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Polydnaviral ankyrin proteins aid parasitic wasp survival by coordinate and selective inhibition of hematopoietic and immune NF-kappa B signaling in insect hosts.

Gueguen G, Kalamarz ME, Ramroop J, Uribe J, Govind S - PLoS Pathog. (2013)

Bottom Line: These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene.Additionally, their maternal expression weakens ventral embryonic patterning.We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, The City College of the City University of New York, New York, New York, United States of America.

ABSTRACT
Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral ankyrins or vankyrins. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous in vivo Drosophila, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. Drosophila NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I²-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.

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Effects of Dif and Dorsal on Antp>GFP expression and on crystal cell number.(A–E) Antp>GFP expression in third instar lymph glands. A. Control. B. Knockdown of Dif (DifRNAi), or C. Dorsal (dlRNAi). Overexpression of D. Dif (Dif), or E. Dorsal (dl). F. Manipulation of Dif or Dorsal levels does not affect the number of Antp>GFP-positive cells (t = −1.45, df = 6.5, p = 0.19 for Antp>DifRNAi, t = 1.45, df = 4.7, p = 0.21 for Antp>dlRNAi, t = −0.62, df = 6.2, p = 0.55 for Antp>Dif and t = −1.29, df = 6.3, p = 0.24 for Antp>dl). G. Quantification of Antp>GFP expression. The intensity of the GFP signal is reduced in Antp>DifRNAi (t = 3.4, df = 7.8, p = 0.01) and Antp>dlRNAi (t = 7.8, df = 5.6, p<0.001) and increased in Antp>Dif (t = −2.4, df = 8, p = 0.04) and Antp>dl (134.6 versus 216.2 - t = −8.2, df = 5.4, p<0.001), compared to controls, N = 5 animals for each genotype. H–I. Anterior lobes of animals H. heterozygous for the deficiency lacking Dif and dl (Df(2L)TW119/+ or Df(2L)J4/+), and I. Dif/dl mutants (Df(2L)TW119/Df(2L)J4) stained for ProPO2 (magenta, crystal cells). (J–K) Crystal cells (black spots) of third instar, J. heterozygous control, and K. Df(2L)TW119/Df(2L)J4 mutant, visualized by incubation at 70°C. L. The average number of sessile crystal cells in the three posterior segments is significantly increased in Dif− dl− mutants (t = −3.4, df = 25.9, p = 0.002. N = 20 for heterozygotes; N = 21 for Dif− dl− mutants). Bars indicate standard deviation. Stars indicate statistical significance relative to controls (* for 0.05<p<0.01, ** for 0.01<p<0.001 and *** for p<0.001).
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ppat-1003580-g003: Effects of Dif and Dorsal on Antp>GFP expression and on crystal cell number.(A–E) Antp>GFP expression in third instar lymph glands. A. Control. B. Knockdown of Dif (DifRNAi), or C. Dorsal (dlRNAi). Overexpression of D. Dif (Dif), or E. Dorsal (dl). F. Manipulation of Dif or Dorsal levels does not affect the number of Antp>GFP-positive cells (t = −1.45, df = 6.5, p = 0.19 for Antp>DifRNAi, t = 1.45, df = 4.7, p = 0.21 for Antp>dlRNAi, t = −0.62, df = 6.2, p = 0.55 for Antp>Dif and t = −1.29, df = 6.3, p = 0.24 for Antp>dl). G. Quantification of Antp>GFP expression. The intensity of the GFP signal is reduced in Antp>DifRNAi (t = 3.4, df = 7.8, p = 0.01) and Antp>dlRNAi (t = 7.8, df = 5.6, p<0.001) and increased in Antp>Dif (t = −2.4, df = 8, p = 0.04) and Antp>dl (134.6 versus 216.2 - t = −8.2, df = 5.4, p<0.001), compared to controls, N = 5 animals for each genotype. H–I. Anterior lobes of animals H. heterozygous for the deficiency lacking Dif and dl (Df(2L)TW119/+ or Df(2L)J4/+), and I. Dif/dl mutants (Df(2L)TW119/Df(2L)J4) stained for ProPO2 (magenta, crystal cells). (J–K) Crystal cells (black spots) of third instar, J. heterozygous control, and K. Df(2L)TW119/Df(2L)J4 mutant, visualized by incubation at 70°C. L. The average number of sessile crystal cells in the three posterior segments is significantly increased in Dif− dl− mutants (t = −3.4, df = 25.9, p = 0.002. N = 20 for heterozygotes; N = 21 for Dif− dl− mutants). Bars indicate standard deviation. Stars indicate statistical significance relative to controls (* for 0.05<p<0.01, ** for 0.01<p<0.001 and *** for p<0.001).

Mentions: To test their individual effects on the niche and on hematopoiesis, we modulated NF-κB levels in the niche. (1) RNAi knockdown with either Antp>GFP, DifRNAi or Antp>GFP, dlRNAi did not yield a significant difference in the number of GFP-positive cells (Fig. 3A–C, F), although, unexpectedly, the intensity of the Antp>GFP signal in cells with RNAi was significantly reduced (Fig. 3A–C, G). Conversely, ectopic expression of Dif or Dorsal in the niche increased Antp>GFP expression (Fig. 3D–E, G). (2) Overexpression of either wild type protein (Antp>dl or Antp>Dif) also showed supernumerary lamellocytes in the lymph gland lobes (Fig. S1G, J–K). (3) We found no significant difference in the number of crystal cells in Antp>GFP, DifRNAi or Antp>GFP, dlRNAi glands, slight increase in Antp>GFP, Dif but no change in Antp>GFP, dl (Fig. S1A–F). However, Df(2L)119/Df(2L)J4 mutants lacking both NF-κB proteins show significantly more crystal cells in the lymph gland and in the sessile compartment, compared to heterozygous controls (Fig. 3H–L), indicating inhibitory and redundant NF-κB roles in crystal cell development. Anti-Antp staining of lobes from Df(2L)119/Df(2L)J4 glands revealed that the niche is specified in the absence of Dif and Dorsal and the Antp protein expression levels appears comparable to those in heterozygous controls (data not shown). Together, these results suggest that Dorsal and Dif can modulate Antp-Gal4 transgene expression in the niche and are required non cell-autonomously for the proportional development of crystal cell and lamellocytic lineages.


Polydnaviral ankyrin proteins aid parasitic wasp survival by coordinate and selective inhibition of hematopoietic and immune NF-kappa B signaling in insect hosts.

Gueguen G, Kalamarz ME, Ramroop J, Uribe J, Govind S - PLoS Pathog. (2013)

Effects of Dif and Dorsal on Antp>GFP expression and on crystal cell number.(A–E) Antp>GFP expression in third instar lymph glands. A. Control. B. Knockdown of Dif (DifRNAi), or C. Dorsal (dlRNAi). Overexpression of D. Dif (Dif), or E. Dorsal (dl). F. Manipulation of Dif or Dorsal levels does not affect the number of Antp>GFP-positive cells (t = −1.45, df = 6.5, p = 0.19 for Antp>DifRNAi, t = 1.45, df = 4.7, p = 0.21 for Antp>dlRNAi, t = −0.62, df = 6.2, p = 0.55 for Antp>Dif and t = −1.29, df = 6.3, p = 0.24 for Antp>dl). G. Quantification of Antp>GFP expression. The intensity of the GFP signal is reduced in Antp>DifRNAi (t = 3.4, df = 7.8, p = 0.01) and Antp>dlRNAi (t = 7.8, df = 5.6, p<0.001) and increased in Antp>Dif (t = −2.4, df = 8, p = 0.04) and Antp>dl (134.6 versus 216.2 - t = −8.2, df = 5.4, p<0.001), compared to controls, N = 5 animals for each genotype. H–I. Anterior lobes of animals H. heterozygous for the deficiency lacking Dif and dl (Df(2L)TW119/+ or Df(2L)J4/+), and I. Dif/dl mutants (Df(2L)TW119/Df(2L)J4) stained for ProPO2 (magenta, crystal cells). (J–K) Crystal cells (black spots) of third instar, J. heterozygous control, and K. Df(2L)TW119/Df(2L)J4 mutant, visualized by incubation at 70°C. L. The average number of sessile crystal cells in the three posterior segments is significantly increased in Dif− dl− mutants (t = −3.4, df = 25.9, p = 0.002. N = 20 for heterozygotes; N = 21 for Dif− dl− mutants). Bars indicate standard deviation. Stars indicate statistical significance relative to controls (* for 0.05<p<0.01, ** for 0.01<p<0.001 and *** for p<0.001).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757122&req=5

ppat-1003580-g003: Effects of Dif and Dorsal on Antp>GFP expression and on crystal cell number.(A–E) Antp>GFP expression in third instar lymph glands. A. Control. B. Knockdown of Dif (DifRNAi), or C. Dorsal (dlRNAi). Overexpression of D. Dif (Dif), or E. Dorsal (dl). F. Manipulation of Dif or Dorsal levels does not affect the number of Antp>GFP-positive cells (t = −1.45, df = 6.5, p = 0.19 for Antp>DifRNAi, t = 1.45, df = 4.7, p = 0.21 for Antp>dlRNAi, t = −0.62, df = 6.2, p = 0.55 for Antp>Dif and t = −1.29, df = 6.3, p = 0.24 for Antp>dl). G. Quantification of Antp>GFP expression. The intensity of the GFP signal is reduced in Antp>DifRNAi (t = 3.4, df = 7.8, p = 0.01) and Antp>dlRNAi (t = 7.8, df = 5.6, p<0.001) and increased in Antp>Dif (t = −2.4, df = 8, p = 0.04) and Antp>dl (134.6 versus 216.2 - t = −8.2, df = 5.4, p<0.001), compared to controls, N = 5 animals for each genotype. H–I. Anterior lobes of animals H. heterozygous for the deficiency lacking Dif and dl (Df(2L)TW119/+ or Df(2L)J4/+), and I. Dif/dl mutants (Df(2L)TW119/Df(2L)J4) stained for ProPO2 (magenta, crystal cells). (J–K) Crystal cells (black spots) of third instar, J. heterozygous control, and K. Df(2L)TW119/Df(2L)J4 mutant, visualized by incubation at 70°C. L. The average number of sessile crystal cells in the three posterior segments is significantly increased in Dif− dl− mutants (t = −3.4, df = 25.9, p = 0.002. N = 20 for heterozygotes; N = 21 for Dif− dl− mutants). Bars indicate standard deviation. Stars indicate statistical significance relative to controls (* for 0.05<p<0.01, ** for 0.01<p<0.001 and *** for p<0.001).
Mentions: To test their individual effects on the niche and on hematopoiesis, we modulated NF-κB levels in the niche. (1) RNAi knockdown with either Antp>GFP, DifRNAi or Antp>GFP, dlRNAi did not yield a significant difference in the number of GFP-positive cells (Fig. 3A–C, F), although, unexpectedly, the intensity of the Antp>GFP signal in cells with RNAi was significantly reduced (Fig. 3A–C, G). Conversely, ectopic expression of Dif or Dorsal in the niche increased Antp>GFP expression (Fig. 3D–E, G). (2) Overexpression of either wild type protein (Antp>dl or Antp>Dif) also showed supernumerary lamellocytes in the lymph gland lobes (Fig. S1G, J–K). (3) We found no significant difference in the number of crystal cells in Antp>GFP, DifRNAi or Antp>GFP, dlRNAi glands, slight increase in Antp>GFP, Dif but no change in Antp>GFP, dl (Fig. S1A–F). However, Df(2L)119/Df(2L)J4 mutants lacking both NF-κB proteins show significantly more crystal cells in the lymph gland and in the sessile compartment, compared to heterozygous controls (Fig. 3H–L), indicating inhibitory and redundant NF-κB roles in crystal cell development. Anti-Antp staining of lobes from Df(2L)119/Df(2L)J4 glands revealed that the niche is specified in the absence of Dif and Dorsal and the Antp protein expression levels appears comparable to those in heterozygous controls (data not shown). Together, these results suggest that Dorsal and Dif can modulate Antp-Gal4 transgene expression in the niche and are required non cell-autonomously for the proportional development of crystal cell and lamellocytic lineages.

Bottom Line: These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene.Additionally, their maternal expression weakens ventral embryonic patterning.We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, The City College of the City University of New York, New York, New York, United States of America.

ABSTRACT
Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral ankyrins or vankyrins. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous in vivo Drosophila, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. Drosophila NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I²-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.

Show MeSH
Related in: MedlinePlus