1. Regulation of germination and infection

For germination on hydrophilic surfaces (e.g. glass), B. cinerea conidia require organic nutrients such as sugars. On the plant cuticle, however, complete germination can occur also in distilled water. Thus, both chemical and physical signals (surface hardness and hydrophobicity) are germination inducers (Fig. 1). We have identified signal transduction components that are required for proper germination induction and pathogenic development, in particular the Ste11-Ste7-BMP1 MAP kinase pathway (Doehlemann et al., 2006; Schamber et al., 2010). With a microarray-based transcriptomics approach, genes were identified that are induced during germination and appressorium differentiation. Many of these genes encode lytic enzymes that are potentially involved in host cell wall penetration, and the majority of them are regulated by the MAP kinase BMP1 (Leroch et al., 2013). The signaling mucin Msb2 has been described in yeast and several plant pathogens as a surface sensor. For B. cinerea, we demonstrated that Msb2 is required for hard surface sensing by germ tubes and appressoria formation, and that it acts upstream of the BMP1 MAP kinase cascade (Fig. 2; Leroch et al., 2015). Our current studies address the role of BMP1-regulated genes during the infection process, and the identification of further signaling components required for pathogenesis.

Fig. 1: Signal transduction pathways leading to germination and infection in <i>B. cinerea</i>. The signaling components shown are described in Doehlemann et al. (2006) and Schamber et al. (2010).

Fig. 2: <i>B. cinerea </i>Msb2 is a hard surface sensor involved in appressorium differentiation. A: Development of wild type and <i>msb2 </i>mutant germlings on bean leaves. Due to lack of surface recognition, the <i>msb2 </i>mutant fails to form appressoria and forms long germ tubes. B: Infection test with detached, intact tomato leaves reveals no infection by the <i>bmp1 </i>mutant, delayed infection by the <i>msb2 </i>mutant, and wild type-like infection by the complemented <i>msb2 </i>mutant (<i>msb2</i>-C). C: Simplified scheme of Msb2, a highly glycosylated protein with a C-terminal transmembrane domain and a short cytoplasmic tail, and its connection with the BMP1 MAPK cascade (Leroch et al., 2015).

We have developed codon-improved GFP and mCherry-encoding genes as live-cell reporters to follow the infection process of B. cinerea with fluorescence and laser scanning microscopy, and to study the expression of infection-related genes (Fig. 3; Leroch et al., 2011).

Fig. 3: Use of codon-improved fluorescent proteins for live-cell imaging and analysis of gene regulation in <i>B. cinerea</i>. A: Mixture of two <i>B. cinerea </i>strains expressing GFP and the red fluorescent mCherry, forming infection cushions on glass. On the lower right, the strains have fused, showing mixed fluorescence B: Germinated conidia with multiple nuclei expressing GFP. C: GFP-labeled <i>B. cinerea </i>hyphae invading <i>Arabidopsis </i>epidermal cells. One cell has already collapsed. D: Activation of the <i>atrB </i>promoter in conidia of an atrB-GFP reporter strain by fungicides (cyprodinil, fludioxonil, fenhexamid, tebuconazol, but not azoxystrobin) and the phytoalexin camalexin (Leroch et al., 2011). E: Visualisation of <i>cutB </i>expresson on onion epidermis cells in a <i>cutB</i>-GFP reporter strain Leroch et al., 2013).

Publications

Leroch M, Mueller N, Hinsenkamp I, Hahn M 2015. The signaling mucin Msb2 regulates surface sensing and host penetration via BMP1-MAP kinase signaling in Botrytis cinerea. Mol. Plant Pathol, in press.

Hahn M, Viaud M, van Kan J 2014. The genome of Botrytis cinera, a ubiquitous broad host range necrotroph. In: Genomics of plant-associated fungi and oomycetes: Dicot pathogens, Dean RA et al. (eds.), Springer Berlin Heidelberg 2014, DOI: 10.1007/978-3-662-44056-8_2.

Leroch M, Kleber A, Silva E, Coenen T, Koppenhöfer D, Shmaryahu A, Valenzuela PDT, Hahn M 2013. Transcriptome profiling of Botrytis cinerea conidial germination reveals upregulation of infection-related genes during the prepenetration stage. Eukaryot Cell 12: 614-626.

Leroch M, Mernke D, Koppenhoefer D, Mosbach A, Doehlemann G, Hahn M 2011. Living colors in the gray mold pathogen Botrytis cinerea: Codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence. Appl Environ Microbiol 77: 2877-2897.

Schamber A, Leroch M, Diwo J, Mendgen K, Hahn M 2010. The role of mitogen-activated protein (MAP) kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea. Mol. Plant Pathol 11: 105-119

Doehlemann, G., Berndt, P and Hahn, M 2006. Different signalling pathways involving a Gα protein, cAMP and a MAP kinase control germination of Botrytis cinerea conidia. Mol. Microbiol 59: 821-835.