Analysis of plant cell death-inducing proteins of the necrotrophic fungal pathogens Botrytis squamosa and Botrytis elliptica.

Fungal plant pathogens secrete proteins that manipulate the host in order to facilitate colonization. Necrotrophs have evolved specialized proteins that actively induce plant cell death by co-opting the programmed cell death machinery of the host. Besides the broad host range pathogen Botrytis cinerea, most other species within the genus Botrytis are restricted to a single host species or a group of closely related hosts. Here, we focused on Botrytis squamosa and B. elliptica, host specific pathogens of onion (Allium cepa) and lily (Lilium spp.), respectively. Despite their occurrence on different hosts, the two fungal species are each other's closest relatives. Therefore, we hypothesize that they share a considerable number of proteins to induce cell death on their respective hosts. In this study, we first confirmed the host-specificity of B. squamosa and B. elliptica. Then we sequenced and assembled high quality genomes. The alignment of these two genomes revealed a high level of synteny with few balanced structural chromosomal arrangements. To assess the cell death-inducing capacity of their secreted proteins, we produced culture filtrates of B. squamosa and B. elliptica that induced cell death responses upon infiltration in host leaves. Protein composition of the culture filtrate was analysed by mass spectrometry, and we identified orthologous proteins that were present in both samples. Subsequently, the expression of the corresponding genes during host infection was compared. RNAseq analysis showed that the majority of the orthogroups of the two sister species display similar expression patterns during infection of their respective host. The analysis of cell death-inducing proteins of B. squamosa and B. elliptica provides insights in the mechanisms used by these two Botrytis species to infect their respective hosts.

Fire Blight Susceptibility in Lilium spp. Correlates to Sensitivity to Botrytis elliptica Secreted Cell Death Inducing Compounds.

Fire blight represents a widespread disease in Lilium spp. and is caused by the necrotrophic Ascomycete Botrytis elliptica. There are >100 Lilium species that fall into distinct phylogenetic groups and these have been used to generate the contemporary commercial genotypes. It is known among lily breeders and growers that different groups of lilies differ in susceptibility to fire blight, but the genetic basis and mechanisms of susceptibility to fire blight are unresolved. The aim of this study was to quantify differences in fire blight susceptibility between plant genotypes and differences in virulence between fungal isolates. To this end we inoculated, in four biological replicates over 2 years, a set of 12 B. elliptica isolates on a panel of 18 lily genotypes representing seven Lilium hybrid groups. A wide spectrum of variation in symptom severity was observed in different isolate-genotype combinations. There was a good correlation between the lesion diameters on leaves and flowers of the Lilium genotypes, although the flowers generally showed faster expanding lesions. It was earlier postulated that B. elliptica pathogenicity on lily is conferred by secreted proteins that induce programmed cell death in lily cells. We selected two aggressive isolates and one mild isolate and collected culture filtrate (CF) samples to compare the cell death inducing activity of their secreted compounds in lily. After leaf infiltration of the CFs, variation was observed in cell death responses between the diverse lilies. The severity of cell death responses upon infiltration of the fungal CF observed among the diverse Lilium hybrid groups correlated well to their fire blight susceptibility. These results support the hypothesis that susceptibility to fire blight in lily is mediated by their sensitivity to B. elliptica effector proteins in a quantitative manner. Cell death-inducing proteins may provide an attractive tool to predict fire blight susceptibility in lily breeding programs.

Red light imaging for programmed cell death visualization and quantification in plant-pathogen interactions.

Studies on plant-pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant-pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.