Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. Pseudomonas spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. Here, we genetically and phenotypically characterize the Pseudomonas fluorescens population in a commercial potato field, where we identify strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens Streptomyces scabies and Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic lipopeptides as key specialized metabolites associated with S. scabies inhibition, which was supported by in planta biocontrol experiments. We show that a single potato field contains a hugely diverse and dynamic population of Pseudomonas bacteria, whose capacity to produce specialized metabolites is shaped both by plant colonization and defined environmental inputs.

RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV’s largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance.

Multisubunit RNA polymerase (Pol) complexes are the core machinery for gene expression in eukaryotes. The enzymes Pol I, Pol II and Pol III transcribe distinct subsets of nuclear genes. This family of nuclear RNA polymerases expanded in terrestrial plants by the duplication of Pol II subunit genes. Two Pol II-related enzymes, Pol IV and Pol V, are highly specialized in the production of regulatory, non-coding RNAs. Pol IV and Pol V are the central players of RNA-directed DNA methylation (RdDM), an RNA interference pathway that represses transposable elements (TEs) and selected genes. Genetic and biochemical analyses of Pol IV/V subunits are now revealing how these enzymes evolved from ancestral Pol II to sustain non-coding RNA biogenesis in silent chromatin. Intriguingly, Pol IV-RdDM regulates genes that influence flowering time, reproductive development, stress responses and plant–pathogen interactions. Pol IV target genes vary among closely related taxa, indicating that these regulatory circuits are often species-specific. Data from crops like maize, rice, tomato and Brassica rapa suggest that dynamic repositioning of TEs, accompanied by Pol IV targeting to TE-proximal genes, leads to the reprogramming of plant gene expression over short evolutionary timescales.

23Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, 24 including the inhibition of plant pathogens. Pseudomonas are one of the most abundant 25 bacterial genera in the soil and rhizosphere and play important roles in promoting plant growth 26 and preventing disease. However, the genetic determinants of this beneficial activity are only 27 partially understood, especially in relation to specialized metabolite production. Here, we 28 genetically and phenotypically characterize the Pseudomonas fluorescens population in 29 commercial potato field soils and identify strong correlations between specialized metabolite 30 biosynthetic pathways and antagonism of the potato pathogens Streptomyces scabies and 31 Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic 32 lipopeptides as key specialized metabolites associated with S. scabies inhibition. We show that 33 a single potato field contains a hugely diverse and dynamic population of Pseudomonas 34 bacteria, whose capacity to produce specialized metabolites is shaped both by plant 35 colonization and defined environmental inputs. 36 2 37 CFC agar and incubated overnight at 28 °C before streaking to single colonies on King's B (KB) 595 agar plates (96). Six isolates were selected at random per soil sample and subjected to 596 phenotypic/genomic analysis. 597 598 Amplicon sequencing 599 Genomic DNA was isolated from 3 g of pooled soil samples using the FastDNA™ SPIN Kit for 600 soil (MP Biomedicals, UK) following the manufacturer's instructions. Genomic DNA 601 concentration and purity was determined by NanoDrop spectrophotometry as above. Microbial 602 16S rRNA genes were amplified from soil DNA samples with barcoded universal prokaryotic 603 23 primers (515F/R806) targeting the V4 region, and then subjected to Illumina® MiSeq 604 sequencing (600-cycle, 2x300 bp) at the DNA Sequencing Facility, Department of Biochemistry, 605

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