Project Abstract

<p>&nbsp;               <b>ABSTRACT</b></p><p>&nbsp;A screen was established for mutants in which the plant defence response is de-repressed. The pathogeninducible isochorismate synthase (ICS1) promoter was fused to firefly luciferase (luc) and a homozygous transgenic line generated in which the ICS1luc fusion is co-regulated with ICS1. This line was mutagenized and M2 seedlings screened for constitutive ICS1luc expression (cie). The cie mutants fall into distinct phenotypic classes based on tissue-specific localization of luciferase activity. One mutant, cie1, that shows constitutive luciferase activity specifically in petioles, was chosen for further analysis. In addition to ICS1, PR and other defence-related genes are constitutively expressed in cie1 plants. The cie1 mutant is also characterized by an increased production of conjugated salicylic acid and reactive oxygen intermediates, as well as spontaneous lesion formation, all confined to petiole tissue. Significantly, defences activated in cie1 are sufficient to prevent infection by a virulent isolate of Hyaloperonospora parasitica, and this enhanced resistance response protects petiole tissue alone. Furthermore, cie1-mediated resistance, along with PR gene expression, is abolished in a sid2-1 mutant background, consistent with a requirement for salicylic acid. A positional cloning approach was used to identify cie1, which carries two point mutations in a gene required for cell wall biosynthesis and actin organization, MUR3. A mur3 knockout mutant also resists infection by H. parasitica in its petioles and this phenotype is complemented by transformation with wild-type MUR3. We propose that perturbed cell wall biosynthesis may activate plant defence and provide a rationale for the cie1 and the mur3 knockout phenotypes. Keywords isochorismate synthase, plant defence, Arabidopsis thaliana, cell wall biosynthesis, luciferase, Hyaloperonospora parasitica. <br></p>

Project Overview

<p> <b>1. INTRODUCTION</b></p><p>Plants have evolved to recognize invading pathogens and activate defence responses that inhibit pathogen growth and prevent disease. Potential plant pathogens can trigger the host immune system at various stages of ingress: (i) as soon as the pathogen makes physical contact with the host (Hardham et al., 2007; Lipka et al., 2005); (ii) through recognition of general elicitors known as pathogen-associated molecular patterns (PAMPs) (Lotze et al., 2007); and (iii) as late as the point of delivery of pathogen effectors into the infected host cell (Wu et al., 2003). The formation of physical and/or chemical barriers may be sufficient to prevent a particular pathogen species from infecting a particular plant species. Successful pathogens have evolved effector proteins to overcome these defences, infect a host plant and cause disease. In turn, plants have evolved resistance (R) genes whose products trigger a battery of defences upon recognition of an effector encoded by a cognate avirulence (Avr) gene expressed by specific pathogen races. In many cases, R protein-mediated resistance is associated with early production of reactive oxygen intermediates (ROIs), followed by an accumulation of salicylic acid (SA) (reviewed by Jones and Dangl, 2006). Recent studies in Arabidopsis thaliana have also established a role for nitric oxide in mediating plant defence (reviewed by Delledonne, 2005). Reactive oxygen intermediates, NO and SA are all thought to act as signalling molecules that activate additional resistance mechanisms; these include the induction of pathogenesisrelated genes and other defence-related genes, as well as the timely activation of a form of programmed cell death known as the hypersensitive response (HR) (reviewed by Nimchuk et al., 2003).<br></p><p> In order to dissect genetically the signalling pathway activated during R protein-mediated defence, various screens have been performed with the model plant Arabidopsis thaliana to identify mutants that are compromised for resistance to avirulent pathogens. Two of these mutants, eds1 (enhanced disease susceptibility) and ndr1 (non-race-specific disease resistance), are fully compromised for resistance mediated by certain R genes (Aarts et al., 1998; Century et al., 1995; Parker et al., 1996). The sgt1b, rar1 (A. thaliana orthologue of barley RAR1: required for Mla-resistance) and pad4 (phytoalexin-deficient) mutants range from full resistance, to partial susceptibility, to complete susceptibility depending on the race of avirulent pathogen tested (Austin et al., 2002; Feys et al., 2001; Glazebrook et al., 1997; Muskett et al., 2002). Two mutants, sid2/eds16 and eds5/sid1 (SA-induction deficient), which are both deficient for pathogeninduced SA production, show partial susceptibility to all avirulent pathogens tested (Nawrath and Metraux, 1999). These results suggest that plant defence is controlled by a branched signalling network rather than a linear pathway. The SID2 gene encodes an enzyme, isochorismate synthase (ICS1), which is involved in SA biosynthesis (Strawn et al., 2007). ICS1 expression is induced by pathogen stress, and SA levels are directly correlated with ICS1 transcript levels (Wildermuth et al., 2001). Pathogen-inducible SA synthesis is therefore likely to be directly controlled through ICS1 expression. Taken together, these results suggest that ICS1 is tightly controlled by the defence signalling network. Genetic screens have also been used to identify mutants that show constitutive defence responses in the absence of pathogen stress. Many groups have focused on recessive mutants as these are most likely loss of function mutations in negative regulators that have evolved to suppress the defence response. These mutants are typically characterized by high levels of SA, constitutive expression of PR genes and enhanced resistance to virulent pathogens. Additionally, two E3 ligases have been shown to be required for the defence response; it is hypothesized that upon pathogen recognition, their role is to ubiquitinate negative regulators to promote their destruction in the proteasome and thus de<small></small>repress defence mechanisms (Gonza´lez-Lamothe et al., 2006; Yang et al., 2006). <br></p><p> Most forward genetic screens for negative regulators have been focused on identifying mutants that show alterations in the activation and/or control of HR. Mutants have been identified that show spreading lesions after pathogen challenge; these include lsd1 (lesion-simulating disease resistance), acd (accelerated cell death) mutants acd1, acd2 and acd11, and vad1 (vascular-associated cell death); these mutants have been classified as propagative lesion-mimics. The rest of the lsd mutants (lsd2, -3, -4, -5, -6, -7), acd5, acd6, and cpr5 (constitutive expresser of PR genes) all produce visible lesions spontaneously; these mutants are known as initiation lesion-mimics. By contrast, the dnd mutants, dnd1 and dnd2 (defence-no-death), hrl1 (hypersensitive responselike) and hlm1 (HR-like lesion mimic) mutants show only microscopic lesions and actually suppress HR when challenged with an avirulent pathogen (reviewed by Lorrain et al., 2003). Some of the genes identified through these screens encode proteins typically associated with signal transduction: lsd1 encodes a zinc finger protein, and dnd1 and dnd2 both encode cyclic nucleotide-gated ion channels (Clough et al., 2000; Dietrich et al., 1997; Jurkowski et al., 2004). Characterization of lesion-mimics cpr5 and vad1 has revealed links between cell death programs involved in defence and those involved in development and/or senescence (Kirik et al., 2001; Lorrain et al., 2004; Yoshida et al., 2002). Identification of mutants such as cpr5 and vad1 suggests that cell death programs controlling various aspects of plant life might be coordinately regulated. Other researchers have screened for mutants that constitutively express defence genes but do not show a lesion-mimic phenotype. This strategy may prevent the identification of mutants that are affected in cell death programs unrelated to defence. Four screens have been performed using the following approaches: both cpr1 and snc1 (suppressor of npr1-1 constitutive) were identified based on their constitutive expression of PR2; the cir mutants (constitutively induced resistance) and cim mutants (constitutive immunity) were identified based on their constitutive expression of PR1 (Bowling et al., 1994; Li et al., 2001; Maleck et al., 2002; Murray et al., 2002). None of the cim or cir mutants have yet been cloned. SNC1 was identified as a gain of function mutation in an R gene homologue from a gene cluster that includes the RPP5/RPP4 (resistance to Peronospora parasitica) gene locus. Genetic crosses showed cpr1 to be allelic to the bal locus. The bal mutation involves epigenetic overexpression of an R gene homologue that is also located within the RPP5/RPP4 R gene cluster. Although cpr1 maps to the same R gene cluster, the cpr1-1 mutant was not found to overexpress any members of this R gene family (Stokes and Richards, 2002; Stokes et al., 2002). <br></p><p> Since recessive mutants that show constitutive defence are also typically characterized by high levels of SA, we hypothesized that ICS1 expression itself is under strong negative regulation. ICS1 is therefore a good target for a genetic screen aimed at identifying genes that negatively regulate the defence response. A genetic screen based on ICS1 expression might also yield other interesting insights into the regulation of the SA-controlled branch of the defence signalling network. In the present study we have identified mutants that show constitutive ICS1 expression (cie) using an ICS1:luciferase promoter–reporter gene fusion. This study also describes the cloning and characterization of one of these mutants, cie1, and demonstrates that cie1 is a novel allele of the previously characterized MUR3 gene involved in cell wall synthesis and cytoskeleton organization (Madson et al., 2003; Tamura et al., 2005). <br></p><p> <b>2. RESULT</b></p><p>Forward genetic screen for mutants that constitutively express ICS1 using an ICS1:luciferase promoter–reporter gene fusion The objective of this study was to identify genes that negatively regulate expression of ICS1 in A. thaliana. We carried out a forward genetic screen to identify A. thaliana mutants that constitutively express ICS1. To rapidly screen thousands of mutagenized A. thaliana plants for constitutive ICS1 expression, we generated a construct, ICS1:luc, consisting of the putative ICS1 promoter region fused to the firefly luciferase reporter gene (details in Experimental procedures). The putative ICS1 promoter region used in this study consists of 3.1 kb upstream of the ICS1 translational start site and includes several predicted pathogen-inducible cis-elements (Tedman-Jones, 2004). The ICS1:luc construct was introduced into A. thaliana wild type Columbia (Col-0) by Agrobacterium tumefaciens-mediated transformation. Ten transformed lines were identified and a single line (C5) that showed a strong increase in luciferase activity in response to pathogen challenge was chosen for further analysis and made homozygous. To determine if the ICS1:luc line (C5) was an accurate reporter of ICS1 expression, a time-course experiment was carried out with Col-0 and Col-0 (ICS1:luc) plants comparing ICS1 and luciferase transcript accumulation after infiltration with an avirulent bacterial pathogen (Figure 1). As expected, ICS1 expression was not affected by insertion of the ICS1:luc transgene. Significantly, ICS1 and luciferase showed a similar pattern of expression in response to Pseudomonas syringae pv. tomato DC3000 carrying AvrRpt2, in Col-0 (ICS1:luc) plants throughout the time-course. <br></p>