Exploring the intersection between opto- and epigenetic networks in the parasitic vine Cuscuta

Plants integrate external signals in their internal regulatory networks and thus govern their reactions to different environments. One of the external signals is light, whose spectral quality plants can distinguish and react to differentially. For instance, plants can tell if they grow in direct sunlight or in the canopy shade of other plants based on the ratio of red (R-) and far-red (FR-) light. Unlike most plants that want to grow away from the shadow and towards the sun, the twining parasitic plant Cuscuta prefers the shadows, which to them is a sign that host plants are nearby that can be infected. Cuscuta’s most noticeable response to light is its coiling around the stems of plants. In R-light coiling is looser or absent which is insufficient to secure the parasite to its host and allow an invasion of the host stem. FR-light, in contrast, anchors Cuscuta in tight coils around the host stem, allowing it to infect the host subsequently. These different behavioral patterns offer potentially some eco-friendly possibilities using light of different color to control the unwanted proliferation of parasites in agricultural landscapes. Our project aims at elucidating how the light signals affect the way the genetic information is translated into the different reactions (optogenetics) and why the parasite has turned the common reaction to R-light into an opposite behaviour. We wanted to in particular investigate how the optogenetic regulation is linked with epigenetic modulation of gene expression. The molecules that absorb specific light colors (or wavelengths) are the chromoproteins. Plant chromoproteins responsible for the detection of R- or FR-light are called phytochromes. To document the kinetics of movement of the parasite under different light conditions, we created time-lapse films using a remotely controlled standard SLR camera. The obtained information that was gathered in this simple way was vital for the downstream experiments and later for their interpretation. Using LED light technology to apply R or FR light with varying intensities and duration of exposure we recorded the changes in the gene expression very accurately. To exclude, that other signals from a living host interfered with the light signaling, we replaced it with wooden sticks. This lowered the biological complexity of the study and enabled us to avoid interfering signal cascades. Our gene expression data showed clear indications that translation of the light signal into a phenotypic response is directly correlated to certain types of transcription factors (interacting proteins that are able to bind to DNA and activate genes) some of which have not been implied in this process before. A surprising result was also that Cuscuta appears to utilize members of the phytochrome protein family than non-parasitic plants do not commonly use, explaining why we have poor knowledge on them. The FR-treatment caused other changes in the methylation signatures on the DNA than the R-treatment, which we interpret indeed as a to date unknown involvement of an epigenetic layer in the parasite’s light responses. Some epigenetic signatures were found in the gene bodies and their promoters, others in neighboring mobile (transposable) genetic elements. Since epigenetic DNA methylation regulation is connected to small regulatory RNAs, their profile and accumulation in Cuscuta’s stems and infective tissue was also analyzed which confirmed that small RNA molecules related to some of the differentially regulated genes accumulated under the respective light condition. Investigations on the correlation with the observed epigenetic changes are ongoing. Alltogether, all data support a strong connection, or even crosstalk, between the regulatory networks involved in light perception and the epigenetic marks. Downstream of the FR-light induced coiling, the production of the infection organ, haustorium, is also regulated by light. Following leads that this actual invasion of the host is regulated by same light cues, we analysed patterns of gene expression in young, developing and mature haustoria to create a basis for comparative analyses of these two separate but functionally linked developmental processes. These studies showed that there are differences in the predominant transcription factors and their target genes and also in the regulatory microRNAs. They have also led to the discovery that the presence of a host as such and, moreover, the type of host have a strong modifying influence on the parasite, showcasing the complexity of the system and highlighting the validity of our initial approach to use a host-free system. In short, we have so far confirmed that Cuscuta offers a unique opportunity to understand how a disruption in cognitive functions in plants can lead to the neofunctionalization of molecules and the evolution of a new lifestyle.

The parasitic vine Cuscuta reacts to long wavelength far-red light with a rotating shoot movement, followed by coiling around host stems and rapid host infection while red light triggers only shoot elongation and results in a strongly decreased tendency to attack and infect host plants. The genome of Cuscuta campestris contains the red light receptor phytochrome, but lacks several important interacting transcription factors, suggesting that alternative, or possibly even novel, signal transduction pathways are behind this behavior. The project will study the molecular responses of Cuscuta to far-red and red light conditions in order to understand the mechanisms underlying the (far-)red light control of infection. Following a lead that points to the regulation of a component of the RNA-directed DNA methylation (RdDM) machinery by red or far-red light, mRNA expression profiles, small RNA composition and genome-wide DNA methylation signatures under red- and far-red-light illumination will be studied in Cuscuta shoots. The integration of these datasets with the phenotypic read-out (infection organ production or lack of infection organ) will help to identify novel key players of the infection process. In parallel, we will characterize in more detail the component of the RdDM machinery, IDN2, that showed light-responsive expression in preliminary studies. In situ hybridization will reveal whether the induction or repression of IDN2 gene transcription is cell- or tissue-type specific and limited to infective tissue. With the help of a reverse genetics approach using host-mediated virus-induced gene silencing, the effect of a loss of function of this gene on the virulence of the parasite will be studied.