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.

Doxorubicin inhibits phosphatidylserine decarboxylase and confers broad-spectrum antifungal activity.

As phospholipids of cell membranes, phosphatidylethanolamine (PE) and phosphatidylserine (PS) play crucial roles in glycerophospholipid metabolism. Broadly, some phospholipid biosynthesis enzymes serve as potential fungicide targets. Therefore, revealing the functions and mechanism of PE biosynthesis in plant pathogens would provide potential targets for crop disease control. We performed analyses including phenotypic characterizations, lipidomics, enzyme activity, site-directed mutagenesis, and chemical inhibition assays to study the function of PS decarboxylase-encoding gene MoPSD2 in rice blast fungus Magnaporthe oryzae. The Mopsd2 mutant was defective in development, lipid metabolism, and plant infection. The PS level increased while PE decreased in Mopsd2, consistent with the enzyme activity. Furthermore, chemical doxorubicin inhibited the enzyme activity of MoPsd2 and showed antifungal activity against 10 phytopathogenic fungi including M. oryzae and reduced disease severity of two crop diseases in the field. Three predicted doxorubicin-interacting residues are important for MoPsd2 functions. Our study demonstrates that MoPsd2 is involved in de novo PE biosynthesis and contributes to the development and plant infection of M. oryzae and that doxorubicin shows broad-spectrum antifungal activity as a fungicide candidate. The study also implicates that bacterium Streptomyces peucetius, which biosynthesizes doxorubicin, could be potentially used as an eco-friendly biocontrol agent.

Identification of propranolol and derivatives that are chemical inhibitors of phosphatidate phosphatase as potential broad-spectrum fungicides.

Plant diseases cause enormous economic losses in agriculture and threaten global food security, and application of agrochemicals is an important method of crop disease control. Exploration of disease-resistance mechanisms and synthesis of highly bioactive agrochemicals are thus important research objectives. Here, we show that propranolol, a phosphatidate phosphatase (Pah) inhibitor, effectively suppresses fungal growth, sporulation, sexual reproduction, and infection of diverse plants. The MoPah1 enzyme activity of the rice blast fungus Magnaporthe oryzae is inhibited by propranolol. Alterations in lipid metabolism are associated with inhibited hyphal growth and appressorium formation caused by propranolol in M. oryzae. Propranolol inhibits a broad spectrum of 12 plant pathogens, effectively inhibiting infection of barley, wheat, maize, tomato, and pear. To improve antifungal capacity, we synthesized a series of propranolol derivatives, one of which shows a 16-fold increase in antifungal ability and binds directly to MoPah1. Propranolol and its derivatives can also reduce the severity of rice blast and Fusarium head blight of wheat in the field. Taken together, our results demonstrate that propranolol suppresses fungal development and infection through mechanisms involved in lipid metabolism. Propranolol and its derivatives may therefore be promising candidates for fungicide development.

The combined formulation of brassinolide and pyraclostrobin increases biomass and seed yield by improving photosynthetic capacity in Arabidopsis thaliana.

In the context of global food crisis, applying the phytohormone-brassinosteroids (BRs) in combination with the fungicide-pyraclostrobin (Pyr) was beneficial for plant quality and productivity in several field trials. However, in addition to the benefits of disease control due to the innate fungicidal activity of Pyr, it remains to be understood whether the coapplication of BL+ Pyr exerts additional growth-promoting effects. For this purpose, the effects of BL treatment, Pyr treatment, and BL+ Pyr treatment in Arabidopsis thaliana were compared. The results showed that the yield increased at a rate of 25.6% in the BL+Pyr group and 9.7% in the BL group, but no significant change was observed in the Pyr group. Furthermore, the BL+Pyr treatment increased the fresh weight of both the leaves and the inflorescences. In contrast, the Pyr and BL treatments only increased the fresh weight of leaves and inflorescences, respectively. Additionally, the BL + Pyr treatment increased the Pn, Gs, Tr, Vc, max, Jmax, VTPU, ETR, Fv'/Fm', ΦPSII, Rd, AYE and Rubisco enzyme activity by 26%, 38%, 40%, 16%, 19%, 15%, 9%, 10%, 17%, 179%, 18% and 32%, respectively. While, these paraments did not change significantly by the BL or Pyr treatments. Treatment with BL + Pyr and Pyr, rather than BL, improved the chlorophyll a and chlorophyll b contents by upregulating genes related to chlorophyll biosynthesis and downregulating genes related to chlorophyll degradation. Additionally, according to transcriptomic and metabolomic analysis, the BL+ Pyr treatment outperformed the individual BL or Pyr treatments in activating the transcription of genes involved in photosynthesis and increasing sugar accumulation. Our results first validated that the combined usage of BL and Pyr exerted striking synergistic effects on enhancing plant biomass and yield by increasing photosynthetic efficiency. These results might provide new understanding for the agricultural effects by the co-application of BL and Pyr, and it might stimulate the efforts to develop new environment-friendly replacement for Pyr to minimize the ecotoxicology of Pyr.

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.