Transient gene expression via agroinfiltration of tobacco leaves has been used to study the interaction between many different resistance (R) proteins and their cognate avirulence (Avr) proteins. In this assay system, R protein activation and downstream signalling results in leaf necrosis, a threshold phenotype that is not well suited to quantification of signal strength. Nor is this assay system suited to the analysis of phytohormone and other signalling pathways that underpin the defence response but do not result in necrosis. A transgenic PR-5 (pathogenesis-related protein-5 E22) promoter: GUS (β-glucuronidase) reporter system has been developed in the Jones laboratory to enable quantitative measurement of defence activation occurring at or below the threshold for necrosis in agroinfiltration assays. In this reporter system, reporter activation is measured in leaf discs (harvested after agroinfiltration) using a MUG (4-methyl-umbelliferyl-β-D-glucuronide)-based fluorescence plate assay to measure β-glucuronidase activity. As expected, β-glucuronidase activity was induced by R/Avr protein interaction and consistent with the presence of salicylic acid (SA), ethylene (ET) and cytokinin (CK) responsive elements in the E22 promoter, β-glucuronidase activity was induced rapidly by SA and ethylene ET, more slowly by CK, but not at all by jasmonic acid (JA). However, JA was found to suppress activation by SA. Salt stress was also found to induce β-glucuronidase activity consistent with the presence of salt responsive elements in the E22 promoter. The E22 promoter also contains abscisic acid (ABA) and drought responsive elements but these have not yet been investigated. The possibility of synergistic or antagonistic crosstalk between phytohormone signalling pathways affecting E22:GUS reporter induction has also not been investigated, apart from the antagonistic interaction between JA and SA and an additive (possibly synergistic) interaction between CK and SA.

The proposed project will use the E22:GUS reporter line to further investigate the role of phytohormones, abiotic stress and pathogen elicitors and effectors in inducing or suppressing defence response activation as follows:

Phytohormone and abiotic stress induction

1) test E22:GUS reporter gene activation in excised leaf disks after 24 hours flotation on solutions containing a range of ABA concentrations (1µM-1mM)

2) test E22:GUS reporter gene activation following drought stress imposed by water deprivation and measuring soil water potential as well as GUS activity over time

3) test E22:GUS reporter gene activation in excised leaf disks after 24 hours flotation on solutions containing the following combinations of phytohormones

     a) 20 µM JA + 1µM-1mM CK compared to 1µM-1mM CK alone

     b) 20 µM JA + 1 mM ethephon (an ET-releasing compound) compared to 1 mM ethephon alone

     c) (depending on results in part 2) 20 µM JA + 1µM-1mM ABA compared to 1µM-1mM ABA alone

     d) 10 µM or 100 µM CK + 1µM-1mM SA compared to 10 µM or 100 µM CK or 1µM-1mM SA alone

     e) 1mM ethephon + 1µM-1mM SA compared to 1mM ethephon or 1µM-1mM SA alone

     f) (depending on 2) 1µM-1mM ABA + 1µM-1mM SA compared to 1µM-1mM ABA alone or 1µM-1mM SA alone

     g) 1mM ethephon + 10 µM or 100 µM CK compared to 1mM ethephon 10 µM CK or 100 µM CK alone

Pathogen elicitors and effectors

1) test E22:GUS reporter gene activation following infiltration of a bacterial flagellin elicitor-active peptide (flg22) over a time course and at various concentrations of flg22 to establish a suitable combination for assaying suppression of flg22-induced defence response by pathogen effectors

2) test the ability of various fax rust (Melampsora lini) effectors (AvrL2, AvrL567, AvrM, AvrM14, AvrP, AvrP123 and AvrP4) and fusarium wilt (Fusarium oxysporum f. sp. lycopersici) effectors (Avr1, Avr2, Avr3, Six5) to suppress PTI (PAMP-triggered immunity) induced by flg22 following agroinfiltration. PAMPs are pathogen-associated molecular patterns recognised by PRRs (pattern recognition receptors) that then activate PTI.

3) test the effect of agroinfiltration of AvrL567 ± LuCKX1 (a cytokinin oxidase targeted by AvrL567) on E22:GUS reporter gene activation in response to 1µM-1mM CK

4) make a kinase activation mutant of the tomato I-3 gene for resistance to fusarium wilt by site-directed mutagenesis and test for E22:GUS reporter gene activation following agroinfiltration. If reporter gene expression is induced then test induction over a time course and at various concentrations of Agrobacterium tumefaciens

6)  test the ability of Fusarium oxysporum Avr1, a known suppressor of ETI (effector-triggered immunity) triggered by Avr2 and Avr3, to suppress signalling in defence signalling pathways triggered by autoactive mutants of resistance proteins i.e. Mla10 (an auto-active truncated derivative of the barley Mla10cc CC-NB-LRR resistance protein), L6tir (an auto-active truncated derivative of the flax L6 TIR-NB-LRR resistance protein), M205 (an auto-active domain swap between tomato Hcr9-9A and Cf-9 eLRR-RLP resistance proteins) and I-3* (the auto-active kinase-activation mutant of the I-3 SRLK resistance protein generated in part 5). Note: CC = coiled-coil; NB = nucleotide binding; LRR = leucine-rich repeat; TIR = Toll and Interleukin-1 receptor cytosolic domain homology; eLRR = extracellular LRR; SRLK = S-receptor-like kinase.

7) test the ability of the I resistance gene to induce E22:GUS reporter gene activation following recognition of Avr1

These assays will provide valuable experience in the use of GUS reporter gene assays to study defence activation and pathogen attack. They will also provide technical expertise with the MUG-based fluorescence assay, transient gene expression via agroinfiltration and site-directed mutagenesis, and convey an understanding of the two main tiers of plant disease resistance (PTI and ETI), including the roles of phytohormones in plant defence, as well as the mechanisms that pathogens use to overcome these defences.