How to tame a parasite - Developing biotechnological pipelines for gene function studies in Cuscuta

This thesis is published on Munin with a CC BY license.Cuscuta is a genus of parasitic plants that form cross-species bridges and connect to the xylem and phloem of a wide variety of host plants. Because of their unique lifestyle, research into processes like the formation of connections in between plant cells and plant immunity would benefit from Cuscuta as a model system. However, a method to study gene functions in a Cuscuta species is still missing. Its development was therefore chosen as the goal of this thesis. In vitro culture techniques for Cuscuta campestris were developed and Agrobacterium transformation of Cuscuta tissue was successfully performed in two different systems, the adhesive disk area close to the parasitic infection organ, and seedlings of C. campestris. As an alternative method for gene function studies in Cuscuta, virus induced gene silencing was tested in C. campestris. Mineral transport across the host-parasite border was studied and the potential use of interspecies plasmodesmata between Cuscuta and its hosts was investigated. The results presented in this thesis allow for first gene function studies in Cuscuta and are good foundations for the development of pipelines for transgenic plant production and gene silencing techniques. Challenges and opportunities with this system are outlined. The conclusions of this thesis will help to “tame” Cuscuta and use its unique physique and strategies for host plant infection to establish a model species for studying plant-cell connections, transport processes and plant immunity

How to tame a parasite - Developing biotechnological pipelines for gene function studies in Cuscuta

Cuscuta is a genus of parasitic plants that form cross-species bridges and connect to the xylem and phloem of a wide variety of host plants. Because of their unique lifestyle, research into processes like the formation of connections in between plant cells and plant immunity would benefit from Cuscuta as a model system. However, a method to study gene functions in a Cuscuta species is still missing. Its development was therefore chosen as the goal of this thesis. In vitro culture techniques for Cuscuta campestris were developed and Agrobacterium transformation of Cuscuta tissue was successfully performed in two different systems, the adhesive disk area close to the parasitic infection organ, and seedlings of C. campestris. As an alternative method for gene function studies in Cuscuta, virus induced gene silencing was tested in C. campestris. Mineral transport across the host-parasite border was studied and the potential use of interspecies plasmodesmata between Cuscuta and its hosts was investigated. The results presented in this thesis allow for first gene function studies in Cuscuta and are good foundations for the development of pipelines for transgenic plant production and gene silencing techniques. Challenges and opportunities with this system are outlined. The conclusions of this thesis will help to “tame” Cuscuta and use its unique physique and strategies for host plant infection to establish a model species for studying plant-cell connections, transport processes and plant immunity

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

The parasitic plant genus Cuscuta is notoriously difficult to transform and to propagate or regenerate in vitro. With it being a substantial threat to many agroecosystems, techniques allowing functional analysis of gene products involved in host interaction and infection mechanisms are, however, in high demand. We set out to explore whether Agrobacterium‐mediated transformation of different plant parts can provide efficient alternatives to the currently scarce and inefficient protocols for transgene expression in Cuscuta. We used fluorescent protein genes on the T‐DNA as markers for transformation efficiency and transformation stability. As a result, we present a novel highly efficient transformation protocol for Cuscuta reflexa cells that exploits the propensity of the infection organ to take up and express transgenes with the T‐DNA. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary transformation vectors with reporter fluorochromes yielded high numbers of transformation events. An overwhelming majority of transformed cells were observed in the cell layer below the adhesive disk’s epidermis, suggesting that these cells are particularly susceptible to infection. Cotransformation of these cells happens frequently when Agrobacterium strains carrying different constructs are applied together. Explants containing transformed tissue expressed the fluorescent markers in in vitro culture for several weeks, offering a future possibility for development of transformed cells into callus. These results are discussed with respect to the future potential of this technique and with respect to the special characteristics of the infection organ that may explain its competence to take up the foreign DNA

A highly efficient protocol for transforming Cuscuta reflexa

The parasitic plant genus Cuscuta is notoriously difficult to transform and to propagate or regenerate in vitro. With it being a substantial threat to many agroecosystems, techniques allowing functional analysis of gene products involved in host interaction and infection mechanisms are, however, in high demand. We set out to explore whether Agrobacterium‐mediated transformation of different plant parts can provide efficient alternatives to the currently scarce and inefficient protocols for transgene expression in Cuscuta. We used fluorescent protein genes on the T‐DNA as markers for transformation efficiency and transformation stability. As a result, we present a novel highly efficient transformation protocol for Cuscuta reflexa cells that exploits the propensity of the infection organ to take up and express transgenes with the T‐DNA. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary transformation vectors with reporter fluorochromes yielded high numbers of transformation events. An overwhelming majority of transformed cells were observed in the cell layer below the adhesive disk’s epidermis, suggesting that these cells are particularly susceptible to infection. Cotransformation of these cells happens frequently when Agrobacterium strains carrying different constructs are applied together. Explants containing transformed tissue expressed the fluorescent markers in in vitro culture for several weeks, offering a future possibility for development of transformed cells into callus. These results are discussed with respect to the future potential of this technique and with respect to the special characteristics of the infection organ that may explain its competence to take up the foreign DNA

The enigma of interspecific plasmodesmata: insight from parasitic plants

Parasitic plants live in intimate physical connection with other plants serving as their hosts. These host plants provide the inorganic and organic compounds that the parasites need for their propagation. The uptake of the macromolecular compounds happens through symplasmic connections in the form of plasmodesmata. In contrast to regular plasmodesmata, which connect genetically identical cells of an individual plant, the plasmodesmata that connect the cells of host and parasite join separate individuals belonging to different species and are therefore termed “interspecific”. The existence of such interspecific plasmodesmata was deduced either indirectly using molecular approaches or observed directly by ultrastructural analyses. Most of this evidence concerns shoot parasitic Cuscuta species and root parasitic Orobanchaceae, which can both infect a large range of phylogenetically distant hosts. The existence of an interspecific chimeric symplast is both striking and unique and, with exceptions being observed in closely related grafted plants, exist only in these parasitic relationships. Considering the recent technical advances and upcoming tools for analyzing parasitic plants, interspecific plasmodesmata in parasite/host connections are a promising system for studying secondary plasmodesmata. For open questions like how their formation is induced, how their positioning is controlled and if they are initiated by one or both bordering cells simultaneously, the parasite/host interface with two adjacent distinguishable genetic systems provides valuable advantages. We summarize here what is known about interspecific plasmodesmata between parasitic plants and their hosts and discuss the potential of the intriguing parasite/host system for deepening our insight into plasmodesmatal structure, function, and development

Selective mineral transport barriers at Cuscuta-host infection sites

The uptake of inorganic nutrients by rootless parasitic plants, which depend on host connections for all nutrient supplies, is largely uncharted. Using X‐ray fluorescence spectroscopy (XRF), we analyzed the element composition of macro‐ and micronutrients at infection sites of the parasitic angiosperm Cuscuta reflexa growing on hosts of the genus Pelargonium. Imaging methods combining XRF with 2‐D or 3‐D (confocal) microscopy show that most of the measured elements are present at similar concentrations in the parasite compared to the host. However, calcium and strontium levels drop pronouncedly at the host/parasite interface, and manganese appears to accumulate in the host tissue surrounding the interface. Chlorine is present in the haustorium at similar levels as in the host tissue but is decreased in the stem of the parasite. Thus, our observations indicate a restricted uptake of calcium, strontium, manganese and chlorine by the parasite. Xylem‐mobile dyes, which can probe for xylem connectivity between host and parasite, provided evidence for an interspecies xylem flow, which in theory would be expected to carry all of the elements indiscriminately. We thus conclude that inorganic nutrient uptake by the parasite Cuscuta is regulated by specific selective barriers whose existence has evaded detection until now