Molecular signals in and from parasitic plants

• Applicant: Dr. Thomas Spallek
• Subject Area: Organismic Interactions, Chemical Ecology and Microbiomes of Plant Systems
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 424122841

The Orobanchaceae family is with more than 2100 described parasitic species one of the globally most widespread lineages of parasitic plants. Infection occurs through a root-borne nutrient withdrawing organ, the haustorium, and reduces host growth. In natural ecosystems, suppression of dominant hosts results in more biodiversity. In agriculture, it causes yield losses. However, only a few Orobanchaceae species are weeds on crops, while more than 85% of Orobanchaceae parasites are classified as euphytoid, meaning they resemble “true” (non-parasitic) plants and perform photosynthesis. Phtheirospermum japonicum (hereafter Phtheirospermum) is one of these euphytoid parasites and emerged as a model system to study parasitism-related gene functions. Research on Phtheirospermum focused on early infection stages so that parasitism in its stricter sense, the withdrawal of nutrients and water, remains elusive. For this reason, I started 2019 a project aiming at “Elucidating the genetic basis of parasitism in the parasitic model plant Phtheirospermum japonicum”. One cornerstone of the project was to screen a mutagenized Phtheirospermum population for mutants that, following successful infection, failed to benefit from parasitism. Screening over 12,000 Phtheirospermum M2 plants resulted in the isolation of four dalton (did not alter growth on host plants, dal1-4) mutants. A bulk-segregant analysis combined with whole-genome sequencing revealed three candidate mutations for dal2. To further narrow down the causal dal2 mutation, I propose the genetic complementation with wild-type loci and generating additional CRISPR/Cas9-induced loss-of-function mutants of the three candidates. Characterization of the dal2 parasitism defect will include quantifying the solute transport from host to parasite and the transcriptome analysis of mature dal2 and wild-type haustoria. In addition, the remaining dalton mutants will be genetically investigated. The second cornerstone of my project addressed the role of small peptides in the inter-specific communication between host and parasite. We have established that a subtilisin-like protease expressed in haustorium tips cleaves peptide precursors in vitro to release the bioactive peptide. The mature peptide was recognized by the host and the parasite. This resulted in reduced host root growth and increased pre-haustoria formation in the parasite. Combined peptide treatments with a known haustorium-inducing factor showed a synergistic effect. I propose determining the role of the peptide in loss-of-function mutants, by RNA-sequencing, and by identifying cognate receptors. Immunolabeling of the peptide during infection is another objective of the proposed research plan. The two-tier approach of forward and reverse genetics has already brought to light novel regulators of parasitism and promises further discoveries with the cloning of the dalton mutants and the characterization of the peptide activity during parasitism.

• DFG Programme: Research Grants