Phytohormones are small signalling molecules that occur in low concentrations that are vital for the regulation of plant growth, development, and reproduction as well as playing an important role in the plant defence response (Pieterse et al., 2009). They regulate the plant defence response against both biotic and abiotic stresses via synergistic and antagonistic actions, referred to as signalling crosstalk (Fujita et al.,
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2006). Traditionally the key defence related phytohormones were SA, JA and ET, with JA and ET viewed as the hormones associated with defence against
necrotrophic pathogens, such as B. cinerea, and SA being involved in defence against biotrophic organisms (Glazebrook, 2005). However, experiments utilising the
quadruple mutant dde2/ein2/pad4/sid2 in Arabidopsis, showed that immunity to the necrotrophic pathogen A. brassicicola was severely compromised compared to single mutants indicating that SA, PAD4, JA and ET, all positively contribute to the defence response of necrotrophs,with network modelling showing a complex interwoven network of the core phytohormones involved in the response to pathogens (Tsuda et al., 2009).
JA has long been known to positively regulate plant defence against B. cinerea with many genetic studies enabling a relatively clear picture of how the JA-mediated signalling pathway brings about resistance. One of the first and possibly the most important JA-mediated signalling mutant found to be more susceptible to B. cinerea
was coi1-1, a loss-of-function mutant that was show to be insensitive to JA due to a
defect in the CORONATINE INSENSITIVE1 (COI1) protein (Thomma et al., 1998; Xie, Feys, James, Nieto-Rostro, & Turner, 1998). COI1 is a F-box protein that has been shown to form part of the Skp1/Cullin/F-box complex (SCFCOI1) in vivo (Devoto et al.,
2002; L. Xu, 2002). The SCFCOI1 complex is a E3 ubiquitin ligase that targets proteins,
specified by the F-box, for degradation by the proteasome. Using a combination of molecular, genetic and biochemical techniques it has been shown JA-Ile is the active hormone and in response to its presence the JAZ proteins are degraded by the proteasome. In addition, the JAZ proteins also interact with the bHLM transcription factor MYC2 (a positive regulator of JA-mediated signalling) (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007).
The JAZ family consists of 12 original members plus JAZ13 (which was added in 2015). They are characterised by three conserved domains, the N terminal domain, which contains an EAR motif in six of the JAZ proteins (JAZ1, JAZ2, JAZ5, JAZ6, JAZ7
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and JAZ8), a ZINC-FINGER EXPRESSED IN INFLORESCENCE MERISTEM (ZIM) domain containing a TIFY (JAZ1-JAZ12) or NAFY (JAZ13) motif, and a C-terminal Jas domain (Thireault et al., 2015; Wager & Browse, 2012). In order to identify the core JA- mediated signalling and to find novel JAZ interactions, Pauwels et al (2010) utilised a tandem affinity purification (TAP) assay (both in the presence and absence of
activate JA) that resulted in isolating NINJA (Novel Interactor of JAZ). This was confirmed, and the domains of interactions were determined, using Y2H and bimolecular fluorescence complementation. Following this they utilized the same techniques to investigate what interacted with NINJA, identifying that the protein TOPLESS (TPL) interacts with NINJA (Pauwels et al., 2010). Results from these key experiments and previous literature led Pauwels et al (2010) to propose the
following model: in the absence of JA-Ile, MYC transcription factors interact with the Jas domain of the JAZ proteins, the JAZ proteins interact through their TIFY motif with domain C of NINJA, with the EAR motif of NINJA interacting with the TPL co- repressors resulting in the repression of transcription of early JA responsive genes. In the presence of JA-Ile, JAZ proteins interact with the ubiquitin ligase SCFCOI1 leading
to ubiquitination and proteosomal JAZ degradation via the 26S proteasome pathway; this is followed by the release of the NINJA–TPL complex from the MYC transcription factors enabling the activation of jasmonate-responsive gene expression (Figure 12)(Pauwels et al., 2010).
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Figure 12. Model for a general function of TPL proteins in plant hormone signalling. A. In the absence of the active form of jasmonate (JA), JA-Ile, MYC factors interact with the JAS domain of the JAZ proteins, which in turn interact through their TIFY domain with the C domain of NINJA, the NINJA EAR motif interacts with TOPLESS (TPL) resulting in the repression of transcription of early JA genes. B In the presence of JA–Ile, the hormone facilitates interaction between JAZ proteins and the ubiquitin ligase SCFCOI1resulting in JAZ proteins being ubiquitinated (yellow circles) and subsequently degraded in the 26S proteasome, releasing transcription factors from inhibition and activating JA-responsive gene transcription. Adapted from (Pauwels et al., 2010).
The JAZ proteins have also been linked to the crosstalk between the JA and ET- mediated pathways, where they physically interact with two transcription factors ETHYLENE INSENSITIVE3 (EIN3) and EIN3-LIKE1 (EIL1) and recruit I6, resulting in the transcriptional activity of EIN3 and EIL1 being repressed (Zhu et al., 2011). In the ET- mediated signalling pathway, perception of ET represses a Raf-like kinase
CONSTITUTIVE ETHYLENE RESPONSE 1 (CTR1), which negatively regulates
downstream ET-mediated signalling events (Kieber, Rothenberg, Roman, Feldmann, & Ecker, 1993). Downstream of CTR1 is the essential positive regulator of ET-
mediated signalling ETHYLENE INSENSITIVE 2 (EIN2), with EIN3 and EIL1 being downstream of this (Alonso et al., 1999; Chao et al., 1997). Zhu et al (2011) propose that EIN3/EIL1 acts as a key integration node where the presence of ET is required for the stabilisation of EIN3/EIL1 and JA is needed for EIN3/EIL1 to be released from the JAZ repressors to enable effective transcriptional reprogramming for defence against necrotrophic pathogens, potentially explaining the synergistic and mutually dependant roles of ET and JA in plant defence (Zhu et al., 2011). In contrast, the crosstalk between the JA and SA-mediated pathways is thought to occur
downstream of the SCFCOI1-JAZ complex, via regulating the accumulation of
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transcription factors, such as ORA59, that target GCC-box motifs in JA-responsive genes (Van der Does et al., 2013).
The experiments above highlight how complex and interwoven the phytohormone signalling pathways are, consisting of a large degree of crosstalk between pathways, which can be either synergistic or antagonistic, much of which mediates and can be mediated by transcription factors helping to fine-tune the defence responsive transcriptional reprogramming. In addition to undergoing massive transcriptional changes in response to pathogens, post-transcriptional changes can radically affect the plants ability to mount an effective plant defence response; here we will particularly focus on differential alternative splicing.