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

PNAS March 27, 2024; 121 (14) e2315982121

Significance

The innate immune system, especially the Toll pathway, plays a vital role in defending against pathogenic microorganisms, including viruses. Nevertheless, 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 remains unclear. In our study, we unveiled the molecular mechanism through which Toll-Dorsal-ZN708 (zinc finger protein 708) mediates the maintenance of homeostasis of a plant arbovirus in the insect vector. Specifically, ZN708 is a newly documented zinc finger protein targeted by Dorsal that mediates the downstream antiviral response. In this study, we also present evidence of the antiviral role of the Toll immune system in an insect vector active against plant arboviruses.

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.

See https://www.pnas.org/doi/10.1073/pnas.2315982121

Figure 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.