Session 10: Plant-Pathogen Interactions
Chair: Roger Innes, Indiana University, USA
email: rinnes@bio.indiana.edu
This session covered three major areas of inquiry, starting off with the structure and function of disease resistance genes, followed by genetic analysis of R-gene signal transduction pathways, and finishing with a talk on systemic acquired resistance. Each speaker provided the following summaries of their talks, for which I am most grateful.Fumiaki Katagiri (University of Maryland, Baltimore County) discussed molecular mechanisms involved in gene-for gene interactions and novel experimental systems for their study. The Pseudomonas syringae avirulence genes, avrRpt2 and avrB, and the corresponding Arabidopsis resistance genes, RPS2 and RPM1, respectively, were used as a model system. Using biolistic transient expression of the avirulence genes in plants, he showed that the avirulence gene products are the only bacterial factors required for elicitation of the specific resistance response in plants as long as the gene products are localized properly. This observation was confirmed in transgenic plants (RPS2 wild type) carrying the avrRpt2 gene linked to the copper-inducible promoter. Such plants showed a copper-dependent HR-like response. Transient expression of the avirulence genes in Arabidopsis protoplasts demonstrated that protoplasts are still capable of specific recognition of the avirulence gene-based signals. The biolistic transient expression assay was also used for analysis of the corresponding resistance genes. The result with one RPS2/RPM1 chimeric gene suggested that the LRR of RPS2 determines the specificity to avrRpt2.
Roger Innes (Indiana University) described the identification of several Arabidopsis genes that are required for resistance mediated by the RPS5 disease resistance gene. RPS5 confers resistance to strains of P. syringae that carry the avirulence gene avrPphB. Randy Warren in his group isolated six mutants that displayed enhanced susceptibility to such strains. Genetic complementation analyses revealed that two of these were rps5 mutants, while the remaining four defined at least three additional genes, which have been designated PBS1, PBS2 and PBS3 for avrPphB susceptible. Mutations in PBS1 reduce resistance mediated by RPS5, but do not affect resistance mediated by other disease resistance genes in Arabidopsis, indicating that the PBS1 protein functions in a pathway specific to RPS5, potentially interacting with RPS5 and/or AvrPphB. In contrast to PBS1, mutations in PBS2 and PBS3 affect several Arabidopsis disease resistance gene pathways, including resistance genes specific to the fungal pathogen Peronospora parasitica. Interestingly, the rps5-1 mutant is phenotypically similar to the PBS2 mutant, affecting multiple disease resistance genes. The RPS5 gene has been isolated and was found to be a member of the NBS-LRR family of disease resistance genes. The rps5-1 mutant contains a single amino acid substitution in the third LRR of RPS5 , suggesting that this region may physically interact with a signal transduction component shared by other disease resistance gene pathways, perhaps PBS2.
Jane Parker (The Sainsbury Laboratory) described her group's work on the RPP5 disease resistance pathway. RPP genes confer resistance to the downy mildew pathogen Peronospora parasitica, with the RPP5 gene specifying resistance to P. parasitica isolate Noco2. RPP5 encodes a protein with a predicted nucleotide binding site (NBS) and leucine rich repeats (LRRs) that exhibits striking structural similarity to the tobacco N and flax L6 resistance proteins and is less similar to the Arabidopsis RPM1 and RPS2 gene products. Like N and L6, the RPP5 N-terminal domain resembles the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 transmembrane receptors. In contrast to N and L6 that produce predicted truncated products by alternative splicing, RPP5 appears to express only a single transcript corresponding to the full-length protein. However, a truncated form structurally similar to those of N and L6 is encoded by one or more other members of the RPP5 gene family that are tightly clustered on chromosome 4. A candidate RPP14 gene that is part of a tightly linked R gene cluster on chromosome 3, also appears to be of the RPP5/ N gene type. Resistance conferred by RPP5, RPP14, and a number of other Arabidopsis R genes is dependent on a second gene, EDS1. The EDS1 gene was cloned by transposon tagging and sequence analysis reveals a novel protein that is highly conserved between different Arabidopsis accessions. Two out of three fast neutron-derived eds1 mutant alleles possess extensive deletions (0.5 and 1 kb, respectively) of the EDS1 open reading frame and several ethyl methane sulphonate-generated mutant alleles are currently being analysed. The EDS1 protein possesses three appropriately spaced sequences that show significant similarity to the consensus motifs comprising a lipase catalytic site. Several site-directed mutations of the catalytic residues have been made to test whether these abolish EDS1 function in transformed eds1 mutant plants. Mutations of EDS1 and a second resistance signalling gene, NDR1, were combined with different Arabidopsis R genes to assess their relative effects. The results so far suggest that the two proteins operate preferentially in distinct resistance signalling pathways. For example, RPP5 requires EDS1 but appears to operate independently of NDR1,
whereas RPS2 requires NDR1 but not EDS1. Further R gene-signalling gene combinations are presently being examined to test the broader requirements for EDS1 and NDR1. EDS1 is also being combined with several other defence response mutations in order to position these in the same or distinct resistance signalling pathways.Allan Shapiro (U.C. Berkeley, and now at the University of Delaware) described work performed in the Staskawicz laboratory on the NDR1 gene of Arabidopsis. NDR1 is essential for numerous bacterial and Peronospora gene-for-gene disease resistance specificities in Arabidopsis. The NDR1 gene was isolated using a positional cloning approach and was found to encode a putative integral membrane protein. The ndr1-1 allele contains a deletion of the promoter and the N-terminal part of the predicted protein, while the ndr1-2 and ndr1-3 alleles contain nonsense mutations. A Northern blot showed that NDR1 message is induced by avirulent but not virulent pathogens at an early time point. Analysis of the ndr1-1 mutant was used to dissect the disease resistance response. This mutant is unable to mount a hypersensitive response (HR) in response to challenge with bacteria containing avrRpt2. However, an enhanced HR was seen in response to avrB or any of three other bacterial avirulence genes. Several other responses normally correlated with disease resistance were evaluated after inoculating ndr1-1 plants with bacteria containing avrRpt2 or avrB. Bacteria containing avrRpt2 failed to induce systemic acquired resistance (SAR) and PR-1 driven transcription, and induced less accumulation of salicylic acid (SA) at an early time point relative to wild type plants. Substantial catch-up in SA levels were seen at a late time point. By contrast, avrB-containing bacteria induced SAR and higher levels of PR-1 driven transcription in ndr1-1 plants as compared to Columbia wild-type. SA levels were comparable to those found in plants inoculated with avrRpt2-containing bacteria. Data was presented on poster 2-16 showing that ndr1-1 plants were partially impaired in induction of PR-1 driven transcription in response to UV-C light (a mimic of the oxidative burst) or low levels of BTH, but not impaired in response to high levels of BTH. A working model incorporating these data would posit that the ndr1-1 plants are partially blocked in disease resistance signal transduction, downstream of the oxidative burst, but upstream of SA production. Mutant screens have been initiated to uncover new genes in this pathway. Data was presented on a screen using a transgenic line carrying ß-glucuronidase (GUS) under PR-1 control. Mutants which show reduced GUS activity in response to challenge with avirulent pathogen have been obtained and are being characterized
Mark Kinkema (Duke University) described his work on characterizing the NPR1 protein. NPR1 regulates a local and systemic defense response against pathogen attack in Arabidopsis, and is required for many of the responses induced by SA. A fusion of NPR1 with the green fluorescent protein (GFP) complements all the npr1 mutant phenotypes in transgenic plants, indicating that NPR1-GFP is functional and, therefore, reflects the endogenous localization of NPR1. NPR1-GFP is localized in the nucleus in response to chemical activators of systemic acquired resistance. Virulent pathogen infection induces the nuclear localization of NPR1-GFP in cells surrounding the infection site, and nuclear localized NPR1-GFP correlates with the expression of the PR gene B-1,3-glucanase. These results suggest that nuclear localization of NPR1 regulates the SA-induced systemic acquired resistance and local acquired resistance defense responses.