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.

磷脂酸（PA）被认为是与蛋白激酶、磷酸酶和 NADPH 氧化酶相互作用的第二信使（Kong等， 2024 ），放大信号以启动植物防御反应（Li 和 Wang， 2019 ）。在水稻中， RBL1的突变会导致 PA 的积累，从而增强多病原体抗性 (Sha et al ., 2023 )。在我们之前的研究中，我们试图通过过表达磷脂酸磷酸水解酶（ PAH ）基因来拯救rbl1突变体。然而， PAH2的过度表达降低了PA水平，但不影响rbl1的抗病性，这促使我们在野生型（WT）背景下测试PA和PAHs在水稻免疫中的作用。在这里，我们发现PAH的敲除会导致 PA 积累并增强水稻和拟南芥的多病原体抗性。

系统发育分析表明多环芳烃在植物中高度保守。水稻 PAH 含有保守的 NLIP 和 LNS2 特征基序（图 1a）。 PAH1和PAH2在所有检查的水稻组织中都有转录，其中叶片中的水平最高（图 1b）。 PAH-GFP 信号主要与内质网 (ER) 标记 HDEL1 共定位（图 1c）。酵母pah1突变体在 37 °C 下是致命的。我们将水稻PAH1和PAH2基因分别转化到WT酵母菌株中，并敲除酵母内源PAH1基因。水稻PAH互补菌株在 37 °C 的诱导培养基 YPGal 上生长良好，但在非诱导培养基 YPD 上生长不良，表明水稻 PAH1 和 PAH2 在酵母中充当 PA 磷酸水解酶（图 1d）。

为了研究水稻PAH的功能，我们通过靶向其第一个外显子对 PAH进行基因组编辑，并获得了pah单突变体（图 1e）。由于pah1和pah2中的所有突变都会导致功能丧失并且没有观察到脱靶，因此我们将pah1-1和pah2-1杂交并创建了pah1pah2双突变体（图 S1 和 S2）。接下来我们测试了水稻pah植物中 ROS 的产生。当受到几丁质的挑战时， pah1pah2叶子表现出强大的 ROS 爆发。表示 ROS 水平的总光子计数显示pah1pah2明显增加（图 1f）。当感染稻瘟病菌Magnaporthe oryzae时， pah1pah2的病斑面积远小于其他品系，仅为 WT 的 46.1%（图 1g）。我们随后测试了水稻pah1pah2品系与水稻白叶枯病病原体米黄单胞菌(Xanthomonas oryzae pv.) 的抗病性。米曲霉（ Xoo ）。同样，与 WT（9.40 厘米）以及pah1-1 （7.02 厘米）和pah2-1 （6.64 厘米）相比， pah1pah2 （1.51 厘米）的病变长度要短得多（图 1h）。总之，真菌和细菌感染测定均表明pah1pah2的抵抗力增强。此外， pah1pah2表现出一定的生长抑制作用， pah1pah2的株高是WT的77.8%（图1i、S1和S3）。水杨酸（SA）在植物防御中发挥着关键作用。与 WT 相比， pah1pah2中的 SA 水平增加了 1.39 倍（图 1j）。

为了研究PAH基因在免疫中的作用是否保守，我们获得了拟南芥pah突变体（Eastmond等， 2010 ），并且pah1pah2幼苗比WT（Col-0）短（图1k）。感染灰葡萄孢后，pah1pah2植物出现较小的病斑，比 WT 减少 28.3%。同样，当接种辣椒疫霉时， pah1pah2 （11.0 mm ² ）的病斑面积仅为WT（70.4 mm ² ）的15.7％，并且pah1和pah2也显示出较小的病斑面积（图1l，m）。我们进一步检查了磷酸化 MAPK (pMAPK) 的水平。在 flg22 和几丁质处理下，拟南芥pah1pah2突变体和pah2中的 pMAPK 水平显着高于 WT（图 1n）。结果与水稻pah1pah2中 ROS 水平的增加一致，这表明通过敲除内质网定位的 PAH 来增加质膜上的 PA，最终增强植物质膜中的免疫反应。

为了研究PAH介导的基因转录和代谢调节，我们进行了 RNA 测序和脂质组学分析。 PR基因和FLS2在水稻pah1pah2中表达上调（图 1o）。此外，对 WT 和pah1pah2植物之间差异表达基因 (DEG) 的基因本体 (GO) 富集分析表明，“对脂质的反应”、“防御反应”和“对真菌/细菌的防御反应”得到了富集（图 1p），与pah1pah2增强的抗病性一致。然后使用层次聚类分析，在拟南芥和水稻中鉴定出三个聚类（图 1q）。在“ pah1pah2基因上调”簇中，许多与JA、ET、SA和IAA相关的激素相关基因的表达水平在pah1pah2品系中同时上调（图1r），表明这些植物激素可能参与pah1pah2系的抗性和生长。 PAH的敲除导致水稻pah1pah2中 PA 的增加和 DAG 的减少（图 1s）。此外，免疫相关基因Chia4a 、 NPR3 、 RBOHE 、 JAZ9和ERF以及负调控植物生长的基因（包括SAUR ）在pah1pah2突变体中表达上调（图 1s）。 SAUR基因的过度表达抑制了植物生长激素的生物合成，其中一些植物生长激素在pah1pah2中上调，从而部分解释了pah1pah2的生长缺陷。 综上所述， PAH基因的敲除改变了植物中的磷脂代谢，积累的PA激活了免疫相关基因的表达，但对植物生长产生负调控。

总之，敲除两个PAH基因可以增强植物的抗性，但会抑制植物的生长，这是一种免疫与生长的权衡。此前，我们使用基因组编辑来打破RBL1中的这种权衡，并生成平衡生长和免疫的等位基因 (Sha et al ., 2023 )。此外，uORF插入启动子以操纵蛋白质翻译、病原体诱导的PAH沉默和最佳天然PAH等位基因是工程化植物PAH以实现多病原体抗性而不造成产量损失的选择（Xiong等， 2022 ；Zhou等， 2018） ）。 PA代谢相关基因PAHs在植物中高度保守， PAHs在其他作物抗多病原体中的作用值得进一步研究。