Parasitic Molecular Strategies to Influence Host Plant Signaling and Gene Transcription

Abstract

Parasitic plants are a constraint on agriculture worldwide. Plants of the genus Cuscuta are obligate holoparasites with a broad host spectrum for nearly all dicotyledonous plants. As leaf- and rootless plants, Cuscuta spp. wind around stems of host plants and penetrate host tissue with haustoria. They directly connect to the vasculature and exhaust water, nutrients and carbohydrates. Thus, the haustorium development and the establishment of a connection to the host represent essential steps in the parasite’s life cycle. To date, little is known concerning the development of such host-parasite connections on molecular level. The aim of this work was to gain knowledge about specific molecular signals of Cuscuta spp. that get sensed by host plants and manipulate hosts towards susceptibility. On the host plant side, the major focus was to identify Cuscuta-derived transcription factor targets or receptors that recognize parasitic molecules and further induce cellular signaling programs related to susceptibility or development. To shed light on the transcriptomic reprogramming during the early stages of infection with Cuscuta spp., a transcriptome analysis by RNA sequencing after infiltration of Cuscuta Extract into N. benthamiana leaves has been performed. Additionally, an intense literature search with a focus on RNA sequencing data providing regulated transcripts of host and parasite genes was initiated. To investigate the molecular cues that might be necessary to switch-on host responses, intracellular processes and the connection to the host vascular system, promoters of upregulated host genes at the Cuscuta spp. infection site have been used to control the expression of the luciferase reporter gene. To establish a promoter:luciferase based bioassay to screen for inducing Cuscuta-derived molecular cues, a promoter was needed to control luciferase expression leading to significantly increased light emission after treatment with Cuscuta Extract. The promoter:luciferase construct of USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTER 25 (pUMAMIT25:luc) that showed a specific reaction to Cuscuta Extract was used to purify the Cuscuta-derived molecular cue. The purification and identification of the Cuscuta-derived molecule is part of an ongoing project. The molecule seems to be difficult to isolate since it did not bind to most of the tested chromatography columns. A characterization showed that the molecular cue had no charge, was hydrophilic and was not of proteidogenous nature. An in silico analysis of the host UMAMIT25 which seems manipulated by C. reflexa revealed its involvement in amino acid transport, that can be relevant for Cuscuta spp. nutrition and growth. Additional beneficial transporter genes like sucrose transporters were analyzed and the corresponding promoter:luciferase constructs showed increased light emission upon Cuscuta Extract treatment. The experiments of this work leave it open whether the molecular cue might be a sucrose gradient, a Cuscuta-derived CLE peptide, a Transcription activator-like (TAL) effector-like or a still elusive molecular trigger.