Periodic Reporting for period 1 - DEF2DEV (Identification of the mode of action of plant defensins during root development and plant defense responses.)
Reporting period: 2019-08-16 to 2021-08-15
Fungal plant pathogens are a major threat to food production worldwide. For example the necrotrophic fungal pathogen Botrytis cinerea can infect over 200 plant species, causing grey mould disease, leading to annual losses of up to $100 billion. Plant defensins (PDFs) constitute a unique class of highly stable antimicrobial peptides and are found throughout the whole plant kingdom. They were originally isolated in the host lab based on their antifungal activity and are regarded as being an important part in plant innate immunity responses directed against fungal pathogens. The antifungal mode of action of PDFs is partly regulated through interaction with a fungal membrane target, such as phospholipids and sphingolipids. Data indicate that their role is not restricted to antimicrobial peptides in the defense response, but also includes important biological functions, such as root development, nodule formation and reproduction. Surprisingly, despite the broad international interest in plant defensins, their mode of action in plant defense responses is still unclear as well as their possible involvement in (other) biological functions.
The project aimed to unravel the biological function and mode of action of the plant defensin AtPDF2.3 and its closest homologue AtPDF2.2. The overall objectives were to find more insights and answers about their mode of action and biological functions, being: (1) their role during root development, (2) their role during plant defense (3) their mode of action in plants and (4) their antifungal mode of action.
The studies in the model plant A. thaliana offer the opportunity to understand both the molecular and physiological mechanism involved in responses of plants to biotic stress. The knowledge obtained will be useful for improving tolerance through genetic engineering and breeding. The envisioned long term goal of this project is to identify novel strategies to cope with biotic stress and regulation of root architecture to be applied on important agricultural crops.
The project aimed to unravel the biological function and mode of action of the plant defensin AtPDF2.3 and its closest homologue AtPDF2.2. The overall objectives were to find more insights and answers about their mode of action and biological functions, being: (1) their role during root development, (2) their role during plant defense (3) their mode of action in plants and (4) their antifungal mode of action.
The studies in the model plant A. thaliana offer the opportunity to understand both the molecular and physiological mechanism involved in responses of plants to biotic stress. The knowledge obtained will be useful for improving tolerance through genetic engineering and breeding. The envisioned long term goal of this project is to identify novel strategies to cope with biotic stress and regulation of root architecture to be applied on important agricultural crops.
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.
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.
Despite the broad international interest in plant defensins, their mode of action in plant defense responses is still unclear as well as their involvement in (other) biological functions. To the best of our knowledge, no other groups are studying in detail the molecular mechanisms of PDFs. The difficulties in unravelling the function of PDFs that we have encountered might be one of the reasons. Nevertheless this project has gathered some interesting data. The expression profiles of PDF2.2 and PDF2.3 were never before studied in such detail within the root. In addition it is the first time that PDFs/PDFLs have been linked to lateral root development. It will be interesting to test this further. The differences in antimicrobial activities between PDF2.2 and PDF2.3 is an unexpected result and it will be highly interesting to use this in the identification in the mode of antimicrobial action.
In addition the project had an important impact on the molecular biology research within the host group. The fellow transferred a large amount of knowledge related to plant molecular biology by optimizing protocols and suggesting new and improved equipment.
In addition the project had an important impact on the molecular biology research within the host group. The fellow transferred a large amount of knowledge related to plant molecular biology by optimizing protocols and suggesting new and improved equipment.