G protein-coupled receptors (GPCRs) are pivotal in mediating diverse physiological and pathological processes, rendering them promising targets for drug discovery. GPCRs account for about 40% of FDA-approved drugs, representing the most successful drug targets. However, only approximately 15% of the 800 human GPCRs are targeted by market drugs, leaving numerous opportunities for drug discovery among the remaining receptors. Cell expression systems play crucial roles in the GPCR drug discovery field, including novel target identification, structural and functional characterization, potential ligand screening, signal pathway elucidation, and drug safety evaluation. Here, we discuss the principles, applications, and limitations of widely used cell expression systems in GPCR-targeted drug discovery, GPCR function investigation, signal pathway characterization, and pharmacological property studies. We also propose three strategies for constructing genome-wide pan-GPCR cell libraries, which will provide a powerful platform for GPCR ligand screening, and facilitate the study of GPCR mechanisms and drug safety evaluation, ultimately accelerating the process of GPCR-targeted drug discovery.

Summary: Rice production is threatened by multiple pathogens. Breeding cultivars with broad‐spectrum disease resistance is necessary to maintain and improve crop production. Previously we found that overexpression of miR160a enhanced rice blast disease resistance. However, it is unclear whether miR160a also regulates resistance against other pathogens, and what the downstream signaling pathways are. Here, we demonstrate that miR160a positively regulates broad‐spectrum resistance against the causative agents of blast, leaf blight and sheath blight in rice. Mutations of miR160a‐targeted Auxin Response Factors result in different alteration of resistance conferred by miR160a. miR160a enhances disease resistance partially by suppressing ARF8, as mutation of ARF8 in MIM160 background partially restores the compromised resistance resulting from MIM160. ARF8 protein binds directly to the promoter and suppresses the expression of WRKY45, which acts as a positive regulator of rice immunity. Mutation of WRKY45 compromises the enhanced blast resistance and bacterial leaf blight resistance conferred by arf8 mutant. Overall, our results reveal that a microRNA coordinates rice broad‐spectrum disease resistance by suppressing multiple target genes that play different roles in disease resistance, and uncover a new regulatory pathway mediated by the miR160a‐ARF8 module. These findings provide new resources to potentially improve disease resistance for breeding in rice.

The flower-infecting fungus Ustilaginoidea virens causes rice false smut, which is a severe emerging disease threatening rice (Oryza sativa) production worldwide. False smut not only reduces yield, but more importantly produces toxins on grains, posing a great threat to food safety. U. virens invades spikelets via the gap between the two bracts (lemma and palea) enclosing the floret and specifically infects the stamen and pistil. Molecular mechanisms for the U. virens–rice interaction are largely unknown. Here, we demonstrate that rice flowers predominantly employ chitin-triggered immunity against U. virens in the lemma and palea, rather than in the stamen and pistil. We identify a crucial U. virens virulence factor, named UvGH18.1, which carries glycoside hydrolase activity. Mechanistically, UvGH18.1 functions by binding to and hydrolyzing immune elicitor chitin and interacting with the chitin receptor CHITIN ELICITOR BINDING PROTEIN (OsCEBiP) and co-receptor CHITIN ELICITOR RECEPTOR KINASE1 (OsCERK1) to impair their chitin-induced dimerization, suppressing host immunity exerted at the lemma and palea for gaining access to the stamen and pistil. Conversely, pretreatment on spikelets with chitin induces a defense response in the lemma and palea, promoting resistance against U. virens. Collectively, our data uncover a mechanism for a U. virens virulence factor and the critical location of the host–pathogen interaction in flowers and provide a potential strategy to control rice false smut disease.

RESISTANCE TO POWDERY MILDEW 8 (RPW8) defines a unique N-terminal coiled-coil domain of nucleotide-binding and leucine-rich repeat immune receptors required for immune signaling in plants. Arabidopsis RPW8.2 is specifically induced by the powdery mildew (PM) fungus ( Golovinomyces cichoracearum ) in the infected epidermal cells to activate immunity. The mechanism of RPW8.2-induction is not well understood. Here, we identify a G. cichoracearum factor delivered to the nucleus of the host cell, named Gc-RPW8.2 interacting protein 1 (GcR8IP1). Ectopic expression of GcR8IP1 in Arabidopsis or Nicotiana benthamiana suppressed host immune responses and enhanced susceptibility to PM. Host-induced gene silencing of GcR8IP1 compromised PM infectivity in susceptible Arabidopsis plants. Co-expression of GcR8IP1 with RPW8.2 increased nuclear localization of RPW8.2, which in turn, promoted transcriptional amplification of RPW8.2 . Thus, RPW8.2-dependent defense strengthening is due to altered partitioning of RPW8.2 by an effector of a PM fungus, which exemplifies an atypical form of effector-triggered immunity.

Natural products represent an important reservoir for GPCR ligand discovery. In this study, we established an integrated workflow combining virtual screening, biophysical validation, functional signaling assays, and transcriptomic profiling to identify reticuline, a dopamine-derived intermediate from the genus of Stephania, as a potential agonist of dopamine D5 receptor (D5R). Molecular docking revealed that most dopamine-derived compounds along the BIA synthetic pathway exhibit predicted binding affinities for the D5R that are lower than that of dopamine. As expected, the reticuline–D5R complex has a favorable predicted binding affinity of −7.9 kcal/mol. As for binding validation, direct interaction between reticuline and D5R was experimentally confirmed using cell membrane chromatography (CMC) and bio-layer interferometry (BLI), yielding a dissociation constant of 1.07 μM. cAMP assay demonstrated that reticuline activates D5R-mediated Gs-cAMP increasement in a concentration-responsive manner, which exhibits agonist-like activity with an EC₅₀ value of 0.07 μM. The transcriptomic profiling further revealed that reticuline treatment induces transcriptional reprogramming in D5R-overexpressing cells, with enrichment of pathways related to ribosome biogenesis, mitochondrial oxidative phosphorylation, and neurodegenerative diseases. In summary, this study demonstrates that reticuline acts as a potential D5R agonist and highlights a systematic natural product-GPCR discovery strategy integrating computational prediction, experimental validation, and transcriptome-level mechanistic exploration.