Light is one of the most important signals that give plants information about the environmental conditions they are exposed to. Light consists of a spectrum of wavelengths that plants can distinguish between and react to differently. 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.
So-called chromoproteins called phytochromes have the ability to absorb specific light colors (or wavelengths) and are thereby able to analyze and indicating to the plant where which kind of light is coming from. The quality of light also determines whether Cuscuta is able to infect a host plant or not. These different behavioral patterns offer potentially some eco-friendly possibilities using wavelength filters of different color to control the unwanted proliferation of parasites in agricultural landscapes. However, this requires that we understand how the perception of light works in a cell and what molecular basis is behind the observed differences between parasitic Cuscuta and a normal plant. Our project aimed at elucidating how the light signals affect the way the genetic information is translated into the different reactions (optogenetics) and what makes the parasite display an opposite behaviour to light. Based on results from preliminary experiments, the optogenetic regulation is possibly linked to a change in the genetic material itself (epigenetic modulation of gene expression. We wanted to particularly investigate how the optogenetic regulation is linked with epigenetic modulation of gene expression.
Time-lapse films using a remotely controlled standard SLR camera helped us to document the kinetics of movement of the parasite under different light conditions, which way was vital for planning the downstream experiments and later for their interpretation. Using LED light technology we mimicked different scenarios for the parasites and recorded their effects on the gene expression in the parasite. Because a living host with its own responses was found to manipulate the gene expression responses from the parastie, we used wood sticks as “host dummies”. This lowered the biological complexity of the study and enabled us to avoid false conclusions from 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 (key 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 other members of the phytochrome protein family than non-parasitic plants typically do. FR- and R-light treatment caused different changes in the methylation signatures on the DNA, which we interpret as a hitherto unknown involvement of an epigenetic control 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, our data support a strong connection, or even crosstalk, between the regulatory networks involved in light perception and the epigenetic marks. We have confirmed that Cuscuta is a unique model for understanding how a disruption in cognitive functions in plants can lead to the neofunctionalization of molecules and the evolution of a new lifestyle. In the future, we hope to be able to disrupt the infection pattern by knocking out individual epigenetic pathway proteins and thus prove the convergence of the different regulatory pathways.
The project has generated 1. comprehensive data on the molecular changes, including mRNA profiles, small RNA profiles and DNA methylation profiles accompanying infection organ development under haustoriogenesis-promoting or preventing illumination schemes; 2. a list of genes whose products are tentative key factors to the differential red/far-red light responses (including putative novel PIFs); 3. a list of small RNAs regulated by R-/FR-light whose apparent involvement in Cuscuta infection makes them future candidates for manipulation of virulence. These novel datasets and findings are presented in a publication that is being revised for publication. Our data support the possibility that DNA methylation links to light perception mechanisms in the parasitic genus Cuscuta. To make up for the shortcoming of not being able to genetically modify Cuscuta, we investigated a range of species from different regions and could document some variation in this genus with respect to the far-red light dependence of haustoriogenesis. This has led to the generation of further genomic and transcriptomic resources that will benefit the parasitic plant society once they have been published.
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.
