Regulation and cytological analysis of the early stages of pathogenic development of Botrytis cinerea
Final Report Abstract
The project aimed at the identification and functional analysis of genes that are regulated by physical and chemical plant signals and which are involved in early pathogenic differentiation of B. cinerea. Comprehensive transcriptome analysis identified genes that are induced by physical and chemical plant signals. These comprised many genes encoding secreted proteins. The mutant approach, in which several of these secreted and BMP1 dependent genes were analyzed for their function was unsuccessful. Only for one gene (Bcarp1) a strong phenotype and reduced virulence could be observed. The regulation of two cutinase genes which are induced during germination by plant lipids was investigated in more detail. While cutA expression is surface independent, cutB expression requires a hard surface. We were able to identify CTF1a as responsible transcription factor for cutA but not for cutB. In addition we could show that CTf1a as well as CTF1b are involved in fatty acid metabolization and peroxisomal function. Unfortunately the transcription factor screen for the hard surface dependent expression of cutB gave no result. Despite this we were able to identify and characterize the signaling mucin Msb2, which likely serves as a hard surface sensor of germlings and hyphae that triggers infection structure formation via activation of the BMP1 MAP kinase pathway. Except for Msb2, no further key regulators connected to BMP1 could be identified. Sho1 could be shown to have only a marginal defect in growth and infection, in contrast to the situation in other plant pathogenic fungi. Because the analysis of early differentiation did not promise major advances in the understanding of Botrytis pathogenesis, we did not continue this project further. The first false sho1 deletion mutants showed a phenotype similar to mutants of the VELVET complex. The mutants showed a normal penetration but a strongly reduced host invasion. We therefore decided to focus on these mutants in comparison to the VELVET complex mutants in order to get an access into the understanding of the later stages of host infection of B. cinerea. By comprehensive quantitative transcriptome and quantitative ‘on planta’ secretome analysis we were able to show that acidification of the host tissue is essential for successful infection and for the expression of virulence- related genes and genes encoding secreted proteins in B. cinerea. The downregulation of proteases in the mutants was correlated with incomplete degradation of cellular host proteins at the infection site, indicating that high protease activity of B. cinerea is required for rapid metabolism of host proteins. Our and other recent studies support a crucial role of in planta secreted proteins for necrotrophic pathogenesis of B. cinerea. To clarify these, the generation and analysis of multiple mutants would be required. For this goal we have recently established the CRISPR/Cas method in B. cinerea, which will be used for sequential inactivation of potential virulence factors.
Publications
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2013. Transcriptome profiling of Botrytis cinerea conidial germination reveals upregulation of infectionrelated genes during the prepenetration stage. Eukaryotic Cell 12:614-626
Leroch M, Kleber A, Silva E, Coenen T, Koppenhöfer D, Shmaryahu A, Valenzuela PDT, Hahn M
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2015. The signaling mucin Msb2 regulates surface sensing and host penetration via BMP1-MAP kinase signaling in Botrytis cinerea. Mol. Plant Pathol. 16:797-798
Leroch M, Mueller N, Hinsenkamp I, Hahn M
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2018. Investigations on VELVET regulatory mutants confirm the role of host tissue acidification and secretion of proteins in the pathogenesis of Botrytis cinerea. New Phytol. 219, 1062-1074
Müller N, Leroch M, Schumacher J, Zimmer D, Könnel A, Klug K, Leisen T, Scheuring D, Sommer F, Mühlhaus T, Schroda M, Hahn M
