A tripartite rheostat controls self-regulated host plant resistance to insects.

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.

The Coiled-Coil and Nucleotide Binding Domains of BROWN PLANTHOPPER RESISTANCE14 Function in Signaling and Resistance against Planthopper in Rice.

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.

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

The brown planthopper (BPH), Nilaparvata lugens (Stål), is a phloem sap-feeding insect. During feeding on rice plants, 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 1,140 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 upregulated 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 the 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, and Nl32 are conserved among insects. The Nl40 family has numerous isoforms produced by alternative splicing, exemplifying rapid evolution and expansion of effector proteins in the BPH. Our results suggest a potential large effector repertoire in BPH and a higher level of effector conservation exist in BPH compared with that in plant pathogens.

A Mucin-Like Protein of Planthopper Is Required for Feeding and Induces Immunity Response in Plants.

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 Ca2+ 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.

Allelic diversity in an NLR gene BPH9 enables rice to combat planthopper variation.

Brown planthopper (BPH), Nilaparvata lugens Stål, is one of the most devastating insect pests of rice (Oryza sativa L.). Currently, 30 BPH-resistance genes have been genetically defined, most of which are clustered on specific chromosome regions. Here, we describe molecular cloning and characterization of a BPH-resistance gene, BPH9, mapped on the long arm of rice chromosome 12 (12L). BPH9 encodes a rare type of nucleotide-binding and leucine-rich repeat (NLR)-containing protein that localizes to the endomembrane system and causes a cell death phenotype. BPH9 activates salicylic acid- and jasmonic acid-signaling pathways in rice plants and confers both antixenosis and antibiosis to BPH. We further demonstrated that the eight BPH-resistance genes that are clustered on chromosome 12L, including the widely used BPH1, are allelic with each other. To honor the priority in the literature, we thus designated this locus as BPH1/9 These eight genes can be classified into four allelotypes, BPH1/9-1, -2, -7, and -9 These allelotypes confer varying levels of resistance to different biotypes of BPH. The coding region of BPH1/9 shows a high level of diversity in rice germplasm. Homologous fragments of the nucleotide-binding (NB) and leucine-rich repeat (LRR) domains exist, which might have served as a repository for generating allele diversity. Our findings reveal a rice plant strategy for modifying the genetic information to gain the upper hand in the struggle against insect herbivores. Further exploration of natural allelic variation and artificial shuffling within this gene may allow breeding to be tailored to control emerging biotypes of BPH.

BAC and RNA sequencing reveal the brown planthopper resistance gene BPH15 in a recombination cold spot that mediates a unique defense mechanism.

BACKGROUND: Brown planthopper (BPH, Nilaparvata lugens Stål), is the most destructive phloem-feeding insect pest of rice (Oryza sativa). The BPH-resistance gene BPH15 has been proved to be effective in controlling the pest and widely applied in rice breeding programs. Nevertheless, molecular mechanism of the resistance remain unclear. In this study, we narrowed down the position of BPH15 on chromosome 4 and investigated the transcriptome of BPH15 rice after BPH attacked. RESULTS: We analyzed 13,000 BC2F2 plants of cross between susceptible rice TN1 and the recombinant inbred line RI93 that carrying the BPH15 gene from original resistant donor B5. BPH15 was mapped to a 0.0269 cM region on chromosome 4, which is 210-kb in the reference genome of Nipponbare. Sequencing bacterial artificial chromosome (BAC) clones that span the BPH15 region revealed that the physical size of BPH15 region in resistant rice B5 is 580-kb, much bigger than the corresponding region in the reference genome of Nipponbare. There were 87 predicted genes in the BPH15 region in resistant rice. The expression profiles of predicted genes were analyzed. Four jacalin-related lectin proteins genes and one LRR protein gene were found constitutively expressed in resistant parent and considered the candidate genes of BPH15. The transcriptomes of resistant BPH15 introgression line and the susceptible recipient line were analyzed using high-throughput RNA sequencing. In total, 2,914 differentially expressed genes (DEGs) were identified. BPH-responsive transcript profiles were distinct between resistant and susceptible plants and between the early stage (6 h after infestation, HAI) and late stage (48 HAI). The key defense mechanism was related to jasmonate signaling, ethylene signaling, receptor kinase, MAPK cascades, Ca(2+) signaling, PR genes, transcription factors, and protein posttranslational modifications. CONCLUSIONS: Our work combined BAC and RNA sequencing to identify candidate genes of BPH15 and revealed the resistance mechanism that it mediated. These results increase our understanding of plant-insect interactions and can be used to protect against this destructive agricultural pest.

Knockout of OsWRKY71 impairs Bph15-mediated resistance against brown planthopper in rice.

The Bph15 gene, known for its ability to confer resistance to the brown planthopper (BPH; Nilaparvata lugens Stål), has been extensively employed in rice breeding. However, the molecular mechanism by which Bph15 provides resistance against BPH in rice remains poorly understood. In this study, we reported that the transcription factor OsWRKY71 was highly responsive to BPH infestation and exhibited early-induced expression in Bph15-NIL (near-isogenic line) plants, and OsWRKY71 was localized in the nucleus of rice protoplasts. The knockout of OsWRKY71 in the Bph15-NIL background by CRISPR-Cas9 technology resulted in an impaired Bph15-mediated resistance against BPH. Transcriptome analysis revealed that the transcript profiles responsive to BPH differed between the wrky71 mutant and Bph15-NIL, and the knockout of OsWRKY71 altered the expression of defense genes. Subsequent quantitative RT-PCR analysis identified three genes, namely sesquiterpene synthase OsSTPS2, EXO70 family gene OsEXO70J1, and disease resistance gene RGA2, which might participate in BPH resistance conferred by OsWRKY71 in Bph15-NIL plants. Our investigation demonstrated the pivotal involvement of OsWRKY71 in Bph15-mediated resistance and provided new insights into the rice defense mechanisms against BPH.

SlbHLH152, a bHLH transcription factor positively regulates iron homeostasis in tomato.

The maintain of iron (Fe) homeostasis is essential for plant survival. In tomato, few transcription factors have been identified as regulators of Fe homeostasis, among which SlbHLH068 induced by iron deficiency, plays an important role. However, the upstream regulator(s) responsible for activating the expression of SlbHLH068 remain(s) unknown. In this study, the bHLH (basic helix-loop-helix) transcription factor SlbHLH152 was identified as an upstream regulator of SlbHLH068 using yeast one-hybrid screening. Deletion of SlbHLH152 led to a significant decline in Fe concentration, which was accompanied by reduced expression of Fe-deficiency-responsive genes. In contrast, SlbHLH152 overexpression plants displayed tolerance to iron deficiency, increased Fe accumulation, and elevated expression of Fe-deficiency-responsive genes. Further analysis indicated that SlbHLH152 directly activates the transcription of SlbHLH068. Taken together, our results suggest that SlbHLH152 may be involved in the regulation of iron homeostasis by directly activating the transcription of SlbHLH068 in tomato.

Bph6 encodes an exocyst-localized protein and confers broad resistance to planthoppers in rice.

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.

Functional Study of Cytochrome P450 Enzymes from the Brown Planthopper (Nilaparvata lugens Stål) to Analyze Its Adaptation to BPH-Resistant Rice.

Plant-insect interactions constitute a complex of system, whereby plants synthesize toxic compounds as the main defense strategy to combat herbivore assault, and insects deploy detoxification systems to cope with toxic plant compounds. Cytochrom P450s are among the main detoxification enzymes employed by insects to combat the chemical defenses of host plants. In this study, we used Nilaparvata lugens (BPH) to constitute an ideal system for studying plant-insect interactions. By feeding BPHs with artificial diets containing ethanol extracts, we show that biotype Y BPHs have a greater ability to metabolize exogenous substrates than biotype 1 BPHs. NlCPR knockdown inhibited the ability of BPHs to feed on YHY15. qRT-PCR was used to screen genes in the P450 family, and upregulation of CYP4C61, CYP6AX1, and CYP6AY1 induced by YHY15 was investigated. When the three P450 genes were knocked down, only CYP4C61 dsRNA treatment was inhibited the ability of BPHs to feed on YHY15. These results indicate that BPH P450 enzymes are a key factor in the physiological functions of BPH when feeding on BPH-resistant rice.

Overexpression of OsRRK1 Changes Leaf Morphology and Defense to Insect in Rice.

It has been reported that the receptor-like cytoplasmic kinases (RLCKs) regulate many biological processes in plants, but only a few members have been functionally characterized. Here, we isolated a rice gene encoding AtRRK1 homology protein kinase, OsRRK1, which belongs to the RLCK VI subfamily. OsRRK1 transcript accumulated in many tissues at low to moderate levels and at high levels in leaves. Overexpression of OsRRK1 (OE-OsRRK1) caused adaxial rolling and erect morphology of rice leaves. In the rolled leaves of OE-OsRRK1 plants, both the number and the size of the bulliform cells are decreased compared to the wild-type (WT) plants. Moreover, the height, tiller number, and seed setting rate were reduced in OE-OsRRK1 plants. In addition, the brown planthopper (BPH), a devastating pest of rice, preferred to settle on WT plants than on the OE-OsRRK1 plants in a two-host choice test, indicating that OE-OsRRK1 conferred an antixenosis resistance to BPH. The analysis of transcriptome sequencing demonstrated that several receptor kinases and transcription factors were differentially expressed in OE-OsRRK1 plants and WT plants. These results indicated that OsRRK1 may play multiple roles in the development and defense of rice, which may facilitate the breeding of novel rice varieties.