Fig 1.
Lysine synthesis pathway in B. tabaci MEAM1.
This figure is adapted from previous work (9, 29). HTHD, 4-hydroxy-2,3,4,5-tetrahydrodipicolinate; THD, 2,3,4,5-tetrahydrodipicolinate; SDAP, N-succinyl-L-2,6-diaminoheptanedioate; SAOP, N-succinyl-L-2-amino-6-oxoheptanedioate; 2,6-DAP, 2,6-diaminopimelate; meso-DAP, meso-2,6-diaminopimelate. Pseudogene is in grey and enriched host genes of bacterial origin are in green. The exchanges of intermediates among Portiera, Hamiltonella and Rickettsia are speculated.
Fig 2.
Effects of Hamiltonella deficiency on lysine gene expression and lysine levels in B. tabaci.
(A) Localization of symbiotic bacteria Portiera (red) and Hamiltonella (green) in the whitefly bacteriocyte, as well as Portiera (red) and Rickettsia (green) in the whitefly ovary. n = 3. (B) Effects of antibiotic treatments on the abundance of symbionts in B. tabaci at 5 d, 10 d, and 15 d after emergence. H, P and R represent Hamiltonella, Portiera and Rickettsia, respectively. n = 12, 10 and 11 for 5 d, 10 d, and 15 d, respectively. (C) Effects of Hamiltonella deficiency on expression of whitefly lysine genes in B. tabaci at 5 d, 10 d, and 15 d after emergence. n = 4. (D) Effects of Hamiltonella deficiency on expression of Portiera lysine gene in B. tabaci at 5 d, 10 d, and 15 d after emergence. n = 4. (E) Effects of Hamiltonella deficiency on expression of Rickettsia lysine genes in B. tabaci at 5 d, 10 d, and 15 d after emergence. n = 4. (F) Effects of Hamiltonella deficiency on lysine levels in B. tabaci at 5 d, 10 d, and 15 d after emergence. n = 6. +HBt and -HBt represent Hamiltonella-infected and Hamiltonella-cured whiteflies, respectively. Data are means ± SEM. The significant differences between treatments are indicated by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001).
Fig 3.
Effects of Hamiltonella deficiency on protein localization in B. tabaci.
Localization of DapB, DapF and LysA proteins (green) in bacteriocytes (A-C) and ovaries (D-F) of female adult whiteflies. DNA was stained with DAPI. +HBt and -HBt represent Hamiltonella-infected and Hamiltonella-cured whiteflies, respectively. n = 3.
Fig 4.
Functional complementation of E. coli lysine auxotrophs.
E. coli K-12 knockout mutant cells were transformed with expression plasmids containing whitefly dapB, dapF, and lysA or the negative control pMD19-T empty vector. The E. coli wild-type K-12, mutant K-12 (-Δ) and mutant K-12 transformants (+Δ) were grown overnight in amino acid-deficient M9 liquid medium at 37°C. All E. coli cells were washed and re-suspended to measure cell density at OD600. Data are means ± SEM. n = 3. Different letters above the bars indicate significant differences between treatments at P < 0.05.
Fig 5.
Effects of silencing horizontally transferred lysA on lysine levels, whitefly fecundity, symbiont titer and Hamiltonella lysA expression in whiteflies infected with Hamiltonella.
(A) Lysine levels in whiteflies at day 3 after whiteflies were microinjected with dslysA. n = 3. (B) Fecundity of female adult whiteflies at day 3 after microinjection with dslysA. n = 22. (C) Effects of silencing horizontally transferred lysA on symbiont titer. n = 11. (D) Expression of Hamiltonella lysA at day 3 after whiteflies were microinjected with dslysA. n = 4. dsGFP and dslysA represent dsGFP-injected and dslysA-injected female adult whiteflies, respectively. Data are means ± SEM. The significant differences between treatments are indicated by asterisks (*P < 0.05; **P < 0.01).
Fig 6.
Effects of silencing horizontally transferred lysA on lysine levels, whitefly fecundity, and symbiont titer in whiteflies lacking Hamiltonella.
(A) Effects of antibiotic treatments on the abundance of symbionts in B. tabaci. n = 10. (B) Lysine levels in Hamiltonella-cured whiteflies at day 3 after whiteflies were microinjected with dslysA. n = 4. (C) Fecundity of Hamiltonella-cured female adult whiteflies at day 3 after microinjection with dslysA. n = 14. (D) Effects of silencing horizontally transferred lysA on symbiont titer in ovarioles of Hamiltonella-cured whiteflies at day 3 after microinjection with dslysA. n = 11. +HBt and -HBt represent Hamiltonella-infected and Hamiltonella-cured whiteflies, respectively. dsGFP and dslysA represent dsGFP-injected and dslysA-injected female adult whiteflies, respectively. Data are means ± SEM. The significant differences between treatments are indicated by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001).
Fig 7.
Schematic overview of how lysine provisioning by horizontally acquired bacteria genes promotes mutual dependence between whitefly and two intracellular symbionts.
The whitefly lifestages consist of egg, nymphs and adult. Whiteflies can synthesize lysine through the cooperation of horizontally transferred genes (HTGs) and two symbionts (one obligate symbiont Portiera and one facultative symbiont Rickettsia). The lysine synthesized through HTGs impacts the fecundity of adult whiteflies and titers of two symbionts.