In this 2 year project the potential role of PDF2.2 and PDF2.3 in plant defense responses and root development was investigated. Within the time frame of this project, 75% fell within the Covid-19 crisis, which had a large impact on the work plan and therefore not all objectives could be achieved. To complement the research an additional PDF-like peptide was investigated on its antimicrobial function and its function during root development.
Several tools were generated for the functional characterization of the candidate genes. Recombinant peptides for both were heterologous produced using the Pichia pastoris system. For in planta characterizations, different transgenic lines for both genes were generated, among which transcriptional and translational reporter lines, constitutive and inducible overexpression lines. In addition CRISPR-Cas lines were created carrying mutations in either PDF2.2 or PDF2.3 (single mutants), or in both (double mutants). This was the first time that in the host lab CRISPR-Cas lines were generated. CRISPR-Cas mutants will increasingly gain more importance as a gene-editing tool, the establishment of this protocol, will benefit the lab in their further research topics.
Functional analysis of AtPDF2.2-2.3 in root development and plant defense:
The influence of both defensins in plant defense was investigated by the use of different pathosystems. One of the main problems was the high variability in the outcome of infections assays, as the progression of the infection is highly dependent on environmental conditions. Therefore additional read-out protocols were generated, based on qPCR mediated detection of pathogen DNA relative to Arabidopsis DNA. This increased the sensitivity of these plant-pathogen infection assays. No strong resistant phenotype could be attributed towards PDF2.3 and PDF2.2 transgenic lines.
Analyzing the expression patterns of both PDF2.2 and PDF2.3 exhibited specific and different expression profiles. Interesting was the association of both PDF2.2 and PDF2.3 with the pericycle, a cell type that is highly associated with lateral root initiation. However our results could not reveal a specific function for PDF2.2 and/or PDF2.3 during root development. Given the association of other PDFs towards abiotic stress, it is possible that their function only becomes relevant under certain environmental conditions. Further investigation is needed to support this hypothesis.
Identification of AtPDF2.2-2.3 antifungal mode of action:
Recombinant produced PDF2.2 and PDF.3 were analyzed on their antimicrobial activity. It could be concluded that although PDF2.2 and PDF2.3 shared some but not all antimicrobial activities. The results of these studies provide an interesting starting point to unravel the specific site of action within these peptides.
A new PDF-like (PDFL) gene with a possible function during lateral root development:
PDFL Bcin3 was selected because of its strong expression in the root system and high upregulation upon B. cinerea infection. Recombinant generated peptide did not exhibit any antimicrobial activity, and no increase plant disease resistance could be detected so far in any of the transgenic lines. However the expression of this peptide coincides with the initiation of lateral root development. In addition overexpression lines exhibit an increase in lateral root primordia density in comparison to control plants.
Exploitation and dissemination
Results generated in the course of the project have been discussed during local seminars and scientific meetings (e.g. group, institute and division seminars). The presentation to a broader to a broader scientific audience during national and international conferences was not possible due to the Covid-19 crisis.
The unclear results of PDF2.2 and PDF2.3 towards plant defense responses and root development do not allow for dissemination of these results. Therefore further experiments are planned in order to understand the major role of this peptides. The obtained results will form the basis of a follow up project.
