Biosynthetic gene clusters in Pseudomonas viridiflava have a fitness cost during Arabidopsis thaliana infection

Specialized or secondary metabolites mediate biotic interactions, including virulence and defense. In plant-pathogenic Pseudomonas, certain specialized metabolites can enhance colonization of plant hosts, yet their broader contribution to plant-microbe interactions and the relative importance of different metabolites remain unclear. Specialized metabolites are products of enzymes encoded in biosynthetic gene clusters (BGCs), whose prediction from genome sequences has become routine but whose functional roles are rarely tested experimentally. Here, we characterize the BGC repertoire of 225 P. viridiflava isolates from Arabidopsis thaliana and assess BGC contributions to fitness in planta and disease severity. The BGC landscape of P. viridiflava was dominated by non-ribosomal peptide synthetase (NRPS) and NRPS-like BGCs, with one-third of families restricted to a single isolate. Transposon mutagenesis coupled with random barcode transposon sequencing (RB-TnSeq) revealed that the majority of BGCs reduce rather than increase fitness during A. thaliana infection, with the magnitude of the fitness cost varying across host genotypes. This cost could be due to exploitation of public goods by cheater mutant strains. In single-isolate plant infections, where public goods are not available, several BGC families were negatively associated with disease severity, which is positively correlated with bacterial growth in this pathosystem, further indicating that BGCs are generally not beneficial in planta. Our findings reveal extensive and largely uncharacterized biosynthetic potential in populations of P. viridiflava and indicate that candidate metabolites are likely not adaptive for direct interactions with the plant, but perhaps for microbe-microbe interactions either in planta or in other ecological niches. IMPORTANCEBacteria living on plant leaves produce a vast array of chemical compounds, called secondary or specialized metabolites, that can mediate their interaction with the plant host or other microorganisms. Some of these compounds are known to directly influence how bacteria interact with plants, but it has been unclear whether this is a general rule. We studied a large collection of closely related leaf-dwelling bacteria that varied in their ability to cause disease, focusing on leaf-associated Pseudomonas viridiflava--a plant pathogen. We found that very few of the gene clusters responsible for making specialized metabolites improved the ability of the bacteria to colonize Arabidopsis thaliana. On the contrary, carrying these gene clusters often reduced bacterial growth and disease severity in plants. Specialized metabolites may instead primarily be important for interacting with other microbes, different host species, or under environmental conditions we did not test. These are questions that remain for future research.