A pentatomomorpha-specific salivary protein activates plant immunity and is critical for insect feeding

Biao Hu, Xiuli Feng, Manru Xu, Yue Huang, Chunyun Guo, Ruikun Yuan, Yiyuan Li, Zhong yan Wei, Jianping Chen, and Zongtao Sun

PNAS; January 31, 2025; 122 (5) e2425190122. https://doi.org/10.1073/pnas.2425190122

Significance

Insects and plants are engaged in a constant battle for survival, with insects frequently employing salivary proteins to subvert plant defenses during feeding. Our research sheds light on this dynamic by identifying a salivary protein, RpSP1, secreted by the stinkbug Riptortus pedestris. This protein exhibits a dual functionality, activating plant defenses while concurrently boosting the insect’s feeding efficiency. Crucially, we have demonstrated that modulating RpSP1 levels, either through overexpression in plants or suppression in pests, can significantly mitigate the soybean staygreen-like syndrome, a widespread and detrimental agricultural challenge. Furthermore, the finding of RpSP1 homologs in Pentatomomorpha implies that similar proteins may play key roles in pest–plant interactions, unveiling a promising target for developing broad-spectrum pest management strategies.

Abstract

The stinkbug Riptortus pedestris, notorious for inducing soybean staygreen-like syndrome, employs a range of salivary proteins to manipulate the host plant for its benefit. Here, we show that RpSP1, a salivary protein specific to Pentatomomorpha, triggers plant defense responses in multiple plant species. RpSP1 interacts with and stabilizes a HSP40 family protein GmSPIP1 and is dependent on GmSPIP1 to induce cell death. We show that a critical 22-amino acid peptide within RpSP1 acts as an intracellular insect-derived elicitor. Furthermore, RpSP1 enhances insect-feeding efficiency. The dual functionality of RpSP1 is highlighted by the significant reduction of soybean staygreen-like syndrome following its overexpression in soybean plants or knockdown in insects. Our findings elucidate the complex molecular interactions between plants and herbivores, positioning RpSP1 as a crucial target for developing advanced pest management strategies with broad implications for agricultural biology.

See https://www.pnas.org/doi/10.1073/pnas.2425190122

Figure 1:

RpSP1 is an elicitor that triggers responses in N. benthamiana defense and G. max. (A and B) Representative N. benthamiana leaves after R. pedestris feeding for 2 d and staining: trypan blue (1 d poststaining) and DAB (1 d poststaining), with arrows indicating cell death areas. (C) Systematic identification of RpSP1 among salivary secretory protein in R. pedestris. The numbers in circles represent proteins that met successively corresponding criteria. (D) Representative N. benthamiana leaves inoculated with Agrobacterium strains carrying RpSP1, with BAX and GFP control. Photos taken 3 d postinfiltration (dpi) and after trypan blue staining. (E–G) Representative soybean leaves inoculated with Agrobacterium strains carrying RpSP1, with BAX and GFP controls. (E) Photos taken 6 dpi, marked with arrows for cell death, with the lower right corner data indicating the number of plants exhibiting positive cell death phenotype. (F) Photos taken of the leaves stained with DAB at 84 h postinfiltration (hpi). (G) Quantification of ROS production areas in the leaves for each group in (F), with means ± SD for three biological replicates. Different lowercase letters above the bars indicate significant differences between groups (P < 0.05, two-sided t test). (H) qRT-PCR quantification of relative transcript levels of defense-related genes in soybean at 84 hpi with RpSP1, using GFP as a control. Means ± SD for three biological replicates is shown. (I–K) Cell death triggered by RpSP1 requires NbHSP90. (I) RpSP1-triggered cell death in TRV-silenced N. benthamiana. Representative photographs were taken at 4 dpi. Data were obtained from three independent experiments, each of which at least used eight TRV-silenced plants per line, with the lower right corner data indicating the number of plants exhibiting positive cell death phenotype. (J) qRT-PCR quantification of relative transcript levels of target gene in TRV-silenced N. benthamiana leaves, normalized to those in TRV:EV lines, using the NbActin gene as the reference gene. Means ± SD obtained from three biological replicates. * at the top of columns indicate significant differences (***P < 0.001) based on two-sided t test. (K) Western blot analysis of RpSP1 transiently expressed in TRV-silenced N. benthamiana leaves.