Jane Glazebrook

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.

A High-Throughput Arabidopsis Reverse Genetics System

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ∼100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae

We performed large-scale mRNA expression profiling using an Affymetrix GeneChip to study Arabidopsis responses to the bacterial pathogen Pseudomonas syringae. The interactions were compatible (virulent bacteria) or incompatible (avirulent bacteria), including a nonhost interaction and interactions mediated by two different avirulence gene–resistance (R) gene combinations. Approximately 2000 of the ∼8000 genes monitored showed reproducible significant expression level changes in at least one of the interactions. Analysis of biological variation suggested that the system behavior of the plant response in an incompatible interaction was robust but that of a compatible interaction was not. A large part of the difference between incompatible and compatible interactions can be explained quantitatively. Despite high similarity between responses mediated by the R genes RPS2 and RPM1 in wild-type plants, RPS2-mediated responses were strongly suppressed by the ndr1 mutation and the NahG transgene, whereas RPM1-mediated responses were not. This finding is consistent with the resistance phenotypes of these plants. We propose a simple quantitative model with a saturating response curve that approximates the overall behavior of this plant-pathogen system.

Characterization of the Early Response of Arabidopsis to Alternaria brassicicola Infection Using Expression Profiling

All tested accessions of Arabidopsis are resistant to the fungal pathogen Alternaria brassicicola. Resistance is compromised by pad3 or coi1 mutations, suggesting that it requires the Arabidopsis phytoalexin camalexin and jasmonic acid (JA)-dependent signaling, respectively. This contrasts with most well-studied Arabidopsis pathogens, which are controlled by salicylic acid-dependent responses and do not benefit from absence of camalexin or JA. Here, mutants with defects in camalexin synthesis (pad1, pad2, pad3, and pad5) or in JA signaling (pad1, coi1) were found to be more susceptible than wild type. Mutants with defects in salicylic acid (pad4 and sid2) or ethylene (ein2) signaling remained resistant. Plant responses to A. brassicicola were characterized using expression profiling. Plants showed dramatic gene expression changes within 12 h, persisting at 24 and 36 h. Wild-type and pad3 plants responded similarly, suggesting that pad3 does not have a major effect on signaling. The response of coi1 plants was quite different. Of the 645 genes induced by A. brassicicola in wild-type and pad3 plants, 265 required COI1 for full expression. It is likely that some of the COI1-dependent genes are important for resistance to A. brassicicola. Responses to A. brassicicola were compared with responses to Pseudomonas syringae infection. Despite the fact that these pathogens are limited by different defense responses, approximately 50% of the induced genes were induced in response to both pathogens. Among these, requirements for COI1 were consistent after infection by either pathogen, suggesting that the regulatory effect of COI1 is similar regardless of the initial stimulus.

Genetic techniques in Rhizobium meliloti.

Publisher Summary This chapter focuses on genetic techniques in Rhizobium meliloti . Rhizobia have been studied extensively because of their ability to form nodules on the roots of leguminous plants and fix atmospheric nitrogen. Many genetic techniques have been developed to facilitate analysis of several species and strains of Rhizobium . Rhizobium meliloti strain SU47, an alfalfa symbiont, has one of the most advanced genetic systems. Two basic methods are used to transfer DNA into and out of R. meliloti in the course of genetic manipulations—transduction and conjugation. Although it is possible to mutagenize R meliloti with chemical mutagens, such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG), transposon mutagenesis has proved to be a very useful tool because mutations induced by transposons are genetically marked by the antibiotic resistance of the transposon and physically marked by the presence of the transposon.

Analysis of the Arabidopsis Defense Response to Pseudomonas Pathogens

We have studied the response of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm) strain ES4326. Several previously unknown Arabidopsis defense-related genes were identified including ones encoding a glutathione-Stransferase, a superoxide dismutase, a lipoxygenase, and two calmodulin-like proteins. Interestingly, mRNA corresponding to each of these genes displayed markedly different patterns of accumulation during the defense response to Psm ES4326. We have isolated three categories of Arabidopsis mutants that show an aberrant defense response to Psm ES4326. Three mutants were isolated that do not mount a hypersensitive response (HR) when infiltrated with Psm ES4326/avrRpt2 but are still able to display an HR in response to other avr genes. At least two of these mutants are allelic and map to chromosome IV. To facilitate the identification of additional Arabidopsis mutants that do not mount an HR in response to an avr gene, we developed a new method that involves vacuum infiltration of seedlings growing in petri plates. We also isolated three mutants that synthesize decreased levels of camalexin, an indole-based Arabidopsis phytoalexin. Two of the three camalexin mutants are significantly more permissive for the growth of Psm ES4326 than wild-type plants. Finally, five Arabidopsis mutants were isolated that display accelerated disease symptoms in response to Psm ES4326. These latter mutants, which were given the name acd for accelerated cell death, were assigned to two complementation groups.

IDENTIFICATION AND CHARACTERIZATION OF AN ARABIDOPSIS ECOTYPE WHICH FAILS TO MOUNT A HYPERSENSITIVE RESPONSE WHEN INFILTRATED WITH PSEUDOMONAS SYRINGAE STRAINS CARRYING A VRRPT2

Effective plant defense responses against particular pathogens often involve a one-for-one correspondence between an avirulence (avr) gene in the pathogen and a resistance gene in the host [1]. Resistance genes are thought to encode receptors that perceive signals generated by avr genes and these specific recognition events are hypothesized to trigger the host defense response, including the so-called hypersensitive response (HR), which involves localized programmed cell death in the plant host in response to infection by an avirulent pathogen. Challenge of resistant plants with high doses of avirulent pathogens results in a visible HR, characterized by formation of dry, necrotic lesions, whereas lower doses lead to an HR that is only detectable microscopically. At the molecular level, several bacterial and fungal avr genes have been cloned, and in a few cases, avrgenerated signals have been identified [2, 3]. In contrast to avr genes, until recently only a single plant resistance gene, the tomato PTO gene, corresponding to a specific avr gene, had been cloned [4]. PTO encodes a serine threonine kinase, indicating that phosphorylation occurs in the signal transduction pathway leading to the HR.