Augmenting rice ANNEXIN expression to counter planthopper NlAnnexin-like5 as an antivirulence strategy against a major crop pest

Xiao-Ya Zhang, Shaoqin Li, Comzit Opachaloemphan, Chuan-Xi Zhang, Sheng Yang He, and Yanjuan Jiang

PNAS; October 9, 2025; 122 (41) e2505698122; https://doi.org/10.1073/pnas.2505698122

Significance

Insect pests represent a major threat to agriculture worldwide. Elucidating the molecular mechanisms by which insect pests attack plants could lead to new pest control strategies, thereby enhancing global food security. The brown planthopper (BPH) is the most destructive insect pest in rice production. In this study, we found that BPH secretes a salivary protein, Nilaparvata lugens Annexin-like5 (NlANX5), to target rice host annexins that are associated with calcium fluxes and activation of multiple rice defense pathways. This knowledge led to employing enhanced expression of rice annexin–encoding genes to successfully defeat the virulence function of NlANX5. Results have significant implications in the development of antivirulence breeding strategies against BPH.

Abstract

The brown planthopper (BPH) is the most devastating insect pest in rice, posing a serious threat to global rice production. One attractive control strategy would be based on the understanding of the virulence mechanisms of BPH at the molecular level and then designing targeted methods to neutralize such mechanisms. Salivary proteins of BPH are important players in mediating rice–BPH interactions. Here, we describe a pivotal role of a watery saliva protein, Nilaparvata lugens Annexin-like5 (NlANX5), in the rice–BPH interaction. RNA interference (RNAi) of NlANX5 greatly compromised BPH feeding performance and survival rate on rice plants. NlANX5-RNAi BPH triggered a rapid calcium ion influx in rice cells. The feeding and survival defects of NlANX5-RNAi BPH can be restored in NlANX5-expressing transgenic rice plants. NlANX5 targets rice annexin (OsANN) proteins, including OsANN2 and OsANN8. Further analysis with NlANX5 and OsANN2 as well as OsANN8 showed that NlANX5 displaces OsANN2 and OsANN8 from the rice cell membrane. The osann2 osann8 mutant rice plants are hypersusceptible to BPH infestation. In contrast, enhanced expression of OsANN2 and OsANN8 genes resulted in robust rice resistance against BPH. This study highlights a successful example of identifying and augmenting the expression of the host targets of a major BPH virulence effector as a promising antivirulence strategy against an important crop pest.

See https://www.pnas.org/doi/10.1073/pnas.2505698122

Figure 1

Characterization of NlANX5. (A) RNA in situ hybridization of NlANN5 in salivary glands isolated from 5th instar BPHs (Upper row). The sense probe was used as a negative control (Lower row). PG, principal gland. AG, accessory gland. APG, a-follicle of the PG. (B) The amino acid sequence of NlANX5. The yellow highlighted amino acid residues indicate the peptides detected in BPH-fed rice sheath tissue by LC–MS analyses. The asterisk indicates the stop codon. The underlined amino acid residues indicate the peptides detected in the phloem exudate of BPH-infested rice by LC–MS analysis. (C) NlANX5 is localized in the plasma membrane (PM), endoreticulum (ER), and nucleus of rice cells. PM protein-mCherry and NlANX5-YFP fusion, ER protein-CFP and NlANX5-YFP, or Nucleus-RFP and NlANX5-YFP proteins were coexpressed in rice protoplasts. The PM marker was the full-length Arabidopsis aquaporin 2A (AtPIP2A) (28), the ER marker was the signal peptide of Arabidopsis wall-associated kinase 2 (AtWAK2) (28), and the nucleus marker was the full-length Arabidopsis ELONGATED HYPOCOTYL 5 (AtHY5). See additional images in SI Appendix, Fig. S2. (D) NlANX5-YFP fusion protein levels in rice protoplasts are detected with anti-GFP (Abmart). Protein samples were extracted from rice protoplasts coexpressing Golgi-CFP and NlANX5-YFP, ER-CFP and NlANX5-YFP, or PM-mCherry and NlANX5-YFP, respectively. Proteins from mock rice protoplasts were included as negative control. Ponceau S staining of the gel shows overall protein loading. Experiments were repeated three times with similar trends.