Abstract
Bacterial canker of Prunus, affecting economically important stone fruit crops including cherry, peach, apricot and plum, is caused by the plant pathogen Pseudomonas syringae (P.s.). Strains from two pathovars—P.s. pv. syringae (Pss) and P.s. pv. morsprunorum race 1 (PsmR1) and 2 (PsmR2)—in three phylogenetically distant clades have convergently evolved to infect Prunus. The bacteria enter woody tissues through wounds and leaf scars, causing black necrotic cankers. Symptoms are also produced on blossom, fruit and leaves. Little is known about the mechanisms P.s. uses to colonise tree hosts such as Prunus. Here, we created transposon (Tn) mutant libraries in one strain of P.s. from each of the three clades and screened the mutants on immature cherry fruit to look for changes in virulence. Mutants (242) with either reduced or enhanced virulence were detected and further characterised by in vitro screens for biofilm formation, swarming ability, and pathogenicity on leaves and cut shoots. In total, 18 genes affecting virulence were selected, and these were involved in diverse functions including motility, type III secretion, membrane transport, amino acid synthesis, DNA repair and primary metabolism. Interestingly, mutation of the effector gene, hopAU1, led to an increase in virulence of Psm R2.
Highlights
Bacterial canker of Prunus which attacks the economically important crop species cherry, peach, apricot and plum is caused by members of at least three clades of Pseudomonas syringae (Ps).s., P.s. pv. syringae (Pss) and P.s. pv. morsprunorum race 1 (Psm R1) and 2 (Psm R2)
The development of disease-resistant cherry tree varieties is a central approach to reducing yield losses and securing fruit supply for the future
Key to developing such varieties is an understanding of how pathogens cause disease and trigger resistance, especially when there is diversity in pathogen genotypes
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. A new approach to mutagenesis, allowing rapid identification of genes improving pathogenic fitness, is transposon sequencing (Tn-seq). This method is based on recent developments in high-throughput sequencing platforms and involves creation of a saturated Mariner transposon insertion library followed by deep-sequencing of populations of bacteria to identify the genes maintained during infection [14]. We describe experiments using the fruit assay as a primary screen of Tn mutants of each of the major cherry canker pathogens Pss, Psm R1 and Psm R2. The mutated genes in a selection of mutants with altered pathogenicity were identified by sequencing and complementation experiments. R2 was found to lead to increased symptom development in all assays
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