Joseph D. Clarke

The Arabidopsis NPR1 Gene That Controls Systemic Acquired Resistance Encodes a Novel Protein Containing Ankyrin Repeats

The Arabidopsis NPR1 gene controls the onset of systemic acquired resistance (SAR), a plant immunity, to a broad spectrum of pathogens that is normally established after a primary exposure to avirulent pathogens. Mutants with defects in NPR1 fail to respond to various SAR-inducing treatments, displaying little expression of pathogenesis-related (PR) genes and exhibiting increased susceptibility to infections. NPR1 was cloned using a map-based approach and was found to encode a novel protein containing ankyrin repeats. The lesion in one npr1 mutant allele disrupted the ankyrin consensus sequence, suggesting that these repeats are important for NPR1 function. Furthermore, transformation of the cloned wild-type NPR1 gene into npr1 mutants not only complemented the mutations, restoring the responsiveness to SAR induction with respect to PR-gene expression and resistance to infections, but also rendered the transgenic plants more resistant to infection by P. syringae in the absence of SAR induction.

The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance.

The cpr5 mutant was identified from a screen for constitutive expression of systemic acquired resistance (SAR). This single recessive mutation also leads to spontaneous expression of chlorotic lesions and reduced trichome development. The cpr5 plants were found to be constitutively resistant to two virulent pathogens, Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2; to have endogenous expression of the pathogenesis-related gene 1 (PR-1); and to have an elevated level of salicylic acid (SA). Lines homozygous for cpr5 and either the SA-degrading bacterial gene nahG or the SA-insensitive mutation npr1 do not express PR-1 or exhibit resistance to P. s. maculicola ES4326. Therefore, we conclude that cpr5 acts upstream of SA in inducing SAR. However, the cpr5 npr1 plants retained heightened resistance to P. parasitica Noco2 and elevated expression of the defensin gene PDF1.2, implying that NPR1-independent resistance signaling also occurs. We conclude that the cpr5 mutation leads to constitutive expression of both an NPR1-dependent and an NPR1-independent SAR pathway. Identification of this mutation indicates that these pathways are connected in early signal transduction steps and that they have overlapping functions in providing resistance.

Roles of Salicylic Acid, Jasmonic Acid, and Ethylene in cpr-Induced Resistance in Arabidopsis

Disease resistance in Arabidopsis is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) function as key signaling molecules. Epistasis analyses were performed between mutants that disrupt these pathways (npr1, eds5, ein2, and jar1) and mutants that constitutively activate these pathways (cpr1, cpr5, and cpr6), allowing exploration of the relationship between the SA- and JA/ET–mediated resistance responses. Two important findings were made. First, the constitutive disease resistance exhibited by cpr1, cpr5, and cpr6 is completely suppressed by the SA-deficient eds5 mutant but is only partially affected by the SA-insensitive npr1 mutant. Moreover, eds5 suppresses the SA-accumulating phenotype of the cpr mutants, whereas npr1 enhances it. These data indicate the existence of an SA-mediated, NPR1-independent resistance response. Second, the ET-insensitive mutation ein2 and the JA-insensitive mutation jar1 suppress the NPR1-independent resistance response exhibited by cpr5 and cpr6. Furthermore, ein2 potentiates SA accumulation in cpr5 and cpr5 npr1 while dampening SA accumulation in cpr6 and cpr6 npr1. These latter results indicate that cpr5 and cpr6 regulate resistance through distinct pathways and that SA-mediated, NPR1-independent resistance works in combination with components of the JA/ET–mediated response pathways.

Uncoupling PR Gene Expression from NPR1 and Bacterial Resistance: Characterization of the Dominant Arabidopsis cpr6-1 Mutant

In Arabidopsis, NPR1 mediates the salicylic acid (SA)–induced expression of pathogenesis-related (PR) genes and systemic acquired resistance (SAR). Here, we report the identification of another component, CPR6, that may function with NPR1 in regulating PR gene expression. The dominant CPR6-1 mutant expresses the SA/NPR1–regulated PR genes (PR-1, BGL2, and PR-5) and displays enhanced resistance to Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 in the absence of SAR induction. cpr6-1–induced PR gene expression is not suppressed in the cpr6-1 npr1-1 double mutant but is suppressed when SA is removed by salicylate hydroxylase. Thus, constitutive PR gene expression in cpr6-1 requires SA but not NPR1. In addition, resistance to P. s. maculicola ES4326 is suppressed in the cpr6-1 npr1-1 double mutant, despite expression of PR-1, BGL2, and PR-5. Resistance to P. s. maculicola ES4326 must therefore be accomplished through unidentified antibacterial gene products that are regulated through NPR1. These results show that CPR6 is an important regulator of multiple signal transduction pathways involved in plant defense.

Identification and Cloning of a Negative Regulator of Systemic Acquired Resistance, SNI1, through a Screen for Suppressors of npr1-1

Systemic acquired resistance (SAR) is a plant immune response induced after a local infection by necrotizing pathogens. The Arabidopsis NPR1 gene is a positive regulator of SAR, essential for transducing the SAR signal salicylic acid (SA). Mutations in the NPR1 gene abolish the SA-induced expression of pathogenesis-related (PR) genes and resistance to pathogens. To identify additional regulators of SAR, we screened for suppressors of npr1-1. In the npr1-1 background, the sni1 (suppressor of npr1-1, inducible 1) mutant shows near wild-type levels of PR1 expression and resistance to pathogens after induction. Restoration of SAR in npr1-1 by the recessive sni1 mutation indicates that wild-type SNI1 may function as a negative regulator of SAR. We cloned the SNI1 gene and found that it encodes a leucine-rich nuclear protein.

A network of rice genes associated with stress response and seed development

We used a systematic approach to build a network of genes associated with developmental and stress responses in rice by identifying interaction domains for 200 proteins from stressed and developing tissues, by measuring the associated gene expression changes in different tissues exposed to a variety of environmental, biological, and chemical stress treatments, and by localizing the cognate genes to regions of stress-tolerance trait genetic loci. The integrated data set suggests that similar genes respond to environmental cues and stresses, and some may also regulate development. We demonstrate that the data can be used to correctly predict gene function in monocots and dicots. As a result, we have identified five genes that contribute to disease resistance in Arabidopsis.

Activation of an EDS1-mediated R-gene pathway in the snc1 mutant leads to constitutive, NPR1-independent pathogen resistance

The Arabidopsis NPR1 protein is an essential regulatory component of systemic acquired resistance (SAR). Mutations in the NPR1 gene completely block the induction of SAR by signals such as salicylic acid (SA). An Arabidopsis mutant, snc1 (suppressor of npr1-1, constitutive 1), was isolated in a screen for suppressors of npr1-1. In the npr1-1 background, the snc1 mutation resulted in constitutive resistance to Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2. High levels of SA were detected in the mutant and shown to be required for manifestation of the snc1 phenotype. The snc1 mutation was mapped to the RPP5 resistance (R) gene cluster and the eds1 mutation that blocks RPP5-mediated resistance suppressed snc1. These data suggest that a RPP5-related resistance pathway is activated constitutively in snc1. This pathway does not employ NPR1 but requires the signal molecule SA and the function of EDS1. Moreover, in snc1, constitutive resistance is conferred in the absence of cell death, which is often associated with R-gene mediated resistance.