Genome-wide identification of Pseudomonas syringae B728a genes required for competitive fitness during colonization of diverse plant hosts, with a focus on toxin efflux transporters

Bacterial colonization of a plant host requires both specialized and general traits. On the leaf surface and in the leaf interior (apoplast), bacteria encounter resources needed to support growth but are also exposed to multiple abiotic and biotic stresses that they must overcome. The foliar plant pathogen Pseudomonas syringae can establish large epiphytic populations on leaf surfaces before moving through stomata or other openings such as wounds into the apoplast, where it can multiply to very high numbers before inciting disease symptoms. Strain B728a is pathogenic to common bean (Phaseolus vulgaris), lima bean (P. lunatus), pepper (Capsicum annuum), Nicotiana benthamiana, and I show that it is rather promiscuous, capable of growth within other plant species. Strains of P. syringae are commonly found as epiphytes on a variety of both host- and non-host plants in agricultural systems, native plants, and in various aquatic environments. P. syringae has thus presumably evolved or acquired a diversity of traits needed for success in these varied habitats, but we lack an understanding of the identity and role of these genes and the degree to which they are linked to fitness in a given context.To examine the context-dependent fitness contributions of genes in P. syringae for growth on or in leaves, I created a barcoded transposon insertional mutant library in strain B728a. Randomly-barcoded TnSeq (RB-TnSeq) enables the same heterogeneous mutant population to be used to interrogate gene contribution to bacterial fitness in a variety of distinct hosts or environments. I determined the contributions of 4,296 nonessential genes to fitness on the leaf surface and in the apoplast of common bean. Genes within the functional categories of amino acid and polysaccharide biosynthesis contributed most to fitness both on the leaf surface and in the apoplast, while genes involved in type III secretion and syringomycin synthesis were important primarily in the apoplast. Interestingly, for most genes no relationship was seen between fitness in planta and either the magnitude of their expression or degree of induction in planta compared in this host plant to in vitro conditions measured in other studies. Given the robust bacterial growth of P. syringae also in lima bean and pepper, I utilized the barcoded transposon library to identify genes that differentially contributed to apoplastic colonization in those hosts. Most genes contributed to apoplastic fitness at similar magnitudes in each of the plant hosts. However, 87 genes significantly differed in their fitness contribution in at least one host, and 50 of these genes were highly important for apoplastic colonization. Genes in the functional categories of amino acid metabolism, phytotoxin biosynthesis, alginate biosynthesis, and cofactor metabolism significantly differed in contributions to fitness in the plant species tested. In addition, six genes that encoded hypothetical proteins contributed differentially to growth in these hosts.Because P. syringae colonizes such a large number of plant species and various habitats it presumably is exposed to a wide variety of inhibitory compounds that it must tolerate. To test the hypothesis that plants would differ in the intensity and nature of chemical defenses against bacterial colonists I used a mexB mutant of P. syringae strain B728a that was blocked in expression of an important efflux pump as an indicator of the chemical defenses of various plants. While MexB was required for full virulence in common bean and pepper, it was dispensable for apoplastic growth in lima bean, Nicotiana benthamiana, sunflower (Helianthus), and tomato (Solanum lycopersicum). Within these plants, B728a grew to varying population sizes, indicating differential susceptibilities among these compatible host plant species. P. syringae strain B728a encodes 668 predicted transport proteins. However, the substrates of a majority of these transporters are unknown. Many of these transporters confer resistance to diverse toxicants present in the environment, but due to a high level of functional redundancy, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. To identify transporters in B728a that are important for tolerance of external toxicants but redundant with MexB, I exposed the WT barcoded transposon mutant library as well as a library constructed in the mexB deletion strain to diverse toxic compounds in vitro to identify mutants with reduced ability to grow in the presence of those compounds. The homologous transporters MexCD and MexEF were partially redundant with MexAB, sharing a subset of substrates tested. I hypothesized that inner membrane transporters contribute to substrate specificity, particularly when functioning cooperatively with outer membrane transporters or porins. In support of this model, the inner membrane transporters Psyr_0228 and Psyr_0541 were highly substrate specific, and contributed to growth independent of mexB genotype. This study of isogenic transposon libraries revealed the substrates of efflux transporters that are masked by the dominant activity of MexAB.These studies provide a more comprehensive insight into the chemical ecology of P. syringae. It is clear that plant species differ in their chemical defenses, as seen in the differential fitness of the hyper-susceptible mexB efflux pump mutant in various host plants. The fitness of various auxotrophs within a transposon library in planta also indicates resources that are often in low amounts in the host, such as amino acids and cofactors. These studies identified genes contributing to competitive fitness in diverse environmental conditions, including tolerance of toxicants that might be encountered by P. syringae. The resulting large datasets represent a valuable tool for the identification of genes required for compatibility with a given plant host. Improving our understanding of genes that contribute to bacterial fitness in diverse environments, including the phyllosphere, will improve our understanding of those habitats and the bacterial traits needed to survive and thrive