Research group Albert
Plant-Plant Interaction of Cuscuta spp. with Host Plants

Research interests

The plasma membrane of plant cells represents a selective barrier for solutes, water, nutrients, exogenous and endogenous molecular cues and signals. Especially the latter are critical to initiate tissue- and cell-specific signaling as triggers which are specifically detected at the cell surface. How get extracellular signals perceived at the plasma membrane and how do the corresponding intracellular signaling programs get activated?

Membrane-bound receptor proteins and receptor complexes are critical to detect signals at the cell surface and to switch-on intracellular signaling cascades. Those receptors are able to recognize both, endogenous signals, e.g. phytohormones such as Brassinosteroide, and exogenous signals, e.g. microbe-associated molecular patterns which signalize danger and activate cellular defense programs in infected host plants. We focus on molecular cues, their perception via membrane-bound receptors and the activation of cellular signaling programs in the context of plant-plant interactions. We are generally interested in the…

 

…molecular mechanisms in plant-plant interactions

In addition to other biotic stresses, parasitic plants pose a further threat to plants and cause crop losses, worldwide. Plant parasites directly connect to the vasculature of host plants thereby stealing water, nutrients, and carbohydrates consequently leading to tremendously reduced biomass and losses in seed yields of the infected host plants.

The major goals of my research team are the elucidation of molecular mechanisms during the interaction of parasitic plants with host plants. Thereby we are following two major routes and try to address both phenomena susceptibility and resistance (see overview in Fig. 1). In susceptible interactions, the parasitic plant hooks up certain, yet unknown, plant developmental signaling pathways to finally abuse it by redirecting and withdrawing nutrients, water and carbohydrates. Up to date nearly nothing is known about how such connection is established while the infection stays unrecognized by the parasitized host.

Complementary, working with host plants that are resistant to an infection by a parasitic plant, we aim to understand this stress-avoidance on molecular level. Does plant resistance against parasitic plants function in a similar manner such as known for plant resistance against microbial pathogens?

In terms of applications, steps to understand the molecular mechanisms behind both susceptibility and resistance, together with the successes in recent breeding efforts will provide fundamental knowledge to further generate resistant crop plants.

 

The plant parasite Cuscuta spp. and its hosts

At present, we are mainly using Cuscuta spp. (Fig. 2) as a model plant parasite to addressing the questions raised above. Living as obligate holoparasites with a very broad host spectrum, plants of the genus Cuscuta possess neither roots nor expanded leaves. To survive, Cuscuta enwinds the stems of host plants and develops haustoria, establishing connections directly to the host’s vascular bundles. This haustorium penetration and accompanying wounding seems to go undetected by most host plants and the parasite succeeds in representing itself as an attractive sink by probably mimicking a part of “host-self”.

How can plants detect plants? We are interested in identifying signals and bioactive molecules on the parasite’s side that trigger visible or measurable responses in the host plant, for either plant development or for defense. On the host side, we study signaling and responses in the infested plant, focusing on perceptual systems that initiate downstream developmental signaling programs and control cellular responses.The chances and possibilities of hosts for defence may differ from those known for plant-microbe interactions. Given their significant evolutionary distance from microbes, over time all higher plants have established an innate immune system to defend against microbial pathogens. By means of pattern recognition receptors (PRRs), plants can sense so-called microbe-associated molecular patterns (MAMPs) and initiate defensive signaling programs that help the plant to restrict pathogen growth.

 

Studying resistance

A chance exception: Cultivated tomato is resistant to Cuscuta reflexa

The relationship between cultivated tomato (Solanum lycopersicum) and Cuscuta reflexa offers a convenient tool for gaining initial insights into how secreted parasitic signaling molecules might trigger a host plant reaction. Tomato is resistant to C. reflexa and actively responds with a clearly visible hypersensitive-like reaction precisely at the penetration site of C. reflexa haustoria (Fig. 3). This strong response leads to tomato cell-wall suberinisation and prevents the host from haustoria penetration, and consequently leading to the death of C. reflexa (Fig. 3). Such defensive reactions are unique to cultivated tomato and do not occur in other plants of the Solanaceae family.

In search for the parasite’s defense-triggering molecules, extracts of C. reflexa stem and haustoria were tested for their capability to trigger typical MAMP responses specific to tomato, such as ethylene production, oxidative burst, or the induction of marker genes.

The ethylene response seemed to work best in tomato but was absent in other related (non-resistant) plants of the Solanaceae (e.g., S. tuberosum, Nicotiana tabacum, or Nicotiana benthamiana), and so it was further used to screen Cuscuta-extracts for defense triggering molecules. Using stepwise purification of crude parasitic extracts by chromatography and monitoring of each purification step for its ability to trigger ethylene production, we purified a small Glycine-Cysteine-rich protein, which is the essential trigger of the ethylene response in cultivated tomato. This protein localizes to the cell walls of Cuscusta and can be found in all parts and organs of the plant. Probably, its original function is related to cell wall stability and flexibility, especially for the middle lamella. More detailed analyses are currently in progress.

How do such defense-triggering molecules get sensed by tomato? After finding a wild tomato species, Solanum pennellii, that is insensitive in the ethylene bio-assay, we screened a collection of ~50 introgression lines (S. lycopersicum x S. pennellii) (Eshed & Zamir, 1995) for ethylene response and mapped one essential locus: On chromosome eight we identified a gene encoding a Leucine-rich repeat receptor-like protein (LRR-RLP), which we termed Cuscuta Receptor 1 (CuRe1). After heterologous expression of the CuRe1 gene in N. benthamiana (usually insensitive to C. reflexa extracts), this plant started to respond by means of ethylene production when treated with crude C. reflexa extracts or with pure fractions of the glycopeptide. This Peptide detection occurred in a highly sensitive and dose-dependent manner. In addition, when infecting transgenic, CuRe1-expressing N. benthamiana plants with C. reflexa, plants were significantly more resistant to parasite invasions demonstrating that CuRe1 contributes to tomato’s resistance against Cuscuta. The first part of this project was published in Science (Hegenauer et al. 2016). A manuscript for the recently identified parasitic molecular pattern, termed as Cuscuta factor, is currently in preparation.

 

Future aims in short and long term

In addition to the locus on tomato chromosome 8, we mapped a second resistance-relevant locus on tomato chromosome 12. We identified a gene-cluster whose genes encode for potential resistance receptor genes. These receptor genes were cloned and are currently under in depth investigation. In comparison to CuRe1 which initiates typical responses of a pattern-triggered immunity, the second locus seems to be rather responsible for resistance comparable to effector-triggered immunity. These findings indicate a second layer of resistance present in tomato also known and described for plant-microbe interaction in the famous zig-zag-model by Jones and Dangl in 2006. Our recent findings as well as ongoing and future research will majorly contribute to create and to substantiate the bigger picture of host plant immunity against parasitic plants and to establish a model similar to the zig-zag model such as described for the interaction of plants with microbes (Jones and Dangl, 2006).

In the long term, we further aim to apply and to transfer our knowledge gained from the tomato-Cuscuta system to other parasitic plant – host systems. Understanding these mechanisms of immunity will deliver ground-breaking insights to create crops resistant to parasitic plant attacks.

 

Studying susceptibility

Screening for parasitic molecular cues and studying their effects to susceptible hosts

The recognition of C. reflexa as “alien” by CuRe1 and other receptors is an exclusive exception of tomato. Usually, the parasite stays unrecognized by its host and actively manipulates it towards susceptibility: parenchymal cells of the host’s vascular bundles start to divide, differentiate and finally connect to the parasitic cells of both Xylem and Phloem. What type of molecular cues initiate and regulate these mechanisms of susceptibility?

In a very recently DFG-funded project (AL 1426/4-1) and related preliminary work, we cloned promoters of host plant genes that are up-regulated in host plants during a Cuscuta infection. Using them as promoter::reporter gene (luciferase) constructs in plant systems, these constructs serve as tools to detect luciferase activity upon treatment with stepwise purified Cuscuta extracts and will finally help to identify new Cuscuta factors (CuFs) that manipulate host plant signaling in a manner of virulence factors. First results indicate the presence of different new CuFs which will be further characterized and identified.

In a complementary project we aim to identify the detection mechanisms for parasitic CuFs in host plants and want to gain knowledge about the susceptibility-related cellular signaling. We will use Arabidopsis receptor mutants for which collections are available in our laboratory. Mutant plants will be grown, infected with Cuscuta and further analyzed in regard to altered susceptibility. Subsidiary, we have diverse Arabidopsis accessions at hand (1001 Genomes project) that we will check in addition. Relevant genes of obtained phenotypes with disturbed susceptibility will be identified either by classical map-based cloning or by using Genome-wide association studies (GWAS).

Novel components of CuF-perception or related signaling components will be further analyzed with well-established methods such as studies on receptor-ligand interaction, localization, protein-protein interaction, etc. Furthermore, we will prove the biological relevance by determining the Cuscuta biomass after growth on mutants or transgenic plants. This will help to decipher the molecular and biological role of novel components required to establish a plant-plant interaction.

 

Future aims in short and long term

Which host plant signaling routes are part of the susceptible plant-plant interaction? Which host plant receptors are involved? Since the parasite is a plant as well, it has all the molecular tools and mechanisms to hook up the required host plant developmental programs and related perception mechanisms. In the long term, we aim to identify the required components such as phytohormones, proteinaceous and low-molecular signaling cues together with the related receptors and signaling cascades that may get sequentially activated in a spatial and temporal order during the long-lasting process of the plant-plant connection. Understanding susceptibility and related signaling will open new perspectives and may help to control parasite growth, e.g. by designing susceptibility-antagonizing substances which act as blockers of a parasite invasion.

In the long-term, it will be a further aim to study the parasite’s strategy of redirecting and withdrawing nutrients, solutes and carbohydrates when the parasite is properly connected to the host. How does the parasite increase the host’s photosynthesis rate, the carbon fixation, nutrient- and water-uptake? What can we learn from this and how could we use such mechanisms to maybe tune crop plants?