Signals and PerCEPtion: diverse roles for C-terminally encoded peptides in symbiosis, root system gravitropism, and pathogen response in Medicago truncatula and Arabidopsis thaliana

Abstract

Plant peptide hormones form part of complex signalling pathways, allowing plants to respond and adapt to biotic and abiotic stresses. This thesis discovers new roles of the C-TERMINALLY ENCODED PEPTIDE (CEP) family and its receptor in the control of nodule number, root gravitropism, and pathogen responses in the model plants, Medicago truncatula (Medicago), and Arabidopsis thaliana (Arabidopsis). Legumes procure nitrogen through a rhizobium-legume symbiosis resulting in specialised root organs called nodules, however, nodule number is controlled tightly to balance the cost of supporting rhizobia with the benefits of nitrogen fixation. The role of Medicago CEP7 in increasing root nodule number is defined here. Unlike other characterised Medicago CEP genes, which are controlled by low nitrogen levels, CEP7 expression requires an intact symbiosis signalling pathway and occurs during rhizobial infection and nodule formation. Using mass spectrometry, I identify the mature in vivo structure of the predominant peptide product of CEP7 (SymCEP7) and demonstrate that its novel hydroxylation pattern is crucial for the positive effect on nodule numbers without inhibiting lateral root number. SymCEP7 interacts with the receptor, COMPACT ROOT ARCHITECTURE 2 (CRA2), in shoot vasculature and increases nodule number when applied to shoots at pM to nM levels, which demonstrates a higher potency for CEPs than previously described. CEP7 is functionally and phylogenetically conserved in other legumes. A different Medicago CEP, CEP1, controls lateral root gravitropism and intersects with auxin production and transport through interactions with the receptor CRA2. cra2 mutants have steeper-angled lateral roots like mutants affected in auxin transport and gravitropism. Physiological approaches demonstrate that CEP1 inhibits lateral root and primary root gravitropism, while cra2 exhibits exaggerated gravitropism. Grafting shows that shoot localised CEP1-CRA2 interactions control lateral root gravitropism. Like CEP7 in controlling nodule number, CEP1 applied to shoots inhibits lateral root gravitropism in the pM to nM range but requires uM range concentrations to inhibit lateral root number via root localised signalling. cra2 mutants also accumulate more shoot auxins and transport more auxin to roots. Consistent with this, CEP1 inhibits shoot-to-root auxin transport in wild type plants. Adding synthetic auxin to wild type shoots mimics the steep cra2 phenotype, while auxin transport inhibitors rescue the cra2 steep-rooted phenotype to wild type levels. The increased auxin transport may explain the steeper root system architecture of cra2. Finally, RNA-Seq analysis of CEP receptor mutants in Medicago and Arabidopsis shows a significant reduction in transcription of three genes involved in the systemic acquired resistance (SAR) response in cra2 and cepr1. I show that CEP receptor mutants are more susceptible to bacterial and fungal pathogens in Arabidopsis and Medicago due to impaired SAR. CEPs stimulate SAR defence mechanisms, which increase pathogen resistance. Overall, this thesis reveals new roles for CEPs in the systemic control of root nodule number, root system architecture, and systemic pathogen defence and demonstrates that CEP-CEP receptor signalling plays a broader role in plant development and defence responses. This thesis also proposes new CEP-CEP receptor downstream targets that may mediate these diverse responses. Show more