Anatomy and functions of LTP interactomes and their relationship to small RNA signals in systemic acquired resistance
Lead Research Organisation:
University of Warwick
Department Name: School of Life Sciences
Abstract
This proposal focuses on a unique form of whole-plant immunity called systemic acquired resistance (SAR). SAR is induced when localized primary infection by microbial pathogens results in the generation of systemically transported mobile signal(s), which prepare the uninfected parts of the plant against future infections by a broad spectrum of pathogens. The mechanisms underlying SAR are remarkably complex and while early studies focused on SAR where the immunizing challenge initiated a classical gene-for-gene interaction, SAR has also been reported following challenges with non-pathogenic or virulent pathogens. Comparative studies are further confounded by different pathosystems and growth conditions. Founded on the assumption that SAR is regulated by universal key signal initiation processes, this proposal addresses exciting new discoveries and deploys new tools to unravel the early mechanisms of SAR induction by classical plant disease resistance protein recognition.
The SAR signal generation and transport occur within a very short and early time frame of 3-6 h after primary infection although the infected leaf appears to continue generating and transmitting the signal over time. This graft transmissible signal(s) moves throughout the plant, probalby through the phloem in a predominantly acropetal manner. Additionally, the mobile signal either comprises proteinaceous component(s) or requires them for movement/functionality. In summary, to qualify as the mobile signal(s), a biomolecule must be essential for SAR, must be physically transported to distal tissue within 4 h of primary infection, and must induce systemic resistance when applied in a localized manner. While the identity of a specific mobile signal remains unresolved, it appears to be conserved between monocots and dicots and, importantly, induces immunity to a diverse collection of pathogens and pests. To date numerous SAR-inducing chemicals, some of which exhibit physical mobility or are considered mobile due to their volatile nature, have been identified. These include, salicylic acid (SA), and its derivative methyl SA (MeSA) azelaic acid (AzA), [glycerol-3-phosphate (G3P), dehydroabietinal (DA), reactive nitrogen and oxygen species, and N-hydroxy pipecolic acid (NHP) amongst others. These chemicals confer systemic resistance when applied exogenously and are required for pathogen-induced SAR.
In recent breakthroughs, and underpinning this collaboration we have (i) developed a novel SAR luciferase reporter to provide spatial-temporal context to SAR establishment and (ii) identified two phased 21 nucleotide RNA (tasi-RNA) derived from Trans-Acting Small Interfering RNA3a (TAS3a) as essential for SAR. Based on their time frame of synthesis (3 hpi) post and systemic movement (4 hpi) we propose that tasi-RNAs function as the elusive early mobile SAR signal. Tasi-RNAs positively regulates the expression of genes encoding the previously identified lipid transfer protein (LTP)-like SAR regulator AZI1 (azelaic acid induced 1), as well as the LTP3, LTP4 and critically A70. LTPs regulate systemic transport of tasi-RNAs and based on the observed systemic mobility of LTPs, their interactions with AZI1, the detection of high molecular weight (HMW) complexes comprising AZI1 and presence of tasi-RNA in AZI1 immunoprecipitates we propose that SAR induction is associated with LTP-containing HMW protein complex-mediated systemic transport of tasi-RNAs. This proposal will characterize the LTP-RNA interactome and its relationship with A70 using computational modeling and functional analyses.
Finally, using these insights we will generate the first comprehensive analysis of the metabolic reconfiguraiton underpinning early SAR events.The knowledge gained here will be important for developing a basic understanding of this unique form of immunity and facilitate its use in developing sustainable and environmentally friendly crop protection strategies.
The SAR signal generation and transport occur within a very short and early time frame of 3-6 h after primary infection although the infected leaf appears to continue generating and transmitting the signal over time. This graft transmissible signal(s) moves throughout the plant, probalby through the phloem in a predominantly acropetal manner. Additionally, the mobile signal either comprises proteinaceous component(s) or requires them for movement/functionality. In summary, to qualify as the mobile signal(s), a biomolecule must be essential for SAR, must be physically transported to distal tissue within 4 h of primary infection, and must induce systemic resistance when applied in a localized manner. While the identity of a specific mobile signal remains unresolved, it appears to be conserved between monocots and dicots and, importantly, induces immunity to a diverse collection of pathogens and pests. To date numerous SAR-inducing chemicals, some of which exhibit physical mobility or are considered mobile due to their volatile nature, have been identified. These include, salicylic acid (SA), and its derivative methyl SA (MeSA) azelaic acid (AzA), [glycerol-3-phosphate (G3P), dehydroabietinal (DA), reactive nitrogen and oxygen species, and N-hydroxy pipecolic acid (NHP) amongst others. These chemicals confer systemic resistance when applied exogenously and are required for pathogen-induced SAR.
In recent breakthroughs, and underpinning this collaboration we have (i) developed a novel SAR luciferase reporter to provide spatial-temporal context to SAR establishment and (ii) identified two phased 21 nucleotide RNA (tasi-RNA) derived from Trans-Acting Small Interfering RNA3a (TAS3a) as essential for SAR. Based on their time frame of synthesis (3 hpi) post and systemic movement (4 hpi) we propose that tasi-RNAs function as the elusive early mobile SAR signal. Tasi-RNAs positively regulates the expression of genes encoding the previously identified lipid transfer protein (LTP)-like SAR regulator AZI1 (azelaic acid induced 1), as well as the LTP3, LTP4 and critically A70. LTPs regulate systemic transport of tasi-RNAs and based on the observed systemic mobility of LTPs, their interactions with AZI1, the detection of high molecular weight (HMW) complexes comprising AZI1 and presence of tasi-RNA in AZI1 immunoprecipitates we propose that SAR induction is associated with LTP-containing HMW protein complex-mediated systemic transport of tasi-RNAs. This proposal will characterize the LTP-RNA interactome and its relationship with A70 using computational modeling and functional analyses.
Finally, using these insights we will generate the first comprehensive analysis of the metabolic reconfiguraiton underpinning early SAR events.The knowledge gained here will be important for developing a basic understanding of this unique form of immunity and facilitate its use in developing sustainable and environmentally friendly crop protection strategies.
Technical Summary
Warwick's main role in this proposal it to (i) undertake A70 reporter screening, generate mutants in key LTP or tasiRNA compromised mutants and (ii) undertake a comprehensive comparative untargeted temporal metabolomics analysis of SAR establishment with complementary development of targeted hormone profiling of jasmonates, cytokinins and auxins to dissect hormonal crosstalk in systemic responding leaves.
Untargeted metabolomics: Using data from our A70::LUC reporter line temporal-spatial sampling strategies will be developed to ensure we capture the earliest metabolic changes in systemic responding leaves. Untargeted metabolomic profiling will use a Dionex UltiMate 3000 UHPLC system with Agilent Eclipse Plus C18 UPLC column (2.1x150 mm, 1.8 um particle size) and outflow routed to a Bruker MaXis II Q-TOF with an electrospray source. Samples will be run in both positive and negative ion mode with appropriate standards. We will us existing analysis pipelines developed for untargeted comparative metabolomics of fungal dieback of ash in Europe (Sollars et al. Nature 2016, Sambles et al. Sci. Data 2017, Sidda et al. Sci Reports 2020).
Once the SAR metabolite landscape is developed and interrogated with existing RNA-seq data we will incorporate a series of signalling mutants that compromise tasiRNA production choosing the most informative timepoint from above to define what discriminating features are altered in these SAR signaling mutants. In addition, we will analyse the TAS3a metabolome and compare to other systemic induced such as G3P and NHP.
Targeted metabolomics.
We will develop comprehensive quantitative hormone profiling methods, concentrating on jasmonates, auxins and cytokinins which are implicated in SAR from our, or other studies. We aim to quantify 15 phytohormone derivatives. This will provide the first systematic insight into phytohormone dynamics in SAR establishment and help interpret outcomes from the global metabolomics and RNA-seq workpackages.
Untargeted metabolomics: Using data from our A70::LUC reporter line temporal-spatial sampling strategies will be developed to ensure we capture the earliest metabolic changes in systemic responding leaves. Untargeted metabolomic profiling will use a Dionex UltiMate 3000 UHPLC system with Agilent Eclipse Plus C18 UPLC column (2.1x150 mm, 1.8 um particle size) and outflow routed to a Bruker MaXis II Q-TOF with an electrospray source. Samples will be run in both positive and negative ion mode with appropriate standards. We will us existing analysis pipelines developed for untargeted comparative metabolomics of fungal dieback of ash in Europe (Sollars et al. Nature 2016, Sambles et al. Sci. Data 2017, Sidda et al. Sci Reports 2020).
Once the SAR metabolite landscape is developed and interrogated with existing RNA-seq data we will incorporate a series of signalling mutants that compromise tasiRNA production choosing the most informative timepoint from above to define what discriminating features are altered in these SAR signaling mutants. In addition, we will analyse the TAS3a metabolome and compare to other systemic induced such as G3P and NHP.
Targeted metabolomics.
We will develop comprehensive quantitative hormone profiling methods, concentrating on jasmonates, auxins and cytokinins which are implicated in SAR from our, or other studies. We aim to quantify 15 phytohormone derivatives. This will provide the first systematic insight into phytohormone dynamics in SAR establishment and help interpret outcomes from the global metabolomics and RNA-seq workpackages.
