ScAnalyzer: an image processing tool to monitor plant disease symptoms and pathogen spread in Arabidopsis thaliana leaves.

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.

Hydathode immunity protects the Arabidopsis leaf vasculature against colonization by bacterial pathogens.

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.

The root-invading pathogen Fusarium oxysporum targets pattern-triggered immunity using both cytoplasmic and apoplastic effectors.

Plant pathogens use effector proteins to promote host colonisation. The mode of action of effectors from root-invading pathogens, such as Fusarium oxysporum (Fo), is poorly understood. Here, we investigated whether Fo effectors suppress pattern-triggered immunity (PTI), and whether they enter host cells during infection. Eight candidate effectors of an Arabidopsis-infecting Fo strain were expressed with and without signal peptide for secretion in Nicotiana benthamiana and their effect on flg22-triggered and chitin-triggered reactive oxidative species (ROS) burst was monitored. To detect uptake, effector biotinylation by an intracellular Arabidopsis-produced biotin ligase was examined following root infection. Four effectors suppressed PTI signalling; two acted intracellularly and two apoplastically. Heterologous expression of a PTI-suppressing effector in Arabidopsis enhanced bacterial susceptibility. Consistent with an intracellular activity, host cell uptake of five effectors, but not of the apoplastically acting ones, was detected in Fo-infected Arabidopsis roots. Multiple Fo effectors targeted PTI signalling, uncovering a surprising overlap in infection strategies between foliar and root pathogens. Extracellular targeting of flg22 signalling by a microbial effector provides a new mechanism on how plant pathogens manipulate their host. Effector translocation appears independent of protein size, charge, presence of conserved motifs or the promoter driving its expression.

Alteration of flavonoid pigmentation patterns during domestication of food crops.

Flavonoids are plant pigments that provide health benefits for human and animal consumers. Understanding why domesticated crops have altered pigmentation patterns and unraveling the molecular/genetic mechanisms that underlie this will facilitate the breeding of new (healthier) varieties. We present an overview of changes in flavonoid pigmentation patterns that have occurred during crop domestication and, where possible, link them to the molecular changes that brought about the new phenotypes. We consider species that lost flavonoid pigmentation in the edible part of the plant at some point during domestication (like cereals). We also consider the converse situation, for example eggplant (aubergine), which instead gained strong anthocyanin accumulation in the skin of the fruit during domestication, and some varieties of citrus and apple that acquired anthocyanins in the fruit flesh. Interestingly, the genes responsible for such changes are sometimes closely linked to, or have pleiotropic effects on, important domestication genes, suggesting accidental and perhaps inevitable changes of anthocyanin patterning during domestication. In other cases, flavonoid pigmentation patterns in domesticated crops are the result of cultural preferences, with examples being found in varieties of citrus, barley, wheat, and maize. Finally, and more recently, in some species, anthocyanins seem to have been the direct target of selection in a second wave of domestication that followed the introduction of industrial food processing.