

Bacteria in the genus Rickettsia, best known as vertebrate pathogens vectored by blood-feeding arthropods, can also be found in phytophagous insects. The presence of closely related bacterial symbionts in evolutionarily distant arthropod hosts presupposes a means of horizontal transmission, but no mechanism for this transmission has been described. Using a combination of experiments with live insects, molecular analyses and microscopy, we found that Rickettsia were transferred from an insect host (the whitefly Bemisia tabaci) to a plant, moved inside the phloem, and could be acquired by other whiteflies. In one experiment, Rickettsia was transferred from the whitefly host to leaves of cotton, basil and black nightshade, where the bacteria were restricted to the phloem cells of the plant. In another experiment, Rickettsia-free adult whiteflies, physically segregated but sharing a cotton leaf with Rickettsia-plus individuals, acquired the Rickettsia at a high rate. Plants can serve as a reservoir for horizontal transmission of Rickettsia, a mechanism which may explain the occurrence of phylogenetically similar symbionts among unrelated phytophagous insect species. This plant-mediated transmission route may also exist in other insect-symbiont systems and, since symbionts may play a critical role in the ecology and evolution of their hosts, serve as an immediate and powerful tool for accelerated evolution.

Figure 1.

Results of PCR amplification of Rickettsia DNA in cotton leaves, using Rickettsia Pgt primers (table 2), demonstrating its presence. Samples 1–8: leaves not exposed to whiteflies; 9–15, 17: leaves exposed to R⁺ whiteflies; 16: NC, negative control—DNA from basil leaves not exposed to whiteflies; 18: NTC, no template control; 19: PC, positive control—DNA from R⁺ whiteflies.

Figure 2.

Results of PCR amplification of Rickettsia DNA in black nightshade and basil leaves demonstrating the presence of Rickettsia DNA. (a) PCR using Rickettsia GltA primers; (b) PCR using Rickettsia 16S rRNA primers; (c) PCR using Rickettsia Pgt primers (table 2). Samples 1–8: black nightshade leaves not exposed to whiteflies; 9–11: black nightshade leaves exposed to R⁺ whiteflies; 12–15: basil leaves not exposed to whiteflies; 16, 17: basil leaves exposed to R⁺ whiteflies; 18: NTC, no template control; 19: PC, positive control—DNA from leaves exposed to R⁺ whiteflies.

Figure 3.

(a,b) PCR amplification of Rickettsia cDNA in cotton leaves using Rickettsia Pgt primers, demonstrating the presence of Rickettsia RNA in two runs. C—cDNA from leaves not exposed to whiteflies; R—cDNA from leaves of five different plants exposed to R⁺ whiteflies; –RT, without the reverse transcriptase; NTC, no template control; PC, positive control—DNA from leaves exposed to R⁺ whiteflies. The PCR on samples R1, R2 and R4 reveal the presence Rickettsia RNA, but the bacterium could not be seen in the absence of reverse transcriptase since DNA digestion was performed after RNA extraction.

Figure 4.

(a-d) FISH of two different cotton leaves with Rickettsia-specific probe for the Phosphoglycerol transferase gene. (a,c) Rickettsia channel; (b,d) overlay of Rickettsia on bright field channels; Rickettsia (red) can be seen in the phloem. Ph, phloem; Xy, xylem.