Our research group investigates proteins or protein families in plants that contribute to polar cell growth and act at the cell surface during the interaction between male and female gametes. In addition, our research focuses on the regulatory role of miRNAs and Argonaute proteins in plant reproduction.
To address questions related to our research topics we established protocols to isolate viable gametes from different flowering plant species, including wheat, Arabidopsis thaliana, maize, and Amborella trichopoda (Sprunck et al., 2005; Leljak-Levanić et al., 2013; Englhart et al., 2017; Chen et al., 2017; Flores-Tornero et al., 2019). From these cells, we performed transcriptomics and small RNAseq to identify candidate genes and small non-coding RNAs that are subsequently tested in functional studies.
Currently, we are investigating the molecular function of a number of protein families in the model plant Arabidopsis thaliana. For example, a small protein family involved in the establishment and maintenance of polar cell growth (Gebert et al., 2008; Kulich et al., 2020) is being investigated to clarify its regulation and to identify potential interaction partners. Other examples are miRNAs and proteins thought to be involved in egg cell specification, and a family of small, secreted cysteine-rich proteins involved in the interaction of gametes.
Methods
We are using a wide range of state-of-the-art methods related to (plant) molecular biology, molecular genetics, cell biology and protein biochemistry. Examples are:
DNA work (DNA isolation, conventional and restriction enzyme independent cloning methods (Gateway®- & Gibson-Assembly-based cloning), PCR, real-time quantitative PCR, PCR-mediated mutagenesis, RNAi, amiRNA, CRISPR/Cas9, Southern Blot, Southwestern Blot, cDNA libraries, etc.)
RNA work (total RNA and mRNA isolation, small RNA isolation, cDNA synthesis, RT-PCR, In vitro-transcription, DIG labeling, In situ hybridization, Northern Blot, etc.)
Gene expression studies (Microarrays, RNAseq, RT-PCR)
Protein work (protein extraction, PAGE, 2D-PAGE, Western Blot, immunoprecipitation, protein phosphorylation, whole-mount immunolocalization, recombinant protein expression, affinity purification, Size Exclusion Chromatography, Äkta pure system, etc.)
Interaction studies (Y1H and Y2H screens, EMSA, Co-immunoprecipitation, etc.)
Microscopy (fluorescence microscopy, DIC microscopy, Confocal Laser Scanning Microscopy, Spinning-disc live-cell imaging, immunofluorescence, etc.)
Transformation of bacteria and yeast
Plant transformation (Agrobacterium-mediated and biolistic) and genetic segregation studies
Plant cell culture (Arabidopsis, wheat, maize, Physcomitrium patens)
Micromanipulation (single cell isolation)