Roger W. Innes

Mitogen-activated protein kinase cascades in plants: a new nomenclature

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, including yeasts, animals and plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. In plants, MAPK cascades are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPK kinases and 60 MAPK kinase kinases. Here, we propose a simplified nomenclature for Arabidopsis MAPKs and MAPK kinases that might also serve as a basis for standard annotation of these gene families in all plants.

Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance

Plants can recognize pathogens through the action of disease resistance (R) genes, which confer resistance to pathogens expressing unique corresponding avirulence (avr) genes. The molecular basis of this gene-for-gene specificity is unknown. The Arabidopsis thaliana RPM1 gene enables dual specificity to pathogens expressing either of two unrelated Pseudomonas syringae avr genes. Despite this function, RPM1 encodes a protein sharing molecular features with recently described single-specificity R genes. Surprisingly, RPM1 is lacking from naturally occurring, disease-susceptible Arabidopsis accessions.

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.

A Yersinia effector and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis.

A Yersinia effector known as YopT and a Pseudomonas avirulence protein known as AvrPphB define a family of 19 proteins involved in bacterial pathogenesis. We show that both YopT and AvrPphB are cysteine proteases, and their proteolytic activities are dependent upon the invariant C/H/D residues conserved in the entire YopT family. YopT cleaves the posttranslationally modified Rho GTPases near their carboxyl termini, releasing them from the membrane. This leads to the disruption of actin cytoskeleton in host cells. The proteolytic activity of AvrPphB is essential for autoproteolytic cleavage of an AvrPphB precursor as well as for eliciting the hypersensitive response in plants. These findings provide new insights into mechanisms of animal and plant pathogenesis.

Negative regulation of defense responses in plants by a conserved MAPKK kinase

The enhanced disease resistance 1 (edr1) mutation of Arabidopsis confers resistance to powdery mildew disease caused by the fungus Erysiphe cichoracearum. Resistance mediated by the edr1 mutation is correlated with induction of several defense responses, including host cell death. Double mutant analysis revealed that all edr1-associated phenotypes are suppressed by mutations that block salicylic acid (SA) perception (nim1) or reduce SA production (pad4 and eds1). The NahG transgene, which lowers endogenous SA levels, also suppressed edr1. In contrast, the ein2 mutation did not suppress edr1-mediated resistance and associated phenotypes, indicating that ethylene and jasmonic acid-induced responses are not required for edr1 resistance. The EDR1 gene was isolated by positional cloning and was found to encode a putative MAP kinase kinase kinase similar to CTR1, a negative regulator of ethylene responses in Arabidopsis. Taken together, these data suggest that EDR1 functions at the top of a MAP kinase cascade that negatively regulates SA-inducible defense responses. Putative orthologs of EDR1 are present in monocots such as rice and barley, indicating that EDR1 may regulate defense responses in a wide range of crop species.

Disease development in ethylene-insensitive Arabidopsis thaliana infected with virulent and avirulent Pseudomonas and Xanthomonas pathogens.

The plant hormone ethylene has been hypothesized to play roles both in disease resistance and in disease susceptibility. These processes were examined by using isogenic virulent and avirulent bacterial pathogens and mutants of Arabidopsis thaliana that were altered in ethylene physiology. Ethylene-insensitive ein1 and ein2 mutants of Arabidopsis were resistant to Pseudomonas syringae pv. tomato made avirulent by the addition of the cloned avirulence genes avrRpt2, avrRpm1, or avrB; this suggests that ethylene is not required for active resistance against avirulent bacteria. In a second set of experiments, susceptibility was monitored with virulent P. s. pv. tomato, P. s. pv. maculicola, or Xanthomonas campestris pv. campestris strains. Wild-type Arabidopsis and ein1 mutants were susceptible to these strains, but ein2 mutants developed only minimal disease symptoms. Despite these reduced symptoms, virulent P. s. pv. tomato grew extensively within ein2 leaves. The Pseudomonas phytotoxin coronatine induces ethylene biosynthesis and diseaselike symptoms on many plant species, but the reduced symptomology of ein2 mutants could not be attributed to insensitivity to coronatine. The enhanced disease tolerance of ein2 plants suggests that ethylene may mediate pathogen-induced damage, but the absence of tolerance in ein1 mutants has yet to be explained.

RPS2, an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2

A molecular genetic approach was used to identify and characterize plant genes that control bacterial disease resistance in Arabidopsis. A screen for mutants with altered resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) expressing the avirulence gene avrRpt2 resulted in the isolation of four susceptible rps (resistance to P. syringae) mutants. The rps mutants lost resistance specifically to bacterial strains expressing avrRpt2 as they retained resistance to Pst strains expressing the avirulence genes avrB or avrRpm1. Genetic analysis indicated that in each of the four rps mutants, susceptibility was due to a single mutation mapping to the same locus on chromosome 4. Identification of a resistance locus with specificity for a single bacterial avirulence gene suggests that this locus, designated RPS2, controls specific recognition of bacteria expressing the avirulence gene avrRpt2. Ecotype Wü-0, a naturally occurring line that is susceptible to Pst strains expressing avrRpt2, appears to lack a functional allele at RPS2, demonstrating that there is natural variation at the RPS2 locus among wild populations of Arabidopsis.

Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease.

Nucleotide binding site–leucine-rich repeat (NBS–LRR) proteins mediate pathogen recognition in both mammals and plants. The molecular mechanisms by which pathogen molecules activate NBS–LRR proteins are poorly understood. Here we show that RPS5, a NBS–LRR protein from Arabidopsis, is activated by AvrPphB, a bacterial protease, via an indirect mechanism. When transiently expressed in Nicotiana benthamiana leaves, full-length RPS5 protein triggered programmed cell death, but only when coexpressed with AvrPphB and a second Arabidopsis protein, PBS1, which is a specific substrate of AvrPphB. Using coimmunoprecipitation analysis, we found that PBS1 is in a complex with the N-terminal coiled coil (CC) domain of RPS5 before exposure to AvrPphB. Deletion of the RPS5 LRR domain caused RPS5 to constitutively activate programmed cell death, even in the absence of AvrPphB and PBS1, and this activation depended on both the CC and NBS domains. The LRR and CC domains both coimmunoprecipitate with the NBS domain but not with each other. Thus, the LRR domain appears to function in part to inhibit RPS5 signaling, and cleavage of PBS1 by AvrPphB appears to release RPS5 from this inhibition. An amino acid substitution in the NBS site of RPS5 that is known to inhibit ATP binding in other NBS–LRR proteins blocked activation of RPS5, whereas a substitution thought to inhibit ATP hydrolysis constitutively activated RPS5. Combined, these data suggest that ATP versus ADP binding functions as a molecular switch that is flipped by cleavage of PBS1.

An Arabidopsis Mutant with Enhanced Resistance to Powdery Mildew

We have identified an Arabidopsis mutant that displays enhanced disease resistance to the fungus Erysiphe cichoracearum, causal agent of powdery mildew. The edr1 mutant does not constitutively express the pathogenesis-related genes PR-1, BGL2, or PR-5 and thus differs from previously described disease-resistant mutants of Arabidopsis. E. cichoracearum conidia (asexual spores) germinated normally and formed extensive hyphae on edr1 plants, indicating that the initial stages of infection were not inhibited. Production of conidiophores on edr1 plants, however, was <16% of that observed on wild-type Arabidopsis. Reduction in sporulation correlated with a more rapid induction of defense responses. Autofluorescent compounds and callose accumulated in edr1 leaves 3 days after inoculation with E. cichoracearum, and dead mesophyll cells accumulated in edr1 leaves starting 5 days after inoculation. Macroscopic patches of dead cells appeared 6 days after inoculation. This resistance phenotype is similar to that conferred by “late-acting” powdery mildew resistance genes of wheat and barley. The edr1 mutation is recessive and maps to chromosome 1 between molecular markers ATEAT1 and NCC1. We speculate that the edr1 mutation derepresses multiple defense responses, making them more easily induced by virulent pathogens.

A disease resistance gene in Arabidopsis with specificity for two different pathogen avirulence genes.

The RPS3 and RPM1 disease resistance loci of Arabidopsis confer resistance to Pseudomonas syringae strains that carry the avirulence genes avrB and avrRpm1, respectively. We have previously shown that RPS3 and RPM1 are closely linked genetically. Here, we show that RPS3 and RPM1 are in fact the same gene. We screened a mutagenized Arabidopsis population with a P. syringae strain carrying avrB and found 12 susceptible mutants. All 12 mutants were also susceptible to an isogenic strain carrying avrRpm1, indicating a loss of both RPS3 and RPM1 functions. No mutants were recovered that lost only RPS3 function. Genetic analysis of four independent mutants revealed that the lesions were in RPS3. Thus, a single gene in Arabidopsis confers resistance that is specific to two distinct pathogen avirulence genes--a gene-for-genes interaction. This observation suggests that the RPS3/RPM1 gene product can bind multiple pathogen ligands, or alternatively, that it does not function as a receptor.