Final Report Summary - OROBANCHE (GENOMICS-BASED INVESTIGATIONS INTO OROBANCHE-HOST INTERACTIONS) Parasitic weeds are among the most destructive and difficult-to-control of all weeds. The main reason for this is that these parasites have the ability to attach themselves to the vascular system of another plant in such a way that the two plant species become physically and physiologically linked. Parasites of the Orobanchaceae attach to host roots, growing undetected underground while they extract host nutrients and deplete the host ability to yield fruit. This is one of the most extreme and interesting examples of plant-plant interaction and is only beginning to be understood at the molecular level. In terms of agricultural impact, effective means to selectively control the various species of parasitic weeds such as Orobanche aegyptiaca and Striga hermonthica are still scarce or lacking despite intense scientific research. The parasite’s complete dependency on the host, and their perfect coordination with host signals for their development, suggests that these highly specialized parasites should be susceptible to novel control strategies that take advantage of the molecular bases of this sophisticated parasitic machinery. However, until now the key processes involved in parasite-host interactions remain poorly understood. The overall objective of research is to increase understanding of the molecular biology of parasitic plant host invasion process in order to generate new approaches to their control. Specific objectives included the characterization of Orobanchaceae gene expression during parasitism, localization of the parasite gene expression and characterization of mRNA movement between hosts and parasitic weeds and study of defense mechanisms in legumes against parasitic weeds. Monica Fernández-Aparicio addressed these objectives while working in the Westwood laboratory at Virginia Tech during the outgoing phase and at IAS-CSIC during the return phase, with an authorized stay at the University of Virginia during this second period. An important element of this research was that it coincided with a key phase of the Parasitic Plant Genome Project (PPGP), a collaborative project that was coordinated by Virginia Tech, and included research teams from the University of Virginia, Penn State University, and the University of California at Davis. The objective of this project was to create the first large scale database of transcriptome data from three related parasite species, Orobanche (syn. Phelipanche) aegyptiaca, Striga hermonthica, and Triphysaria versicolor, which represent holoparasitism, obligate hemiparasitism, and facultative hemiparasitism, respectively. Monica Fernández-Aparicio joined the PPGP during the stage when RNA samples were being generated for sequencing. In terms of contributing to the PPGP project, her work focused on generating protocols, tissue and RNA purification for sequencing of parasitic plant stages during haustorium development and host root penetration process. She developed aseptic cultures of P. aegyptiaca haustorial induced seedlings, the stage in which seedlings are in the process of haustorial induction, she developed early established P. aegyptiaca-host and Striga hermonthica-host connection and prepared for laser micro dissection. The resulting data is deposited for public use on the PPGP website, (http://ppgp.huck.psu.edu). In addition, no existing protocols for genetic transformation existed before this project for weeds belonging to Orobanche and Striga genera. These parasitic weeds do not grow as a normal seedling but his independent development is reduced to a few millimeters long infective radicle in which the haustorium will differentiate in order to attach and connect with the host vasculature. Without host conection the seedling inevitable will decay in few days. The rapid expiration of the haustorial competent stage in Orobanche and Striga seedlings and their permanent connection with the host afterwards, constituted a difficulty on the generation of transformation protocols in the lab. To this end, Monica Fernández-Aparicio developed systems for genetic transformation of O. aegyptiaca (outgoing phase) and S. hermonthica and Striga gesnerioides (return phase) that used in vitro parasitic root cultures as a starting point. Genetic transformation of the parasitic tissue was performed using Agrobacterium rhizogenes. Another critical step that she successfully addressed was the regeneration of lateral parasitic haustoria by inoculating transgenic parasitic roots onto host plants growing in rhizotron system. This system allows functional characterization of genes required for development of haustorium. Due to the haustorium constitutes the key feature in the parasitic plant life cycle the knowledge of essential genes for its development could contribute to the design of new control measures for parasitic plants. In addition to the above activities, she participated in the PPGP’s team by attending meetings and discussions, training opportunities, and screening for potential host and microbial contaminations of the assembled transcriptomes The existence of the PPGP sequence database enabled a number of other research directions. Among the first interesting genes discovered in the database were those related to germination signals. The parasites Orobanche and Striga only undergo seed germination after stimulation by specific chemical compounds secreted by host roots. Studies were initiated to characterize parasite ability to synthesize and perceive these signals. The main class of germination stimulants are strigolactones, which are plant hormones that control plant architecture and function in rhizosphere signalling. Research revealed that specific strigolactones act singly or in combinations to stimulate different Orobanche species, necessitating that parasites have a sophisticated system for strigolactones detection. This project initiated the process of characterizing putative strigolactones receptors in parasitic weeds. These genes have been sequenced from a wide range of Orobanche species with the goal of correlating differences in gene structure with differing sensitivities to strigolactones combinations. Finally, the sequence data revealed that Orobanche and Striga appear to contain copies of all genes currently known to act in strigolactone synthesis and perception. The strigolactone gene transcripts appear to encode functional proteins and are co-ordinately regulated during life stages of the parasites. These results suggest that the germination trigger is more complicated than a simple presence or absence of strigolactones. In addition to approaches derived directly from the availability of new sequences, Monica Fernández-Aparicio pursued other approaches to characterizing molecular host-parasite interactions. A proteomic study was followed to identify a) proteins secreted by the haustorial competent- in vitro grown O. aegyptiaca tubercle when exposed to the aseptically grown Arabidopsis roots, identifying a small set of proteins that could present the potential to manipulate processes in their host plants promoting either infection or trigger defense reactions; and b) identification of a set of 150 proteins from O. aegyptiaca seedlings with differential abundance when exposed to host root exudates (containing haustorial induced factors), non-host-derived root exudates, and absence of host. Another outcome of the project has been the identification of a horizontal gene transfer event from a legume ancestor to O. aegyptiaca. This is among the more intriguing cases of horizontal gene transfer in plants because the transferred gene, an albumin 1 KNOTTIN-like protein gene, is related to anti-insecticidal proteins so its acquisition from a host may carry a fitness benefit. The gene appears to be expressed and function in O. aegyptiaca and homologs were sequenced from several related species, suggesting that the gene has been retained through several speciation events. Regarding legume defense against parasitic plants, the molecular study of individual components of resistance was studied in the interaction between the legume plant cowpea and Striga gesneriodes. Gene silencing was conducted on the cowpea peroxidase, cationic peroxidase, narbonin, small heat shock protein, transcriptional activator-GRF, aquaporing NIP1 and lipoxygenase genes and its effects screening for modulation of the hypersensitive response developed by the resistant cowpea cultivar B301 in response to infestation by S. gesnerioides race 3. In addition the interaction of RSG3-301 immune receptor with RIN4 was tested using yeast two hybrid assay. In summary, the research project has contributed multiple new insights into the parasitic plants and their hosts. The work conducted as part of this project has helped to transition the field of parasitic plant research from one of limited options to one where the full range of modern scientific approaches in genomics and molecular biology are available to researchers. We expect that this work will contribute to an acceleration in the pace of scientific discovery, and hence the development of more effective parasitic weed control strategies.