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The brown planthopper (BPH) and white-backed planthopper (WBPH) are the most destructive insect pests of rice, and they pose serious threats to rice production throughout Asia. Thus, there are urgent needs to identify resistance-conferring genes and to breed planthopper-resistant rice varieties. Here we report the map-based cloning and functional analysis of Bph6, a gene that confers resistance to planthoppers in rice. Bph6 encodes a previously uncharacterized protein that localizes to exocysts and interacts with the exocyst subunit OsEXO70E1. Bph6 expression increases exocytosis and participates in cell wall maintenance and reinforcement. A coordinated cytokinin, salicylic acid and jasmonic acid signaling pathway is activated in Bph6-carrying plants, which display broad resistance to all tested BPH biotypes and to WBPH without sacrificing yield, as these plants were found to maintain a high level of performance in a field that was heavily infested with BPH. Our results suggest that a superior resistance gene that evolved long ago in a region where planthoppers are found year round could be very valuable for controlling agricultural insect pests.

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.

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.

Analysis of complex biological functions usually requires tissue-specific genetic manipulations in multicellular organisms. The C. elegans germline plays regulatory roles not only in reproduction, but also in metabolism, stress response and ageing. Previous studies have used mutants of rrf-1, which encodes an RNA-directed RNA polymerase, as a germline-specific RNAi tool. However, the rrf-1 mutants showed RNAi activities in somatic tissues. Here we constructed a germline-specific RNAi strain by combining an indel mutation of rde-1, which encodes an Argonaute protein that functions cell autonomously to ensure RNAi efficiency, and a single copy rde-1 transgene driven by the sun-1 germline-specific promoter. The germline RNAi efficiency and specificity are confirmed by RNAi phenocopy of known mutations, knockdown of GFP reporter expression, as well as quantitative RT-PCR measurement of tissue-specific mRNAs upon RNAi knockdown. The germline-specific RNAi strain shows no obvious deficiencies in reproduction, lipid accumulation, thermo-tolerance and life span compared to wild-type animals. By screening an RNAi sub-library of phosphatase genes, we identified novel regulators of thermo-tolerance. Together, we have created a useful tool that can facilitate the genetic analysis of germline-specific functions in C. elegans.

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Highlights d Longevity of daf-2 rsks-1 is mediated by translational repression of cyc-2.1 d Germline inhibition of cyc-2.1 activates intestinal UPR mt and AMPK to extend lifespan d Increased GLD-1 represses germline cyc-2.1 translation in the daf-2 rsks-1 mutant d Translational regulation of cyc-2.1 and UPR mt contribute to longevity of daf-2 rsks-1

Fly Dicer-2 exhibits a non–RNA interference function in the posttranscriptional modulation of Toll protein expression and Toll signaling.