To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [(35)S]GTPγS binding/Gα(q/11) immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (∼50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.

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As with the majority of the hemibiotrophic fungal pathogens, the rice blast fungus Magnaporthe oryzae uses highly specialized infection structures called appressoria for plant penetration. Appressoria differentiated from germ tubes rely on enormous turgor pressure to directly penetrate the plant cell, in which process lipid metabolism plays a critical role. In this study, we characterized the MoPAH1 gene in M. oryzae, encoding a putative highly conserved phosphatidate phosphatase. The expression of MoPAH1 was up-regulated during plant infection. The MoPah1 protein is expressed at all developmental and infection stages, and is localized to the cytoplasm. Disruption of MoPAH1 causes pleiotropic defects in vegetative growth, sporulation, and heat tolerance. The lipid profile is significantly altered in the Mopah1 mutant. Lipidomics assays showed that the level of phosphatidic acid (PA) was increased in the mutant, which had reduced levels of diacylglycerol and triacylglycerol. Using a PA biosensor, we showed that the increased level of PA in the Mopah1 mutant was primarily accumulated in the vacuole. The Mopah1 mutant was blocked in both conidiation and the formation of appressorium-like structures at hyphal tips. It was nonpathogenic and failed to cause any blast lesions on rice and barley seedlings. RNA sequencing analysis revealed that MoPah1 regulates the expression of transcription factors critical for various developmental and infection-related processes. The Mopah1 mutant was reduced in the expression and phosphorylation of Pmk1 MAP kinase and delayed in autophagy. Our study demonstrates that MoPah1 is necessary for lipid metabolism, fungal development, and pathogenicity in M. oryzae.

The discovery and application of genome editing introduce a new era of plant breeding, giving researchers efficient tools for the precise engineering of crop genomes 1 . Here, we demonstrate the power of genome editing for engineering broad-spectrum disease resistance in rice (Oryza sativa). We first isolated a lesion mimic mutant (LMM) from a mutagenized rice population, demonstrated that a 29-bp deletion in a gene we named RESISTANCE TO BLAST1 (RBL1) caused this phenotype and showed that this mutation caused a ca. 20-fold reduction in yield. RBL1encodes a cytidine diphosphate diacylglycerol (CDP-DAG) synthase required for phospholipid biosynthesis 2 . Mutation of RBL1 results in reduced levels of phosphatidylinositol (PI) and its derivative PI(4,5)P2. Rice PI(4,5)P2 is enriched in cellular structures specifically associated with effector secretion and fungal infection, suggesting a role as a disease susceptibility factor 3 . Using targeted mutagenesis, we obtained an allele of RBL1, named RBL12, which confers broad-spectrum resistance but does not decrease yield in a model rice variety as assessed in small-scale field trials. Our study has demonstrated the usefulness of editing of an LMM gene, a strategy relevant to diverse LMM genes and crops. Main textGenome editing has been widely used in functional studies of genes but its potential for crop improvement has not yet been broadly utilized 4 . Plant diseases cause severe losses in agriculture, threatening global food security 5 . Rice blast alone, caused by the fungal pathogen Magnaporthe oryzae, results in annual yield losses that are sufficient to feed more than 60 million people worldwide 6 . Given this cost, cultivating crops with resistance to diseases, particularly broad-spectrum resistance, is highly desirable 7 . Despite the importance of this goal, only a limited number of broad-spectrum resistance genes have been cloned and used in the field, such as rice Xa21 (ref. 8 ), bsr-d1 (ref. 9 ), Pigm 10 , IPA1 (ref. 11 ), ROD1 (ref. 12 ), UMP1 (ref. 13 ), wheat Lr34 (ref. 14 ) and PsIPK1 (ref. 15 ), and barley mlo 16,17 .LMMs form hypersensitive response-like lesions (a form of programmed cell death) in the absence of pathogens 18 . LMMs often confer durable and broad-spectrum resistance, representing a potential source for breeding resistance to diseases. However, LMMs are usually associated with reduced yield, and therefore the use of the genes conferring LMM phenotypes (hereafter referred to as LMM genes) has not been fully exploited in plant breeding due to the lack of useful alleles.Phospholipids are essential components of biological membranes and are involved in various biological processes, including development and response to biotic and abiotic stress 19 . In phospholipid biosynthesis (Extended Data Fig. 1), phosphatidic acid (PA) and cytidine triphosphate (CTP) are converted to CDP-DAG by CDP-DAG synthases (CDSs). CDP-DAG and Myo-inositol are used to produce PI by phosphatidylinositol synthases (PISs) 20 . PI is added to by a varied number of p...