Plants, unlike animals, lack mobile immune cells. They rely on individual cells to detect and respond to pathogens, primarily in the apoplast (extracellular space). Pattern-triggered immunity (PTI) is initiated when plant cell surface receptors (PRRs) recognize microbe-associated molecular patterns (MAMPs). While many MAMP-induced pathways are known, the precise mechanisms limiting pathogen growth in the apoplast remain unclear. *Pseudomonas syringae*, a significant plant pathogen, uses its type III secretion system (T3SS) to deliver effector proteins into plant cells, suppressing PTI. T3SS gene expression is induced by plant-exuded metabolites, such as sugars and amino acids. Previous research showed that an Arabidopsis mutant lacking MAP KINASE PHOSPHATASE1 (MKP1) exudes fewer T3SS-inducing metabolites and exhibits increased resistance to *P. syringae*. This highlights the importance of metabolite abundance in determining infection outcomes. This study aims to investigate MAMP-induced metabolic changes in the Arabidopsis leaf apoplast to identify potential causal factors in the MAMP-induced restriction of T3SS deployment by *P. syringae*. Understanding this mechanism could lead to novel strategies for enhancing plant disease resistance.

The plant immune system initiates pattern-triggered immunity (PTI) when pattern recognition receptors (PRRs) detect conserved microbial features called microbe- or pathogen-associated molecular patterns (M/PAMPs). In animals, MAMP recognition activates mobile immune cells, while plants use individual cells to mitigate threats locally. *Pseudomonas syringae*, a significant plant pathogen, relies on a type III secretion system (T3SS) to deliver proteins that suppress PTI. T3SS gene expression is induced by host-derived metabolites like sugars and amino acids. Studies have shown that reducing the abundance of T3SS-inducing metabolites enhances plant resistance. For example, Arabidopsis mutants deficient in MKP1 exude lower amounts of these metabolites and exhibit increased resistance to *P. syringae*. Prior research has demonstrated that plant immunity can directly or indirectly restrict the injection of type III effectors by *P. syringae*, and transcriptome analyses indicate that MAMPs inhibit T3SS gene expression. This body of work establishes the crucial role of host-derived metabolites in regulating *P. syringae* virulence and suggests that manipulating these metabolites could be a valuable strategy for improving plant disease resistance.

The researchers used a combination of genetic and metabolomic approaches. They first verified that PTI restricts the expression of T3SS genes in *P. syringae* using immunoblotting to detect AvrPto (a type III effector) levels in Arabidopsis leaves pre-treated with MAMPs (flg22 and elf26). They confirmed receptor-dependence by testing *fls2* and *efr* mutants. A quadruple knockout (QKO) mutant deficient in defense hormones (salicylic acid, ethylene, and jasmonic acid) was used as a control to demonstrate the role of PTI. Next, they isolated apoplastic wash fluid (AWF) from mock- and MAMP-treated Arabidopsis leaves and tested its effect on T3SS deployment and bacterial growth. A malate dehydrogenase (MDH) assay confirmed that the AWF isolation procedure did not cause substantial cytoplasmic leakage. The pH of AWF was measured and found not to be causal for flg22-induced restriction of T3SS genes. GC-MS was used to profile MAMP-induced changes in the Arabidopsis leaf apoplast metabolome. Absolute concentrations of metabolites were determined using internal standards. The effect of adding proline to mock- and flg22-AWF on T3SS deployment and bacterial growth was evaluated. The rate of proline removal from the leaf apoplast was examined using ¹³C-labelled proline. Arabidopsis mutants lacking functional ProT1, ProT2, or ProT3 genes were used to investigate the role of proline transporters. A *putA* (proline utilization A) deletion mutant of *P. syringae* was used to study the role of proline catabolism in T3SS gene expression and virulence. Finally, quantitative real-time PCR (qRT-PCR) was used to measure the expression of LHT1 in response to flg22 treatment. Statistical analysis was performed using Microsoft Excel, Metaboanalyst 5.0, R, and Jamovi software.

The study confirmed that PTI restricts the expression of *P. syringae* T3SS genes, and this restriction was absent in a PTI-deficient quadruple knockout (QKO) mutant. Apoplastic wash fluid (AWF) from flg22-treated leaves inhibited *P. syringae* type III secretion and growth, while AWF from QKO leaves did not. Metabolomics analysis revealed that proline levels were significantly decreased in the apoplast of flg22-treated leaves, a change not observed in QKO leaves. Adding proline to flg22-AWF restored T3SS expression and bacterial growth. Flg22 stimulated an increased rate of proline removal from the leaf apoplast, a process dependent on LHT1. The *P. syringae* proline utilization gene *putA* was essential for maximal T3SS deployment and bacterial growth during Arabidopsis infection. Arabidopsis *prot2* mutants, which exhibit elevated apoplastic proline levels, were more susceptible to *P. syringae* infection. However, flg22-induced proline depletion and growth inhibition of *P. syringae* were maintained in *prot2* mutants, indicating that another transporter is involved in this process. The *Arabidopsis* amino acid transporter LHT1 was shown to be necessary for flg22-triggered depletion of apoplastic proline, and LHT1-mediated depletion of apoplastic proline directly contributes to PTI against *P. syringae* infection, even in the absence of salicylic acid (SA).

This research demonstrates that the depletion of a single extracellular metabolite, proline, significantly contributes to plant immunity against *P. syringae*. The study reveals a novel mechanism of plant disease resistance, focusing on the dynamic regulation of apoplastic proline levels rather than solely on antimicrobial compound accumulation or physical barriers. Proline's role in various cellular processes, including osmotic pressure regulation and nutrient availability, is well-known. This study underscores the dynamic nature of extracellular proline levels in Arabidopsis leaves, influenced by both baseline uptake mechanisms and MAMP-triggered responses. The findings highlight the crucial role of LHT1, an amino acid transporter, in mediating the flg22-induced depletion of apoplastic proline. The results also emphasize the importance of proline as a nutrient and virulence-inducing signal for *P. syringae*, with proline catabolism via PutA being necessary for proline-induced T3SS expression. The study suggests that depletion of proline, possibly below a threshold level, contributes to the reduction of T3SS induction and consequently, bacterial virulence. The data suggest a model where PTI-mediated proline depletion acts as a double-edged sword, simultaneously limiting proline availability as a nutrient source and disrupting the virulence signal that stimulates T3SS.

This study reveals a novel mechanism of plant immunity where the transporter-mediated depletion of extracellular proline directly contributes to pattern-triggered immunity against *Pseudomonas syringae*. The amino acid transporter LHT1 plays a crucial role in this process. The findings highlight the importance of manipulating metabolite levels for enhancing plant disease resistance. Future research should focus on elucidating the precise regulatory mechanisms controlling LHT1 activity following MAMP perception, investigating the broader impact of apoplastic proline restriction on plant-microbe interactions beyond *P. syringae*, and exploring the potential of targeting proline metabolism as a strategy for disease control.

The study primarily focuses on *Pseudomonas syringae* pv. tomato DC3000 and Arabidopsis. The generalizability of the findings to other plant-pathogen interactions needs further investigation. The metabolomics analysis used a single time point, potentially missing other MAMP-induced metabolic changes. While the study strongly suggests a causal link between proline depletion and reduced virulence, it cannot definitively rule out the possibility that nutrient limitation also contributes to reduced bacterial growth.