Figure 1.
Western blot analysis of r-bursicon proteins in non-reduced (A) and reduced (B) SDS-PAGE identified with an anti-His-Tag antibody.
The positions of the r-burs α−β heterodimer, burs α−α and burs β−β homodimers are indicated. Lanes annotated α, β and α+β indicate separate expression of burs α and burs β and co-expression of α+β. The monomers are not present under non-reducing conditions (A). Numbers on far left indicate positions of molecular weight standards.
Figure 2.
Transcription expression profiles of burs α and burs β subunits and AMP genes in pharate and newly-emerged adults.
qPCR analysis (A) of bursicon α and β gene expression in pharate (PA) and eclosed adults (A) 0–24 h after eclosion; qPCR analysis (B) of AMP gene expression in pharate and eclosed adults 0–24 h after eclosion; Transcription expression (C) of AMP genes in 24 h-old adults injected with blank vector transfected sample, r-burs α−α homodimer, r-burs β−β homodimer and r-burs α−β heterodimer, respectively.
Figure 3.
Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in adults.
Separate groups of adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with 60 ng/0.5 µl of r-burs α−β, burs α−α, or burs β−β. The control flies were injected with the purified cell culture transfected with blank pcDNA 3.1 vector. After the indicated incubation periods, expression of the AMP genes was determined by qPCR. (A) AMP transcript levels in wild type adults. For bacterial inhibition assay (B), neck-ligated wild type flies were injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, respectively, homogenized and centrifuged for 15 min at 16,000 g. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count. The histograms show the means ± SEM, n = 3 biologically independent experiments.
Figure 4.
Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in larva FB.
FB was dissected from early wondering 3rd instar larvae and incubated with r-burs α−α or burs β−β homodimer or r-burs α+β heterodimer for 0.5, 1 and 3 h. The control group received the blank vector transfected sample. After incubation, RNA was extracted for qPCR analysis of 4 representative AMP genes (A). For bacterial inhibition assay, FB was homogenized and centrifuged at 16000 g for 20 min at 4°C. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.
Figure 5.
AMP transcript levels in rk4 mutant.
Newly emerged adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, as described in Figure 3. RNA was extracted for qPCR analysis of 11 representative genes (A). Larva FB from the mutant was also used to assay the effect of mutation on burs α−α and burs β−β homodimer induced AMP expression (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.
Figure 6.
Western blot analysis of NF-κB transcription factor Relish activation in wild type larvae and adults.
(A) Neck ligated flies were treated with indicated r-burs α−β, burs α−α, burs β−β or blank vector transfected sample. After the indicated incubation periods the flies were homogenized and then subjected to Western blot analysis using anti-Relish polyclonal antibodies. Bands labeled actin show protein loading controls. (B) Third instar larval fat body preparations were treated with the indicated bursicon proteins. After incubation for the indicated times the FB preparations were homogenized and subjected to Western blot analysis using anti-Relish monoclonal antibody. (C) qPCR analysis of 3 representative genes in the wild type and Relish mutant RelE20 flies. (D) RT-PCR analysis shows the presence of Relish transcripts in the wild type controls, but not the mutant RelE20. The histograms show the means ± SEM, n = 3 biologically independent experiments. Actin loading controls are present in the bottom of A and B.
Figure 7.
A partly hypothetical model of bursicon signaling pathways, showing the known interaction between burs α−β heterodimer and its receptor, DLGR2, and interactions between burs α−α and burs β−β homodimers and a hypothesized receptor(s) leading to the transcription of AMP genes.
The model also shows the Imd pathway and the position of Relish. Once activated, Relish translocates into the nucleus, where it promotes transcription of genes encoding AMPs.