ABSTRACT
BACKGROUND: Plants are known to be infected by a wide range of pathogenic microbes. To study plant diseases caused by microbes, it is imperative to be able to monitor disease symptoms and microbial colonization in a quantitative and objective manner. In contrast to more traditional measures that use manual assignments of disease categories, image processing provides a more accurate and objective quantification of plant disease symptoms. Besides monitoring disease symptoms, computational image processing provides additional information on the spatial localization of pathogenic microbes in different plant tissues. RESULTS: Here we report on an image analysis tool called ScAnalyzer to monitor disease symptoms and bacterial spread in Arabidopsis thaliana leaves. Thereto, detached leaves are assembled in a grid and scanned, which enables automated separation of individual samples. A pixel color threshold is used to segment healthy (green) from chlorotic (yellow) leaf areas. The spread of luminescence-tagged bacteria is monitored via light-sensitive films, which are processed in a similar manner as the leaf scans. We show that this tool is able to capture previously identified differences in susceptibility of the model plant A. thaliana to the bacterial pathogen Xanthomonas campestris pv. campestris. Moreover, we show that the ScAnalyzer pipeline provides a more detailed assessment of bacterial spread within plant leaves than previously used methods. Finally, by combining the disease symptom values with bacterial spread values from the same leaves, we show that bacterial spread precedes visual disease symptoms. CONCLUSION: Taken together, we present an automated script to monitor plant disease symptoms and microbial spread in A. thaliana leaves. The freely available software ( https://github.com/MolPlantPathology/ScAnalyzer ) has the potential to standardize the analysis of disease assays between different groups.
ABSTRACT
Plants prevent disease by passively and actively protecting potential entry routes against invading microbes. For example, the plant immune system actively guards roots, wounds, and stomata. How plants prevent vascular disease upon bacterial entry via guttation fluids excreted from specialized glands at the leaf margin remains largely unknown. These so-called hydathodes release xylem sap when root pressure is too high. By studying hydathode colonization by both hydathode-adapted (Xanthomonas campestris pv. campestris) and non-adapted pathogenic bacteria (Pseudomonas syringae pv. tomato) in immunocompromised Arabidopsis mutants, we show that the immune hubs BAK1 and EDS1-PAD4-ADR1 restrict bacterial multiplication in hydathodes. Both immune hubs effectively confine bacterial pathogens to hydathodes and lower the number of successful escape events of an hydathode-adapted pathogen toward the xylem. A second layer of defense, which is dependent on the plant hormones' pipecolic acid and to a lesser extent on salicylic acid, reduces the vascular spread of the pathogen. Thus, besides glands, hydathodes represent a potent first line of defense against leaf-invading microbes.
