Summary Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient‐limited T3SS‐inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non‐genetic origin, and the T3SSHIGH and T3SSLOW subpopulations show differences in virulence. T3SS bistability requires the transcriptional activator HrpL, the double negative regulatory loop established by HrpV and HrpG, and may be enhanced through a positive feedback loop involving HrpA, the main component of the T3SS pilus. To our knowledge, this is the first example of phenotypic heterogeneity in the expression of virulence determinants during colonization of a non‐mammalian host.

Plants encode numerous intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize pathogen-derived effectors or their activity to activate defenses. MiRNA regulate NLR genes in many species, often triggering the production of phasiRNAs. Most such examples involve genes encoding NLR carrying coiled-coil domains, although a few include genes encoding NLRs carrying a Toll/interleukin-1 domain (TNL). Here, we characterize the role of miR825-5p in Arabidopsis, using a combination of bioinformatics, transgenic plants with altered miRNA levels and/or reporters, small RNA and virulence assays. We demonstrate that miR825-5p downregulates TNL MIST1 by targeting for endonucleolytic cleavage the sequence coding for TIR2, a highly conserved amino acid motif, linked to a catalytic residue essential for immune function. MiR825-5p acts as a negative regulator of basal resistance against Pseudomonas syringae. MiR825-5p triggers the production from MIST1 of a large number of phased siRNAs that can mediate cleavage of both MIST1 and additional TNL gene transcripts, potentially acting as a regulatory hub. MiR825-5p is expressed in unchallenged leaves and transcriptionally downregulated in response to PAMPs. Our results show that miR825-5p, which is required for full expression of PTI, establishes a link between PAMP perception and expression of uncharacterized TNL genes.