Genome-wide identification of Pseudomonas syringae genes required for fitness during colonization of the leaf surface and apoplast

Abstract

The foliar plant pathogen Pseudomonas syringae can establish large epiphytic populations on leaf surfaces before apoplastic colonization. However, the bacterial genes that contribute to these lifestyles have not been completely defined. The fitness contributions of 4,296 genes in P. syringae pv. syringae B728a were determined by genome-wide fitness profiling with a randomly barcoded transposon mutant library that was grown on the leaf surface and in the apoplast of the susceptible plant Phaseolus vulgaris Genes within the functional categories of amino acid and polysaccharide (including alginate) biosynthesis contributed most to fitness both on the leaf surface (epiphytic) and in the leaf interior (apoplast), while genes involved in type III secretion system and syringomycin synthesis were primarily important in the apoplast. Numerous other genes that had not been previously associated with in planta growth were also required for maximum epiphytic or apoplastic fitness. Fourteen hypothetical proteins and uncategorized glycosyltransferases were also required for maximum competitive fitness in and on leaves. For most genes, no relationship was seen between fitness in planta and either the magnitude of their expression in planta or degree of induction in planta compared to in vitro conditions measured in other studies. A lack of association of gene expression and fitness has important implications for the interpretation of transcriptional information and our broad understanding of plant-microbe interactions.

Keywords: endophytes; epiphytes; gene expression.

Fig. 1.

Rank-ordered mean gene fitness values for each condition in which P. syringae was grown. Fitness values for independent replicate experiments are shown in gray, while mean fitness values are plotted in black. Gene-fitness values are calculated as the log₂ ratio of the barcode counts following growth in a given condition compared to the barcode counts from midlog phase cultures following library outgrowth and before inoculation. Fitness values are normalized across the genome so the typical gene has a fitness value of 0. Black lines at fitness values of −2 and 2 are used to indicate strong phenotypes; for example, a value of −2 indicates that mutants in that gene were 25% as fit as the typical strain in the mutant library. In each dataset, fitness values less than −2 or greater than 2 are more than 3 SDs from the mean (∼0).

Fig. 2.

Genes with significant contributions to competitive fitness in the experimental conditions tested. (A) Venn diagram of genes with average fitness values less than −2, and t < −3 for at least 2 experimental replicates. (B) Venn diagram of genes with average fitness values less than −1, and t < −2.5 for at least 2 experimental replicates.

Fig. 3.

Fitness contributions of genes involved in phytotoxin synthesis and transport, the T3SS, and polysaccharide synthesis and regulation are required for apoplastic colonization. Gene fitness values are shown from 3 replicate epiphytic experiments (epi), 3 replicate apoplastic experiments (apo), and 2 replicate experiments in KB medium. An expanded version of this figure containing gene names and loci can be found in the SI Appendix. The treatment ordering and corresponding dendrogram are calculated based on these genes only.

Fig. 4.

Apoplastic growth of B728a and deletion strains in bean. Growth of the amino acid auxotrophs ∆trpA and ∆hisD, type III regulatory mutant ∆hrpL, and syringomycin mutant ∆syrP. Apoplastic fitness of deletion mutants of glycosyltransferase genes Psyr_0532 and Psyr_0920, and hypothetical protein eftA. Error bars represent the SEM. At each time point, strains labeled with the same letter are not significantly different (Tukey’s honest significant difference test, P < 0.05).

Fig. 5.

The magnitude of fitness contributions of genes in P. syringae do not correlate well with their absolute level of expression (Upper) or fold-change of these genes in planta compared to that in hrp-inducing MM or KB (Lower). Epiphytic (Left) and apoplastic (Center) gene expression in B728a is a measure of fluorescence in microarrays (27). A comparison of B728a apoplastic gene fitness to expression of orthologs in the closely related strain DC3000, measured in A. thaliana, reveals a similar trend (Right). DC3000 gene expression is calculated as the number of reads per million (28). Fold-change in gene expression was calculated as a log₂ of the ratio of gene expression estimated from microarray fluorescence (B728a) or RNA-seq (DC3000) in planta relative to that in MM or in KB (27). The Pearson correlation coefficients are shown.