Insights into plant immunity signaling: The bacterial competitive index angle

The interaction between a bacterial pathogen and its potential plant host develops from a complex combination of bacterial and plant elements, which determines either the establishment of resistance or the development of disease. The use of virulence assays based on competitive index in mixed infections constitutes a powerful tool for the analysis of bacterial virulence factors. In this work, we describe how the use of competitive index assays also constitutes an alternative approach for the analysis of plant immunity, to determine the contribution of different elements to bacterial recognition or immunity signaling.Key words: competitive index, mixed infections, pathogen, plant immunity, defence response, effector-triggered immunityThe type III secretion system (T3SS) allows Gram negative bacterial pathogens to deliver a set of effector proteins into the host cell. The plant pathogen Pseudomonas syringae employs a large inventory of type III-secreted effectors (T3SEs) to suppress plant immunity. Individual mutation of effector genes has traditionally failed to provide a relevant virulence phenotype, a fact generally associated to a high degree of functional redundancy between T3SEs, which also hinders the characterisation of effector activities within the plant cell. 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 We have previously established that the use of competitive index in mixed infections provides an accurate and sensitive manner of establishing virulence phenotypes for single effector mutants for which other assays have failed,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.²Open in a separate windowFigure 1Basis for the increased sensitivity and acuracy of CI assays. A mixed inoculum with equal amounts of wild type and query strain is inoculated within the same plant (A), allowing a direct comparison between the replication values of both strains within the same infection (B). On the contrary, in regular individual infections, the values obtained from different plants have to be pulled first and compared afterwards (B), thus accumulating experimental and plant-to-plant variation.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.^3,4 Detection of such effectors by NB-LRR proteins determines effector-triggered immunity (ETI),³ an amplified version of PTI, which usually crosses the threshold inducing the hypersensitive response (HR), a localized cell death response. Furthermore, bacteria have evolved effectors that suppress ETI, some of which can in turn be recognized by the plant, thus triggering a secondary ETI.⁴ Selection favors new plant NB-LRRs that can recognize such secondary, newly acquired effectors.R-gene-mediated defences are usually associated with the accumulation of salycilic acid (SA),⁵ although SA-independent pathways such as that dependent on EDS1 (enhanced disease susceptibility-1),⁶ can also mediate ETI and trigger an HR.⁷ Although regulating independent pathways, EDS1 and SA have been recently described to function redundantly to regulate R-gene-mediated signaling.⁸