Genome editing of a rice CDP-DAG synthase confers multipathogen resistance.

The discovery and application of genome editing introduced a new era of plant breeding by giving researchers efficient tools for the precise engineering of crop genomes1. 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. We then demonstrated that a 29-base-pair deletion in a gene we named RESISTANCE TO BLAST1 (RBL1) caused broad-spectrum disease resistance and showed that this mutation caused an approximately 20-fold reduction in yield. RBL1 encodes a cytidine diphosphate diacylglycerol synthase that is required for phospholipid biosynthesis2. Mutation of RBL1 results in reduced levels of phosphatidylinositol and its derivative phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In rice, PtdIns(4,5)P2 is enriched in cellular structures that are specifically associated with effector secretion and fungal infection, suggesting that it has a role as a disease-susceptibility factor3. By using targeted genome editing, we obtained an allele of RBL1, named RBL1Δ12, which confers broad-spectrum disease resistance but does not decrease yield in a model rice variety, as assessed in small-scale field trials. Our study has demonstrated the benefits of editing an LMM gene, a strategy relevant to diverse LMM genes and crops.

Inhibitor of cardiolipin biosynthesis-related enzyme MoGep4 confers broad-spectrum anti-fungal activity.

Plant pathogens cause devastating diseases, leading to serious losses to agriculture. Mechanistic understanding of pathogenesis of plant pathogens lays the foundation for the development of fungicides for disease control. Mitophagy, a specific form of autophagy, is important for fungal virulence. The role of cardiolipin, mitochondrial signature phospholipid, in mitophagy and pathogenesis is largely unknown in plant pathogenic fungi. The functions of enzymes involved in cardiolipin biosynthesis and relevant inhibitors were assessed using a set of assays, including genetic deletion, plant infection, lipidomics, chemical-protein interaction, chemical inhibition, and field trials. Our results showed that the cardiolipin biosynthesis-related gene MoGEP4 of the rice blast fungus Magnaporthe oryzae regulates growth, conidiation, cardiolipin biosynthesis, and virulence. Mechanistically, MoGep4 regulated mitophagy and Mps1-MAPK phosphorylation, which are required for virulence. Chemical alexidine dihydrochloride (AXD) inhibited the enzyme activity of MoGep4, cardiolipin biosynthesis and mitophagy. Importantly, AXD efficiently inhibited the growth of 10 plant pathogens and controlled rice blast and Fusarium head blight in the field. Our study demonstrated that MoGep4 regulates mitophagy, Mps1 phosphorylation and pathogenesis in M. oryzae. In addition, we found that the MoGep4 inhibitor, AXD, displays broad-spectrum antifungal activity and is a promising candidate for fungicide development.

Effector translocation and rational design of disease resistance.

The effector repertoire of a pathogen is dynamically evolving. However, the effector translocation mechanism, partly elucidated recently, may be conserved. By targeting the effector translocation machinery, rather than the individual evolving effector, rational design of durable and broad-spectrum disease resistance can be achieved, facilitated by genome-editing and artificial intelligence-enabling technologies.

BsR1, a broad-spectrum antibacterial peptide with potential for plant protection.

IMPORTANCE: This study addresses the critical need for new antibacterial drugs in the face of bacterial multidrug resistance resulting from antibiotic overuse. It highlights the significance of antimicrobial peptides as essential components of innate immunity in animals and plants, which have been proven effective against multidrug-resistant bacteria and are difficult to develop resistance against. This study successfully synthesizes a broad-spectrum antibacterial peptide, BsR1, with strong inhibitory activities against various Gram-positive and Gram-negative bacteria. BsR1 demonstrates favorable stability and a mode of action that damages bacterial cell membranes, leading to cell death. It also exhibits biological safety and shows potential in enhancing disease resistance in rice. This research offers a novel approach and potential medication for antibacterial drug development, presenting a valuable tool in combating pathogenic microorganisms, particularly in plants.

The MoPah1 phosphatidate phosphatase is involved in lipid metabolism, development, and pathogenesis in Magnaporthe oryzae.

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 genome of the rice variety LTH provides insight into its universal susceptibility mechanism to worldwide rice blast fungal strains.

The widely used rice variety Lijiangxintuanheigu (LTH) shows a universal susceptibility to thousands of Magnaporthe oryzae isolates, the causal agent of devastating rice blast, making LTH an ideal line in resistance (R) gene cloning. However, the underlying genetic mechanism of the universal susceptibility has not been fully revealed because of the lack of a high-quality genome. Here, we took a genomic approach together with experimental assays to investigate LTH's universal susceptibility to rice blast. Using Nanopore long reads, we assembled a chromosome-level genome. Millions of genomic variants were detected by comparing LTH with 10 other rice varieties, of which large-effect variants could affect plant immunity. Gene family analyses show that the number of R genes and leucine-rich repeat receptor-like protein kinase (LRR-RLK)-encoding genes decrease significantly in LTH. Rice blast resistance genes called Pi genes are either absent or disrupted by genomic variations. Additionally, residual R genes of LTH are likely under weak pathogen selection pressure, and other plant defense-related genes are weakly induced by rice blast. In contrast, the pattern-triggered immunity (PTI) of LTH is normal, as demonstrated by experimental assays. Therefore, we conclude that weak effector-trigger immunity (ETI)-mediated primarily by Pi genes but not PTI results in the universal susceptibility of LTH to rice blast. The attenuated ETI of LTH may be also associated with reduced numbers of R genes and LRR-RLKs, and minimally functional residual defense-related genes. Finally, we demonstrate the use of the LTH genome by rapid cloning of the Pi gene Piak from a resistant variety.