The brown planthopper, Nilaparvata lugens, is a pest that threatens rice (Oryza sativa) production worldwide. While feeding on rice plants, planthoppers secrete saliva, which plays crucial roles in nutrient ingestion and modulating plant defense responses, although the specific functions of salivary proteins remain largely unknown. We identified an N. lugens-secreted mucin-like protein (NlMLP) by transcriptome and proteome analyses and characterized its function, both in brown planthopper and in plants. NlMLP is highly expressed in salivary glands and is secreted into rice during feeding. Inhibition of NlMLP expression in planthoppers disturbs the formation of salivary sheaths, thereby reducing their performance. In plants, NlMLP induces cell death, the expression of defense-related genes, and callose deposition. These defense responses are related to Ca 2+ mobilization and the MEK2 MAP kinase and jasmonic acid signaling pathways. The active region of NlMLP that elicits plant responses is located in its carboxyl terminus. Our work provides a detailed characterization of a salivary protein from a piercing-sucking insect other than aphids. Our finding that the protein functions in plant immune responses offers new insights into the mechanism underlying interactions between plants and herbivorous insects.

BROWN PLANTHOPPER RESISTANCE14 (BPH14), the first planthopper resistance gene isolated via map-based cloning in rice (Oryza sativa), encodes a coiled-coil, nucleotide binding site, leucine-rich repeat (CC-NB-LRR) protein. Several planthopper and aphid resistance genes encoding proteins with similar structures have recently been identified. Here, we analyzed the functions of the domains of BPH14 to identify molecular mechanisms underpinning BPH14-mediated planthopper resistance. The CC or NB domains alone or in combination (CC-NB [CN]) conferred a similar level of brown planthopper resistance to that of full-length (FL) BPH14. Both domains activated the salicylic acid signaling pathway and defense gene expression. In rice protoplasts and Nicotiana benthamiana leaves, these domains increased reactive oxygen species levels without triggering cell death. Additionally, the resistance domains and FL BPH14 protein formed homocomplexes that interacted with transcription factors WRKY46 and WRKY72. In rice protoplasts, the expression of FL BPH14 or its CC, NB, and CN domains increased the accumulation of WRKY46 and WRKY72 as well as WRKY46-and WRKY72-dependent transactivation activity. WRKY46 and WRKY72 bind to the promoters of the receptor-like cytoplasmic kinase gene RLCK281 and the callose synthase gene LOC_Os01g67364.1, whose transactivation activity is dependent on WRKY46 or WRKY72. These findings shed light on this important insect resistance mechanism.

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.

The brown planthopper (BPH) is the most devastating insect pest of rice. The rice gene BPH15 confers resistance to BPH. MicroRNAs (miRNAs) regulate a spectrum of development and defense response processes in plants. In this study, we analyzed six miRNA profiles of a BPH15 introgression line (P15) and a susceptible recipient line (PC) at three time points (0 h, 6 h and 48 h) after BPH attack, and identified 464 known miRNAs and 183 potential novel miRNAs. Before the BPH feeding, we identified 23 miRNAs differentially expressed in P15 and PC. We speculated that the resistant plant is in a priming state by the regulation of miRNAs. After the BPH feeding, 104 miRNAs were found to be expressed differentially in P15 (68 in P15-6/P15-0, 36 in P15-48/P15-0), and 80 miRNAs were found expressed differentially in PC (32 in PC-6/PC-0, 48 in PC-48/PC-0), which illustrated that miRNA expression is activated upon attack. These miRNAs regulate different pathways that contribute to the basal defense and specific resistance of rice to the BPH. Our study provides additional data for scientists to further explore the mechanism of plant defense against insect attack and to find a way for efficient insect control.

Plants deploy receptor-like kinases and nucleotide-binding leucine-rich repeat receptors to confer host plant resistance (HPR) to herbivores1. These gene-for-gene interactions between insects and their hosts have been proposed for more than 50 years2. However, the molecular and cellular mechanisms that underlie HPR have been elusive, as the identity and sensing mechanisms of insect avirulence effectors have remained unknown. Here we identify an insect salivary protein perceived by a plant immune receptor. The BPH14-interacting salivary protein (BISP) from the brown planthopper (Nilaparvata lugens Stål) is secreted into rice (Oryza sativa) during feeding. In susceptible plants, BISP targets O. satvia RLCK185 (OsRLCK185; hereafter Os is used to denote O. satvia-related proteins or genes) to suppress basal defences. In resistant plants, the nucleotide-binding leucine-rich repeat receptor BPH14 directly binds BISP to activate HPR. Constitutive activation of Bph14-mediated immunity is detrimental to plant growth and productivity. The fine-tuning of Bph14-mediated HPR is achieved through direct binding of BISP and BPH14 to the selective autophagy cargo receptor OsNBR1, which delivers BISP to OsATG8 for degradation. Autophagy therefore controls BISP levels. In Bph14 plants, autophagy restores cellular homeostasis by downregulating HPR when feeding by brown planthoppers ceases. We identify an insect saliva protein sensed by a plant immune receptor and discover a three-way interaction system that offers opportunities for developing high-yield, insect-resistant crops.