Secretome Analysis and In Planta Expression of Salivary Proteins Identify Candidate Effectors from the Brown Planthopper Nilaparvata lugens

Abstract

The brown planthopper (BPH), Nilaparvata lugens (Stål), is a phloem sap-feeding insect. During the feeding on rice plant, BPH secretes salivary proteins with potential effector functions, which may play a critical role in the plant-insect interactions. However, a limited number of BPH effector proteins have been identified to date. Here, we sequenced the salivary gland transcriptomes of five BPH populations and subsequently established a N. lugens secretome consisting of 1140 protein-encoding genes. Secretome analysis revealed the presence of both conserved and rapidly evolving salivary proteins. A screen for potential effectors that elicit responses in the plant was performed via the transient expression analysis of 64 BPH salivary proteins in Nicotiana benthamiana leaves and rice protoplasts. The salivary proteins Nl12, Nl16, Nl28, and Nl43 induced cell death, whereas Nl40 induced chlorosis and Nl32 induced a dwarf phenotype in N. benthamiana, indicating effector properties of these proteins. Ectopic expression of the six salivary proteins in N. benthamiana up-regulated expression of defense-related genes and callose deposition. Tissue expression analysis showed a higher expression level of the six candidate effectors in salivary glands than in other tissues. Subcellular localization and analysis of domain required for cell death showed a diverse structure of the six effectors. Nl28, Nl40 and Nl43 are N. lugens-specific, in contrast, Nl12, Nl16, Nl32 are conserved among insects. Nl40 family has numerous isoforms produced by alternative splicing, exemplifying rapid evolution and expansion of effector proteins in the brown planthopper. Our results suggest a potential large effector repertoire in BPH and a higher level of effector conservation exist in BPH compared to that in plant pathogens.

How does saliva function in planthopper–host interactions?

“…The C‐terminal region of this protein induces defense responses in plant cells, including cell death, the expression of pathogen‐responsive genes, and callose deposition (Shangguan et al, ). Salivary sheaths directly contact with plant cell after secretion and are easier sharped by natural selection (Rao et al, ). Because of their “forward” role in herbivore–plant interactions, salivary sheaths may undergo a higher evolution rate than other salivary components.…”

“…Suppression of ANX‐5 has negative effects on planthopper feeding (Huang et al, ). Interestingly, ANXs, which constitute the second largest protein family in N. lugens (Rao et al, ), do not possess typical annexin structure. These secretory ANX structures have so far been found only in planthopper species, indicating that ANXs were important in planthopper–plant coevolution.…”

“…Among 64 tested salivary proteins, 6 were identified as candidate effector proteins (Table ). The salivary proteins Nl12, Nl16, Nl28, and Nl43 induced cell death in N. benthamiana , whereas Nl40 induced chlorosis and Nl32 induced a dwarf phenotype (Rao et al, ). However, the exact function of these candidate effectors and recognizing mechanism in plants remain to be elucidated.…”

Gene silencing of Diaphorina citri candidate effectors promotes changes in feeding behaviors

“…Many insect effectors have been described over the years as from saliva or salivary gland proteomic and transcriptomic data [27][28][29][30][31] . To unveil the actions of effector proteins on insect-hosts interactions, RNA interference (RNAi) has been applied.…”

Salivary DNase II from Laodelphax striatellus acts as an effector that suppresses plant defence

“…S4c). This suggested that the salivary DNase II may function differently from other salivary effectors such as Nl12, Nl40, Nl16, Nl28 and Nl43 (Rao et al, 2019).…”