Plant immunity relies on the perception of microbe-associated molecular patterns (MAMPs) from invading microbes to induce defense responses that suppress attempted infections. It has been proposed that MAMP-triggered immunity (MTI) suppresses bacterial infections by suppressing the onset of bacterial virulence. However, the mechanisms by which plants exert this action are poorly understood. Here, we showed that MAMP perception in Arabidopsis (Arabidopsis thaliana) induces the accumulation of free amino acids in a salicylic acid (SA)-dependent manner. When co-infiltrated with Glutamine and Serine, two of the MAMP-induced highly accumulating amino acids, Pseudomonas syringae pv. tomato DC3000 expressed low levels of virulence genes and failed to produce robust infections in otherwise susceptible plants. When applied exogenously, Glutamine and Serine directly suppressed bacterial virulence and growth, bypassing MAMP perception and SA signaling. In addition, an increased level of endogenous Glutamine in the leaf apoplast of a gain-of-function mutant of Glutamine Dumper-1 rescued the partially compromised bacterial virulence- and growth-suppressing phenotype of the SA-induced deficient-2 (sid2) mutant. Our data suggest that MTI suppresses bacterial infections by delaying the onset of virulence with an excess of amino acids at the early stages of infection.

Plants live under the constant challenge of microbes that probe the environment in search of potential hosts. Plant cells perceive microbe-associated molecular patterns (MAMPs) from incoming microbes and activate defense responses that suppress attempted infections. Despite the substantial progress made in understanding MAMP-triggered signaling pathways, the downstream mechanisms that suppress bacterial growth and disease remain poorly understood. Here, we uncover how MAMP perception in Arabidopsis (Arabidopsis thaliana) elicits dynamic changes in extracellular concentrations of free L-amino acids (AA). Within the first 3 h of MAMP perception, a fast and transient inhibition of AA uptake produces a transient increase in extracellular AA concentrations. Within 4 and 12 h of MAMP perception, a sustained enhanced uptake activity decreases the extracellular concentrations of AA. Gene expression analysis showed that salicylic acid-mediated signaling contributes to inducing the expression of AA/H+ symporters responsible for the MAMP-induced enhanced uptake. A screening of loss-of-function mutants identified the AA/H+ symporter lysin/histidine transporter-1 as an important contributor to MAMP-induced enhanced uptake of AA. Infection assays in lht1-1 seedlings revealed that high concentrations of extracellular AA promote bacterial growth in the absence of induced defense elicitation but contribute to suppressing bacterial growth upon MAMP perception. Overall, the data presented in this study reveal a mechanistic connection between MAMP-induced plant defense and suppression of bacterial growth through the modulation of AA transport activity.

The root microbiome structure ensures optimal plant host health and fitness, and it is, at least in part, defined by the plant genotype. It is well documented that root-secreted amino acids promote microbial chemotaxis and growth in the rhizosphere. However, whether the plant-mediated re-uptake of amino acids contributes to maintaining optimal levels of amino acids in the root exudates, and, in turn, microbial growth and metabolism, remains to be established. Here, we show that Lysine-Histidine Transporter-1 (LHT1), an amino acid inward transporter expressed in Arabidopsis thaliana roots, limits the growth of the plant-growth-promoting bacteria Pseudomonas simiae WCS417r (Ps WCS417r). The amino acid profiling of the lht1 mutant root exudates showed increased levels of glutamine, among other amino acids. Interestingly, lht1 exudates or Gln-supplemented wild-type exudates enhance Ps WCS417r growth. However, despite promoting bacterial growth and robust root colonization, lht1 exudates and Gln-supplemented wild-type exudates inhibited plant growth in a Ps WCS417r-dependent manner. The transcriptional analysis of defense and growth marker genes revealed that plant growth inhibition was not linked to the elicitation of plant defense but likely to the impact of Ps WCS417r amino acids metabolism on auxin signaling. These data suggest that an excess of amino acids in the rhizosphere impacts Ps WCS417r metabolism, which, in turn, inhibits plant growth. Together, these results show that LHT1 regulates the amino-acid-mediated interaction between plants and Ps WCS417r and suggest a complex relationship between root-exuded amino acids, root colonization by beneficial bacteria, bacterial metabolism, and plant growth promotion.

CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprogramming of Cas9 specificity using customizable small non-coding RNAs, and to the possibility of editing several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource-intensive. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxy genes. We found that each proxy predicted the occurrence of mutations in each surrogate gene with efficiencies ranging from 68% to 100%. The selection strategy laid out here provides a framework to facilitate the identification of multiplex edited plants, thus aiding in the study of gene function when functional redundancy hinders the effort to define gene-function-phenotype links.

Bacterial pathogens adapt their metabolism to the plant environment to successfully colonize their hosts. In our efforts to uncover the metabolic pathways that contribute to the colonization of Arabidopsis thaliana leaves by Pseudomonas syringae pv tomato DC3000 (Pst DC3000), we created iPst19, an ensemble of 100 genome-scale network reconstructions of Pst DC3000 metabolism. We developed a novel approach for gene essentiality screens, leveraging the predictive power of iPst19 to identify core and ancillary condition-specific essential genes. Constraining the metabolic flux of iPst19 with Pst DC3000 gene expression data obtained from naïve-infected or pre-immunized-infected plants, revealed changes in bacterial metabolism imposed by plant immunity. Machine learning analysis revealed that among other amino acids, branched-chain amino acids (BCAAs) metabolism significantly contributed to the overall metabolic status of each gene-expression-contextualized iPst19 simulation. These predictions were tested and confirmed experimentally. Pst DC3000 growth and gene expression analysis showed that BCAAs suppress virulence gene expression in vitro without affecting bacterial growth. In planta, however, an excess of BCAAs suppress the expression of virulence genes at the early stages of infection and significantly impair the colonization of Arabidopsis leaves. Our findings suggesting that BCAAs catabolism is necessary to express virulence and colonize the host. Overall, this study provides valuable insights into how plant immunity impacts Pst DC3000 metabolism, and how bacterial metabolism impacts the expression of virulence.

Root exudation is an important trait that enables plants to shape their interactions with soil-borne organisms. Amino acids present in root exudates play i