This proposal is focused on examination of two plant interactions: parasitic with Orobanche, and symbiosis with arbuscular mycorrhiza fungi (AMF), and the involvement of a newly define plant hormones, strigolactones (SLs), in these plant interactions. In addition to strigolactones role in regulation of above-ground plant architecture, they are also known to be secreted from roots, and to be a signal for seed germination of the parasitic plants Orobanche. Moreover, secreted strigolactones were recognized as inducers of AMFhyphae branching. The present work was aimed at Generation of RNAi mutants of both tomato and Medicago, targeting multiple genes that may be involved in strigolactone production, carotenoid biosynthesis pathway, Pi signaling or other metabolic pathways, and hence affect AMF colonization and/or Orobanche resistance. Following the newly formed and existing RNAi mutants were examined for AMF colonization and Orobanche resistance. At the first phase of this project Orobanche seed germination assays and AMF colonization were examined in intact plants. These assays were shown to be effective and resulted with enhancement of Orobanche seed germination and AMF colonization in WT tomato plants, whereas roots of strigolactones impaired lines did not result with Orobanche seed germination and mycorrhiza colonization. Unexpectedly, root organ cultures (ROC) that were produced from the same wild type (WT) and mutant lines did not induce the Orobanche seed germination and AMFhyphal branching. This implies that under in vitro conditions ROC cultures are missing an important component for induction of Orobanche seed germination and AMFhyphal branching. In another line of experiments we have tested transgenic lines of Medicagotruncatula for AMFhuyphal branching and Orobanche seed germination assays. These lines included lines silenced for a GRAS transcription factor (RNAi 1845), an NBS-LRR type resistance gene (RNAi 1847), a kinase (RNAi 2403) and a protein of unknown function (RNAi 2417). In all cases, five independent transgenic root lines showed altered AMFphenotypes with reduced or aberrant colonization patterns. Following, we transformed tomato plants with the M. truncatulaTC 127050 PhosphoinositidekinaseRNAi construct. Transgenic lines that contained GUS constructs were used as control. All transgenic lines showed reduced level of Orobanche seed germination, masking any strigoalctones-specific effect. The research demonstrated that SLs production may not be examined in ROC –based bioassays. It was shown by the 3 independent assays employed in this project that none of the recognized characters of SLs may be reflected in these bioassays. However, when the whole plant root exudates were examined, SLs activity in root exudates was demonstrated. Hence, it can be concluded that the presence of an intact shoot, and possibly, shoot factors, may be necessary for production of SLs in roots. Another point of interest that rises from these results is that the presence of SLs is not necessary for AMF completion of life cycle. Hence, it may be concluded that SLs are important for AMFhyphal branching, before symbiosis, but not essential for AMF colonization and life cycle completion under ROC system conditions.
Targeting of Strigolacatones Associated Pathways for Conferring Orobanche Resistant Traits in Tomato and Medicago