Competitive index in mixed infections: a sensitive and accurate assay for the genetic analysis of Pseudomonas syringae–plant interactions

Abstract

Mixed infections have been broadly applied to the study of bacterial pathogens in animals. However, the application of mixed infection-based methods in plant pathogens has been very limited. An important factor for this limitation is the different dynamics that mixed infections have been reported to show in the different types of models. Reports in systemic animal infections have shown that any bacterium has the same probability of multiplying within a mixed infection than in a single infection. However, in plant pathogens, bacterial growth in a mixed infection does not seem to reflect growth in a single infection, as growth interference takes place between the co-inoculated strains. Here we show that growth interference in mixed infection between different Pseudomonas syringae strains is not intrinsic to growth within a plant host, but dependent on the dose of inoculation. We also show that the minimal inoculation dose required to avoid interference depends on the aggressiveness of the pathogen as well as the type of virulence factor that differentiates the co-inoculated strains. This study establishes the basis for the use of mixed infection-based applications to the study of phytopathogenic bacteria. Analysis of the virulence of a type III effector mutant and an hrp regulatory mutant illustrate the increased accuracy and sensitivity of competitive index assays vs. regular growth assays. Several applications of this assay are addressed, and potential implications for this and other mixed infection-based methods are discussed.

Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae, Creates a Binding Site for Host 14-3-3 Proteins

“…To assess the functional relevance of 14-3-3 binding in HopQ1 virulence, we employed a competitive index (CI) assay (Macho et al, 2007). Plasmids expressing HopQ1 variants were introduced to P. syringae pv tomato DC3000D28E, a strain deficient in 28 effectors.…”

“…The bacteria were grown at 28°C and after 2 d replicated onto plates containing either kanamycin or gentamicin, which enabled strain differentiation and cfu counting. The CI was calculated as described previously (Macho et al, 2007). CI was defined as the ratio of the strain carrying wild-type hopQ1 to the strain expressing hopQ1S51A within the output samples, divided by the corresponding ratios in the input inocula.…”

Insights into plant immunity signaling

“…2,9 The versatility that mixed infections have demonstrated as a methodology for genetic analysis of virulence suggests that they are potentially adaptable to other models of bacteria-plant interactions. So far, we have taken advantage of the high sensitivity and accuracy offered by CI assays to determine the contribution of many effector proteins to bacterial growth within the host plant.…”

“…This problem has led researchers to use alternative approaches to overcome functional redundancy, including the generation of polymutants lacking several effector genes, ectopic expression of effectors in heterologous strains lacking the corresponding homolog, as well as the generation of transgenic plants expressing a given effector. 1 2 thus providing an alternative to previously used approaches for the analysis of effector function within the context of the infection. This increase in sensitivity and accuracy is due to the direct comparison between growth of the co-inoculated strains within the same infection ( Fig.…”

“…1), which replicate as they would in individual infections under the appropriate experimental settings. 2 Plant immunity can be triggered by a group of conserved microbial molecules known as PAMPs (pathogen-associated molecular patterns). PAMP-triggered immunity (PTI) can be suppressed by effectors which in turn can be recognized by nucleotide binding-leucine rich repeat (NB-LRR) proteins, encoded by resistance genes or R genes.…”