Knockout of phosphatidate phosphohydrolase genes confers broad‐spectrum disease resistance in plants

Phosphatidic acid (PA) is considered a second messenger that interacts with protein kinases, phosphatases and NADPH oxidases (Kong et al., 2024), amplifying the signal to initiate plant defence responses (Li and Wang, 2019). In rice, mutation of RBL1 causes the accumulation of PA, enhancing multipathogen resistance (Sha et al., 2023). In our previous study, we attempted to rescue rbl1 mutant by overexpressing phosphatidate phosphohydrolase (PAH) genes. However, overexpression of PAH2 reduced the PA level but did not affect the disease resistance of rbl1, which prompted us to test the role of PA and PAHs in rice immunity in the wild-type (WT) background. Here, we identified that knockout of PAHs caused PA accumulation and enhanced multipathogen resistance in rice and Arabidopsis. Phylogenetic analyses reveal that PAHs are highly conserved in plants. Rice PAHs contain conserved NLIP and LNS2 signature motifs (Figure 1a). PAH1 and PAH2 were transcribed in all rice tissues examined, with the highest level in the leaf (Figure 1b). PAH-GFP signals predominantly co-localized with the endoplasmic reticulum (ER) marker HDEL1 (Figure 1c). The yeast pah1 mutant is lethal at 37 °C. We transformed rice PAH1 and PAH2 genes into the WT yeast strain, respectively, and knocked out the yeast endogenous PAH1 gene. The rice PAH complementation strains grew well on the induction medium YPGal but not on the non-inducing medium YPD at 37 °C, indicating that rice PAH1 and PAH2 function as PA phosphohydrolases in yeast (Figure 1d). To investigate the functions of rice PAHs, we genome-edited PAHs by targeting their first exons and obtained pah single mutants (Figure 1e). Since all mutations in pah1 and pah2 led to loss-of-function and no off-targets were observed, we crossed pah1-1 and pah2-1 and created the pah1pah2 double mutant (Figures S1 and S2). We next tested ROS production in rice pah plants. The pah1pah2 leaves exhibited a robust ROS burst when challenged with chitin. The total photon counts, denoting the ROS level, showed an obvious increase in pah1pah2 (Figure 1f). When infected with rice blast fungus Magnaporthe oryzae, the lesion area of pah1pah2 was much smaller than other lines and only 46.1% of the WT (Figure 1g). We subsequently tested the disease resistance of rice pah1pah2 lines with the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). Similarly, the lesion length was much shorter in pah1pah2 (1.51 cm) compared with the WT (9.40 cm) as well as the pah1-1 (7.02 cm) and pah2-1 (6.64 cm) (Figure 1h). In summary, both fungal and bacterial infection assays demonstrate enhanced resistance of pah1pah2. Besides, pah1pah2 exhibited some growth inhibition, and the plant height of pah1pah2 was 77.8% of the WT (Figures 1i, S1 and S3). Salicylic acid (SA) plays key roles in plant defence. The SA level increased 1.39-fold in pah1pah2 compared with the WT (Figure 1j). To investigate whether the role of the PAH genes in immunity is conserved, we obtained Arabidopsis pah mutants (Eastmond et al., 2010), and the pah1pah2 seedlings were shorter than the WT (Col-0) (Figure 1k). Infection with Botrytis cinerea, pah1pah2 plants developed smaller lesions, a 28.3% reduction than the WT. Similarly, when inoculated with Phytophthora capsici, the lesion area of pah1pah2 (11.0 mm2) was only 15.7% of the WT (70.4 mm2), and smaller lesion areas were also shown in pah1 and pah2 (Figure 1l,m). We further examined the levels of phosphorylated MAPKs (pMAPKs). Under flg22 and chitin treatments, the levels of pMAPKs were significantly higher in the Arabidopsis pah1pah2 mutants as well as pah2 than that in the WT (Figure 1n). The results are consistent with increased ROS levels in rice pah1pah2 , which indicates that increased PA at the plasma membrane by knockout of the ER-localized PAHs ultimately enhances plant immune responses in the plasma membrane. To investigate PAH-mediated regulation of gene transcription and metabolism, we performed RNA-seq and lipidomics analyses. PR genes and FLS2 were upregulated in rice pah1pah2 (Figure 1o). Furthermore, Gene Ontology (GO) enrichment analyses of differentially expressed genes (DEGs) between WT and pah1pah2 plants showed that ‘response to lipid’, ‘defense response’ and ‘defense response to fungus/bacterium’ were enriched (Figure 1p), which are consistent with the enhanced disease resistance of pah1pah2. Then using the hierarchical clustering analyses, three clusters were identified in Arabidopsis and rice (Figure 1q). In the ‘genes upregulated in pah1pah2’ cluster, the expression levels of many hormone-related genes that are involved in JA, ET, SA and IAA were simultaneously upregulated in pah1pah2 lines (Figure 1r), indicating that these plant hormones are likely involved in resistance and growth of pah1pah2 lines. Knockout of PAHs resulted in the increase of PA and decrease of DAG in rice pah1pah2 (Figure 1s). Additionally, immunity-related genes, Chia4a, NPR3, RBOHE, JAZ9 and ERF, and genes negatively regulating plant growth, including SAUR, were upregulated in pah1pah2 mutants (Figure 1s). Overexpression of the SAUR genes inhibited the biosynthesis of plant growth hormones, some of which were upregulated in pah1pah2, thus partially explaining the growth defects of pah1pah2. In summary, knockout of PAH genes alters phospholipid metabolism in plants, and accumulated PA activates the expression of immunity-related genes, but negatively regulates plant growth. In conclusion, knockout of both PAH genes enhances plant resistance but inhibits plant growth, an immunity-growth tradeoff. Previously, we used genome editing to break this tradeoff in RBL1 and generated alleles that balance growth and immunity (Sha et al., 2023). Moreover, uORF insertion into the promoter to manipulate protein translation, pathogen-induced silencing of PAHs and optimal natural PAH alleles are options to engineer plant PAHs for multipathogen resistance without yield penalty (Xiong et al., 2022; Zhou et al., 2018). The PA metabolism-related genes PAHs are highly conserved in plants, and the role of PAHs in multipathogen resistance in other crops is worthy of further investigation. This work was supported by Biological Breeding-National Science and Technology Major Projects (2023ZD04070), the Key R&D Program of Hubei Province (2023BBB171), NNSFC (32172373) and FRFCU (2662023PY006, AML2023A05, 2662024ZKPY001) to G.L. G.S. is supported by the GPKLMSDC (MSDC2023-02). This work was also supported by the Hubei Hongshan Laboratory. G.L., G.S., Q.G., and X.H. are coinventors on a patent no. ZL202311556377.0. The remaining authors declare no conflict of interest. G.L. and G.S. designed the experiments. G.S., L.Y., Q.G., W.Y., X.H., Z.G., T.C., R.T., G.C., Y.L., X.S., M.L., F.X., K.X., G.C., H.H., J.L., and Q.L. performed the experiments. G.L., G.S., X.H., and Q.G. drafted and revised the manuscript. All authors read and approved the final manuscript. The data that support the findings of this study are openly available in NCBI genebank at https://submit.ncbi.nlm.nih.gov/subs/sra/SUB14395780/overview, reference number PRJNA1104162. Figure S1 The plant height phenotype of five pah1pah2 mutants. Figure S2 No mutations were observed in predicted off-target sites in the pah1pah2 double mutant. Figure S3 The rice pah1pah2 plants showed growth defects. Table S1 Primers used in this study Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.