Supplementary Material from “The inherited bacterial symbiont Hamiltonella influences the sex ratio of an insect host”

Supplementary "Materials and Methods", Table S1, Table S2, Figure S1-Figure S7

PaLCuCNV C2 interacts with RPS27A and promotes the performance of whitefly.

(A) In vitro GST pull-down assays. MBP or MBP-PaL-C2 fusion proteins were pull-down by GST or GST-NtRPS27A fusion protein. GST beads were washed and proteins were analyzed by SDS-PAGE western blot. Associated proteins were detected by anti-MBP antibody and gels were stained with Coomassie Brilliant Blue to monitor GST and GST fusion proteins. (B) In vivo BiFC analysis of PaL-C2 interaction with NtRPS27A or ubiquitin. Nuclei of tobacco leaf epidermal cells were marked with a RFP fusion protein which is located in nucleus. Bars = 20 mm. (C) Subcellular localization of PaL-C2, NtRPS27A and ubiquitin. Nuclei of tobacco leaf epidermal cells were marked with a RFP fusion protein. Bars = 20 mm. (D) Survival rate of adult whitefly on control and PaLCuCNV-infected tobacco plants. Values are means±SE, n = 30. (E) Daily number of eggs laid by per female whitefly on control empty-vector-inoculated and PaLCuCNV -infected tobacco plants. Values are means±SE, n = 30. (F) Survival rate of non-viruliferous and viruliferous adult whiteflies on cotton plants. Values are means±SE, n = 30. (G) Daily number of eggs laid by per non-viruliferous and viruliferous adult female whiteflies on cotton plants. Values are means±SE, n = 30. Asterisks indicate significant differences between different treatments (P < 0.05; Student’s t test for all experiments). All experiments were repeated three times with similar results.</p

Localization of C2 and NtRPS27A and their interaction.

(A) Structure of RPS27A. (B) Interaction between SH2-C2 and NtRPS27A in the yeast two-hybrid system. Yeast strain Y2H Gold co-transformed with the indicated plasmids was spotted on synthetic medium SD-Leu-Trp-His with x-α-gal and 2 mM 3-amino-1,2,4-triazole. The empty vectors pGBKT7 and pGADT7 were used as negative controls. (C) In vivo BiFC analysis of SH2-C2 interaction with NtRPS27A. Nuclei of tobacco leaf epidermal cells were marked with a RFP fusion protein that is located in Nuclei. Bars = 20 mm. (D) and (E) In vitro GST pull-down assays. MBP or MBP-SH2-C2 fusion proteins were pull-down by GST, GST-NtRPS27A, GST-ubiquitin or GST-ubiquitin32-76 fusion proteins. GST beads were washed and proteins were analyzed by SDS-PAGE western blot. Associated proteins were detected by anti-MBP antibody and gels were stained with Coomassie Brilliant Blue to monitor GST and GST fusion proteins. (F) Subcellular localization of SH2-C2 and different segments of NtRPS27A. Nuclei of tobacco leaf epidermal cells were marked with a RFP fusion protein that is located in Nuclei. Bars = 20 mm.</p

Interaction between TYLCV C2 and AtRPS27A affects whitefly performance.

(A) In vitro GST pull-down assays. MBP or MBP- C2 fusion proteins were pull-down by GST or GST-AtRPS27A fusion protein. GST beads were washed and proteins were analyzed by SDS-PAGE western blot. Associated proteins were detected by anti-MBP antibody and gels were stained with Coomassie Brilliant Blue to monitor GST and GST fusion proteins. (B) In vivo BiFC analysis of C2 interaction with NtRPS27A. Nuclei of tobacco leaf epidermal cells were marked with a RFP fusion protein. Bars = 20 mm. (C) Subcellular localization of C2 and AtRPS27A. Nuclei of tobacco leaf epidermal cells were marked with a RFP fusion protein. Bars = 20 mm. (D) Survival rate of adult whitefly on wild type Arabidopsis plants and the transgenic C2 expressing Arabidopsis plants. Values are means± SE, n = 30. (E) Daily number of eggs laid by per female whitefly on wild type Arabidopsis plants and the transgenic Arabidopsis plants expressing C2. Values are means±SE, n = 30. Asterisks indicate significant differences between different treatments (P < 0.05; Student’s t test for all experiments). All experiments were repeated three times with similar results.</p

RPS27A and C2 protein regulate JAZ1 degradation to manipulate plant defense.

(A) Expression of RPS27A gene in RPS27A-silencing tobacco plants. Values are means±SE, n = 8. (B) Survival rate of whitefly on control and rps27A-silenced tobacco. Values are means±SE, n = 30. (C) Daily number of eggs laid by per female whitefly on control empty-vector-inoculated and rps27A-silenced tobacco plants. Values are means±SE, n = 30. (D) Relative expression of RPS27A gene in RPS27A overexpressing (OE-RPS27A) plants. Values are means±SE, n = 8. (E) Survival rate of adult whitefly on control and RPS27A overexpressing (OE-RPS27A) tobacco plants. Values are means±SE, n = 30. (F) Daily number of eggs laid by per female whitefly on control and RPS27A overexpressing (OE-RPS27A) plants. Values are means±SE, n = 30. (G) Expression of terpene-related genes [epi-arisotolchene synthase (EAS), epi-arisotolchene synthase 12 (EAS12), terpenoid cyclase (Cyclase)] in control and RPS27A-silencing tobacco plants. Values are means±SE, n = 8. (H) Expression levels of MYC2 in control and RPS27A-silencing tobacco plants. Values are means±SE, n = 8. (I) Effects of whitefly infestation on content of RPS27A protein. (J) & (K) RPS27A-silencing and C2 overexpression affect the degradation of JAZ1 protein. Accumulation of JAZ1 protein in control, whiteflies infested, rps27A-silencing or C2 expressing plants was detected by western blot. Same amount of leave samples were collected and JAZ1 protein content was detected by western blot. Asterisks indicate significant differences between different treatments (P < 0.05; Student’s t test for all experiments). All experiments were repeated twice with similar results.</p

TYLCV infection subverts plant JA signaling pathway to benefit whitefly.

(A) Survival rate of adult whitefly on control and TYLCV-infected tobacco plants. Values are means±SE, n = 30. (B) Daily number of eggs laid per female whitefly on control and TYLCV-infected tobacco plants. Values are means±SE, n = 30. (C) Survival rate of non-viruliferous and viruliferous adult whiteflies on cotton plants. Values are means±SE, n = 30. (D) Daily number of eggs laid per non-viruliferous and viruliferous adult female whiteflies on cotton plants. Values are means±SE, n = 30. (E) Expression of JA synthesis-related genes [allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxophytodienoate reductase 3 (OPR3)] in healthy (H), whitefly infestation (W), TYLCV infection (V) plants and plants with both TYLCV and whiteflies (VW). Values are means±SE, n = 8. (F) JA content in tobacco plants after different treatments: healthy (H), whitefly infestation (W), TYLCV infection (V) plants and plants with both TYLCV and whiteflies (VW) and wounding plants. Values are means±SE, n>6. (G) Expression of genes related to terpene syntheses [epi-arisotolchene synthase (EAS), epi-arisotolchene syntheses 12 (EAS12), terpenoid cyclase (cyclase)] in healthy (H), whitefly infestation (W), TYLCV infection (V) plants and plants with both TYLCV and whiteflies (VW). Values are means±SE, n = 8. (H) Expression levels of MYC2 in healthy (H), whitefly infestation (W), TYLCV infection (V) plants and plants with both TYLCV and whiteflies (VW). Values are means±SE, n = 8. (I) Expression level of genes related to nicotine syntheses in healthy (H), whitefly infestation (W), TYLCV infection (V) plants and plants with both TYLCV and whiteflies (VW). Values are means±SE, n = 8. Asterisks or different letters above the bars indicate significant differences between different treatments (P < 0.05; Student’s t test for all experiments). All experiments were repeated three times with similar results.</p

Model for the role of TYLCV C2 in regulating plant defense.

(A) When plants are infested by whiteflies, JAZ1 protein can be degraded by ubiquitination via El ubiquitin-activating enzyme (E1), E2 ubiquitin-conjugating enzymes (E2) and E3 ubiquitin–protein ligase (E3) in 26S proteasomes. Then the MYC2 transcription factor is released from JAZ1-MYC2 protein complex, binds to the promoter of defense genes and activates the expression of down-stream defense genes, such as epi-arisotolchene synthase (EAS), and terpene synthase (TPS). As a consequence, plant defense is triggered. (B) When TYLCV and whitefly co-infect plants, TYLCV C2 protein competitively bind to ubiquitin, which lead to the decrease of JAZ1 protein ubiquitination. As a consequence, the MYC2 is bound to JAZ1 and unable to induce the gene expression of down-stream defense genes.</p

TYLCV C2 promotes whitefly performance by suppressing plant JA signaling.

(A) Survival rate of adult whitefly on wild type tobacco plants (WT) and the transgenic C2 expressing tobacco plants (C2). Values are means±SE, n = 30. (B) Daily number of eggs laid by per female whitefly on wild type tobacco plants and C2 expressing tobacco plants. Values are means±SE, n = 30. (C) Expression of genes related to JA synthesis [allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxophytodienoate reductase 3 (OPR3)] in wild type (WT) and C2 expressing tobacco plants. Values are means±SE, n = 8. (D) Expression level of MYC2 in wild type (WT) and C2 expressing tobacco plants. Values are means±SE, n = 8. (E) Expression of genes related to terpene synthesis [epi-arisotolchene synthase (EAS), epi-arisotolchene synthase 12 (EAS12), terpenoid cyclase (cyclase)] in wild type (WT) and C2 expressing tobacco plants. Values are means±SE, n = 8. (F) Expression levels of genes related to nicotine syntheses in wild type (WT) and C2 expressing tobacco plants. Values are means±SE, n = 8. Asterisks indicate significant differences between different treatments (P < 0.05; Student’s t test for all experiments). All experiments were repeated three times with similar results.</p