Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Apr 2;121(14):e2315982121.
doi: 10.1073/pnas.2315982121. Epub 2024 Mar 27.

Maintenance of persistent transmission of a plant arbovirus in its insect vector mediated by the Toll-Dorsal immune pathway

Yu-Juan He  1 , Gang Lu  1 , Bo-Jie Xu  2 , Qian-Zhuo Mao  1 , Yu-Hua Qi  1 , Gao-Yang Jiao  1 , Hai-Tao Weng  1 , Yan-Zhen Tian  1 , Hai-Jian Huang  1 , Chuan-Xi Zhang  1 , Jian-Ping Chen  1 , Jun-Min Li  1
Affiliations

Maintenance of persistent transmission of a plant arbovirus in its insect vector mediated by the Toll-Dorsal immune pathway

Yu-Juan He et al. Proc Natl Acad Sci U S A. .

Abstract

Throughout evolution, arboviruses have developed various strategies to counteract the host's innate immune defenses to maintain persistent transmission. Recent studies have shown that, in addition to bacteria and fungi, the innate Toll-Dorsal immune system also plays an essential role in preventing viral infections in invertebrates. However, whether the classical Toll immune pathway is involved in maintaining the homeostatic process to ensure the persistent and propagative transmission of arboviruses in insect vectors remain unclear. In this study, we revealed that the transcription factor Dorsal is actively involved in the antiviral defense of an insect vector (Laodelphax striatellus) by regulating the target gene, zinc finger protein 708 (LsZN708), which mediates downstream immune-related effectors against infection with the plant virus (Rice stripe virus, RSV). In contrast, an antidefense strategy involving the use of the nonstructural-protein (NS4) to antagonize host antiviral defense through competitive binding to Dorsal from the MSK2 kinase was employed by RSV; this competitive binding inhibited Dorsal phosphorylation and reduced the antiviral response of the host insect. Our study revealed the molecular mechanism through which Toll-Dorsal-ZN708 mediates the maintenance of an arbovirus homeostasis in insect vectors. Specifically, ZN708 is a newly documented zinc finger protein targeted by Dorsal that mediates the downstream antiviral response. This study will contribute to our understanding of the successful transmission and spread of arboviruses in plant or invertebrate hosts.

Keywords: Laodelphax striatellus; Rice stripe virus; dorsal; toll immune pathway; zinc finger protein.

PubMed Disclaimer

Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
LsDorsal knockdown promotes RSV replication and transmission in L. striatellus. (A and B) Detection for the transcript levels of LsDorsal injected with crude extracts of RSV-free and RSV-infected L. striatellus at various time points. (C–E) Effects of LsDorsal knockdown on the expression levels of RSV-NP in L. striatellus treated with dsLsDorsal and RSV crude extracts for transcripts at 3 and 6 dpi (C), protein at 6 dpi (D), and ratio of virus acquisition (3 dpi) and transmission (E). (F) Immunofluorescence staining of RSV NP in the SG and ovary of L. striatellus at 6 dpi after treatment with dsGFP or dsLsDorsal and RSV crude extracts. (Scale bar, 50 μm.) (G) Identification of Dorsal phosphorylation site in L. striatellus based on its homologous to Rel (Homo sapiens) and p65 (Drosophila melanogaster). (H) Phosphorylation level of Dorsal protein in the nucleus of L. striatellus at different time points after RSV infection. “VF 3 dpi” indicated that nonviruliferous L. striatellus were injected with RSV-free insect crude extracts for 3 d. “RSV 3 dpi” and “RSV 6 dpi” indicated that nonviruliferous L. striatellus were injected with RSV-infected insect crude extracts for 3 d and 6 d, respectively. H3 and GAPDH antibodies represent specific marker of the cell nucleus and cytoplasm, respectively. Three biological replicates were performed for each of the experiment (10 to 15 L. striatellus for each of the replicate). The t test method was used for significance analysis. * represents significant difference (P < 0.05), ** and *** represent extremely significant difference (P < 0.01 and P < 0.001). The error bars represent the SE of the mean.
Fig. 2.
Fig. 2.
LsDorsal binds to the promoter of the zinc finger protein LsZN708 and regulates its expression. (A and B) Evaluation for the binding ability of LsDorsal to the promoters of eight candidate genes in RSV-free (A) and RSV-infected L. striatellus (B) by Chip-qPCR. (C) Comparative analysis for the LsDorsal binding ability to the LsZN708 promoter in nonviruliferous and viruliferous L. striatellus. (D) Verification for the binding of LsDorsal to the promoter of the Zinc Finger Protein LsZN708 using yeast one-hybrid assay. pAbAi-LsZN708 indicated that the promoter of LsZN708 was constructed to pAbAi vector. AD-LsDorsal and AD-RHD-n suggested that the full length of genes was constructed to pGAD-T7 vector. pAbAi-P53 and AD-T7-REC represented a group of positive control. pAbAi-LsZN708 and AD-T7 represented a group of negative control. ABA screening concentration was set at 50 ng/μL (inhibiting self-activation). The self-activation assay was performed on selective medium SD/-Ura. The different combinations of constructs transformed into yeast cells were grown on selective medium SD/-Leu, and interactions were detected on SD/-Leu/-ABA (50). Pictures were taken after 3 d of incubation at 30 °C. IgG antibody was used as a negative control, and three biological replicates were performed. (E and F) Effects of LsDorsal knockdown on the expression levels of transcript (E) and protein (F) of LsZN708 in the viruliferous L. striatellus injected with dsLsDorsal. (G and H) Effects of LsDorsal knockdown on the transcripts of LsZN708 in nonviruliferous L. striatellus treated with dsGFP or dsLsDorsal and RSV crude extracts at 3 and 6 dpi, respectively. Three biological replicates were performed for each experiment. The t test method was used for significance analysis. * represents significant difference (P < 0.05), ** and *** represent extremely significant difference (P < 0.01 and P < 0.001), n.s. means no significance. The error bars represent the SE of the mean.
Fig. 3.
Fig. 3.
LsZN708 knockdown promotes RSV replication and transmission in L. striatellus. (A) Transcription level of LsZN708 in nonviruliferous and viruliferous L. striatellus. (B and C) Relative transcription levels of RSV-NP in viruliferous L. striatellus treated with dsGFP or dsLsZN708 at 3 dpi and 6 dpi, respectively. (D–F) Effects of dsLsZN708 knockdown on the expression levels of RSV-NP in L. striatellus treated with dsLsZN708 and RSV crude extracts for transcripts at 3 and 6 dpi (D), protein at 6 dpi (E), and ratio of virus acquisition (3 dpi) and transmission (F). (G) Immunofluorescence staining of RSV NP in the SG of L. striatellus treated with dsLsZN708 or dsLsDorsal and RSV crude extracts at 6 dpi. The scale bar represents 50 μm. Three biological replicates were conducted for each experiment. Significance was determined using the t test method. * represents significant difference (P < 0.05), ** and *** represent extremely significant difference (P < 0.01 and P < 0.001). The error bars represent the SE of the mean.
Fig. 4.
Fig. 4.
Downstream immune-related effectors of toll pathway that potentially involved in the antiviral response of L. striatellus against RSV infection. (A and B) KEGG pathway enrichment analysis of the common DEG in nonviruliferous L. striatellus treated with a dsLsDorsal or dsLsZN708 and RSV crude extracts at 3 dpi (A) and 6 dpi (B). Significant differences were indicated when log2 (fold change) ratio was ≥1 and P ≤ 0.05. (C) The interaction between LsZN708 and autophagy protein LsAtg8 through Y2H assay. The different combinations of constructs transformed into yeast cells were grown on selective medium SD/-Leu/-Trp, and interactions were detected on SD/-Ade/-His/-Leu/-Trp. The images were taken after 3 d of incubation at 30 °C. (D) The interaction between LsZN708 and LsAtg8 protein through an in vitro pull-down assay. MBP-ZN708 fusion protein was used to pull-down with His-Atg8. MBP was used as negative control and His-Atg8 was further detected with anti-His antibody. (E–H) Effects of LsZN708 or LsDorsal knockdown on the transcription levels of autophagy-related genes (LsAtg8, LsAtg5 and LsAtg3) in nonviruliferous L. striatellus treated with dsLsZN708 (E and F) or dsLsDorsal (G and H) and RSV crude extracts at 3 or 6 dpi. (I) Protein level of LsAtg8II in viruliferous L. striatellus treated with QNZ inhibitor for 48 h (DMSO was used as control). Three biological replicates were performed for each experiment. Significance analysis was performed using the t test method. * represents a significant difference (P < 0.05), ** and *** represent extremely significant differences (P < 0.01 and P < 0.001). The error bars represent the SE of the mean.
Fig. 5.
Fig. 5.
Nonstructural protein NS4 of RSV (RSV NS4) participates in the viral counterdefense strategy through the inhibition of LsDorsal phosphorylation. (A) Interaction of RSV NS4 with LsDorsal or RHD-n domain (301 to 339 aa) of LsDorsal was confirmed by Y2H assay. (B) BIFC assays verified the interaction between LsDorsal and NS4 in the cell nucleus and cytoplasm of Nicotiana benthamiana leaves. (C) The interaction between LsDorsal and NS4 was confirmed by an in vitro pull-down assay. MBP-Dorsal and MBP-RHD-n proteins were used to pull-down with His-NS4. His-NS4 was further detected with anti-His antibody. (D) The Co-IP assay confirmed the interaction between LsDorsal and NS4 of L. striatellus in vivo. The crude extracts of L. striatellus were prepared and immunoprecipitated by ProteinG-NS4 combinations. The coimmunoprecipitated proteins were detected with LsDorsal antibody. (E and F) Effect of NS4 knockdown on the transcription (E) and phosphorylation (F) levels of LsDorsal in viruliferous L. striatellus (dsGFP as control). (G) Phosphorylation level of LsDorsal in nonviruliferous L. striatellus treated with purified NS4 protein (GFP protein as control). (H) Phosphorylation level of LsDorsal in nonviruliferous L. striatellus treated with dsNS4 and RSV crude extracts at 5 dpi. (I–K) Effect of NS4 knockdown on the protein level of p-Dorsal in the nucleus and cytoplasm (I), the protein level of LsZN708 (J), and relative transcription levels of autophagy genes LsAtg8, LsAtg3, LsAtg12 and LsSqstm1 (K) in viruliferous L. striatellus. Three biological replicates were performed for each experiment. The t test method was used for significance analysis. * represents significant difference (P < 0.05), * and *** represent extremely significant difference (P < 0.01 and P < 0.001), n.s. means no significance. The error bars represent the SE of the mean.
Fig. 6.
Fig. 6.
RSV NS4 competes with LsMSK2 for LsDorsal binding. (A) Interaction between RHD-n domain of LsDorsal and the C-terminus of LsMSK2 kinase confirmed through Y2H assay. (B) An in vitro pull-down assay verified the interaction between LsRHD-n and C-terminus of LsMSK2. GST-RHD-n protein were used to pull-down with His-MSK2-C. His-MSK2-C was further detected with anti-His antibody. (C) The phosphorylation of LsDorsal protein by LsMSK2 kinase protein in vitro. (D) Binding ability of LsMSK2-C to LsRHD-n with the increasing concentration of NS4 protein. (E) Binding ability of NS4 to LsRHD-n with the increasing concentration of LsMSK2-C. (F) Effect of LsMSK2 knockdown on the accumulation level of RSV in viruliferous L. striatellus treated with dsGFP and dsLsMSK2. (G) Effect of LsMSK2 knockdown on the accumulation level of RSV in L. striatellus treated with dsGFP or dsLsMSK2 and RSV crude extracts at 6 dpi. Three biological replicates were performed for each experiment. The t test method was used for significance analysis. * represents significant difference (P < 0.05), ** and *** represent extremely significant difference (P < 0.01 and P < 0.001). The error bars represent the SE of the mean.
Fig. 7.
Fig. 7.
The schematic diagram indicating toll-dorsal pathway mediated homeostasis for the persistent transmission of RSV in L. striatellus. The Toll pathway is activated by the interaction between the Toll receptor of L. striatellus and RSV NP. Then, Dorsal participated in the antiviral defense of L. striatellus by regulating the target gene LsZN708 and induced the downstream immune response pathways (such as autophagy) against RSV infection (Left). Conversely, RSV also developed antidefense strategy by using its NS4 protein to antagonize the host Toll antiviral defense through competitively binding to LsDorsal with LsMSK2 kinase, resulting in the inhibition of LsDorsal phosphorylation and translocation to the nucleus (Right).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Janeway C. A. Jr., Medzhitov R., Innate immune recognition. Annu. Rev. Immunol. 20, 197–216 (2002). - PubMed
    1. Nakamoto M., et al. , Virus recognition by Toll-7 activates antiviral autophagy in Drosophila. Immunity 36, 658–667 (2012). - PMC - PubMed
    1. Zambon R. A., Nandakumar M., Vakharia V. N., Wu L. P., The Toll pathway is important for an antiviral response in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 102, 7257–7262 (2005). - PMC - PubMed
    1. Bang I. S., JAK/STAT signaling in insect innate immunity. Entomol. Res. 49, 339–353 (2019).
    1. Zhao W., et al. , The nucleocapsid protein of rice stripe virus in cell nuclei of vector insect regulates viral replication. Protein Cell. 13, 360–378 (2022). - PMC - PubMed

Substances

LinkOut - more resources