Douglas Dahlbeck

Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster

In tomato, resistance to Pseudomonas syringae pv. tomato (Pst) strains expressing the avirulence gene avrPto requires the presence of at least two host genes, designated Pto and Prf. Here we report that Prf encodes a protein with leucine-zipper, nucleotide-binding, and leucine-rich repeat motifs, as are found in a number of resistance gene products from other plants. prf mutant alleles (4) were found to carry alterations within the Prf coding sequence. A genomic fragment containing Prf complemented a prf mutant tomato line both for resistance to Pst strains expressing avrPto and for sensitivity to the insecticide Fenthion. Prf resides in the middle of the Pto gene cluster, 24 kb from the Pto gene and 500 bp from the Fen gene.

A pathogen-inducible endogenous siRNA in plant immunity

RNA interference, mediated by small interfering RNAs (siRNAs), is a conserved regulatory process that has evolved as an antiviral defense mechanism in plants and animals. It is not known whether host cells also use siRNAs as an antibacterial defense mechanism in eukaryotes. Here, we report the discovery of an endogenous siRNA, nat-siRNAATGB2, that is specifically induced by the bacterial pathogen Pseudomonas syringae carrying effector avrRpt2. We demonstrate that the biogenesis of this siRNA requires DCL1, HYL1, HEN1, RDR6, NRPD1A, and SGS3. Its induction also depends on the cognate host disease resistance gene RPS2 and the NDR1 gene that is required for RPS2-specified resistance. This siRNA contributes to RPS2-mediated race-specific disease resistance by repressing PPRL, a putative negative regulator of the RPS2 resistance pathway.

Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance

Plant diseases cause massive losses in agriculture. Increasing the natural defenses of plants may reduce the impact of phytopathogens on agricultural productivity. Pattern-recognition receptors (PRRs) detect microbes by recognizing conserved pathogen-associated molecular patterns (PAMPs). Although the overall importance of PAMP-triggered immunity for plant defense is established, it has not been used to confer disease resistance in crops. We report that activity of a PRR is retained after its transfer between two plant families. Expression of EFR (ref. 4), a PRR from the cruciferous plant Arabidopsis thaliana, confers responsiveness to bacterial elongation factor Tu in the solanaceous plants Nicotiana benthamiana and tomato (Solanum lycopersicum), making them more resistant to a range of phytopathogenic bacteria from different genera. Our results in controlled laboratory conditions suggest that heterologous expression of PAMP recognition systems could be used to engineer broad-spectrum disease resistance to important bacterial pathogens, potentially enabling more durable and sustainable resistance in the field.

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.

Gene-for-gene relationships specifying disease resistance in Xanthomonas campestris pv. vesicatoria - pepper interactions.

In this study, we describe the cloning and characterization of three avirulence genes from X.c. pv. vesicatoria. We present evidence that these avirulence genes restrict the host range of X.c. pv. vesicatoria strains

Genetic and molecular evidence that the Pseudomonas syringae type III effector protein AvrRpt2 is a cysteine protease.

Upon delivery to the plant cell during infection, the Pseudomonas syringae effector protein AvrRpt2 undergoes proteolytic processing, enhances pathogen virulence and causes the elimination of the Arabidopsis RIN4 protein. A structure‐prediction method was employed in order to investigate possible biochemical functions of AvrRpt2. Results of a secondary structure prediction algorithm suggest that the functional C‐terminal portion of AvrRpt2 is a cysteine protease. Mutation of predicted catalytic residues within this portion of AvrRpt2 abolished in planta processing, elimination of Arabidopsis RIN4, and the ability to trigger an RPS2‐specific resistance response. These data indicate that AvrRpt2 is most likely a sequence divergent cysteine protease whose activity is required for elimination of RIN4 during infection.

Activation of an Arabidopsis Resistance Protein Is Specified by the in Planta Association of Its Leucine-Rich Repeat Domain with the Cognate Oomycete Effector

The Arabidopsis disease resistance protein RPP1 recognizes the ATR1 effector protein from Hyaloperonospora arabidopsidis. This works shows that the molecular basis of recognition is mediated by the in planta association of the LRR domain of the RPP1 protein with the ATR1 effector protein. The in planta association of specific alleles of ATR1 leads to the activation of plant immune responses. Activation of plant immunity relies on recognition of pathogen effectors by several classes of plant resistance proteins. To discover the underlying molecular mechanisms of effector recognition by the Arabidopsis thaliana RECOGNITION OF PERONOSPORA PARASITICA1 (RPP1) resistance protein, we adopted an Agrobacterium tumefaciens–mediated transient protein expression system in tobacco (Nicotiana tabacum), which allowed us to perform coimmunoprecipitation experiments and mutational analyses. Herein, we demonstrate that RPP1 associates with its cognate effector ARABIDOPSIS THALIANA RECOGNIZED1 (ATR1) in a recognition-specific manner and that this association is a prerequisite step in the induction of the hypersensitive cell death response of host tissue. The leucine-rich repeat (LRR) domain of RPP1 mediates the interaction with ATR1, while the Toll/Interleukin1 Receptor (TIR) domain facilitates the induction of the hypersensitive cell death response. Additionally, we demonstrate that mutations in the TIR and nucleotide binding site domains, which exhibit loss of function for the induction of the hypersensitive response, are still able to associate with the effector in planta. Thus, our data suggest molecular epistasis between signaling activity of the TIR domain and the recognition function of the LRR and allow us to propose a model for ATR1 recognition by RPP1.

NPK1, an MEKK1-like Mitogen-Activated Protein Kinase Kinase Kinase, Regulates Innate Immunity and Development in Plants

Mitogen-activated protein kinase (MAPK) cascades are rapidly activated upon plant recognition of invading pathogens. Here, we describe the use of virus-induced gene silencing (VIGS) to study the role of candidate plant MAP kinase kinase kinase (MAPKKK) homologs of human MEKK1 in pathogen-resistance pathways. We demonstrate that silencing expression of a tobacco MAPKKK, Nicotiana Protein Kinase 1 (NPK1), interferes with the function of the disease-resistance genes N, Bs2, and Rx, but does not affect Pto- and Cf4-mediated resistance. Further, NPK1-silenced plants also exhibit reduced cell size, defective cytokinesis, and an overall dwarf phenotype. Our results provide evidence that NPK1 functions in the regulation of N-, Bs2-, and Rx-mediated resistance responses and may play a role in one or more MAPK cascades, regulating multiple cellular processes.

Direct biochemical evidence for type III secretion-dependent translocation of the AvrBs2 effector protein into plant cells

The calmodulin-dependent adenylate cyclase domain (Cya) of the Bordetella pertussis cyclolysin was used as a reporter protein to study the direct translocation of the Xanthomonas effector protein, AvrBs2, into the plant host cell. Adenylate cyclase activity (production of cAMP) depends on the presence of eukaryotic plant calmodulin and is only active after translocation from the prokaryotic cell into the eukaryotic plant cell. Here, we show that infection of pepper plants by Xanthomonas campestris pv. vesicatoria strains expressing the AvrBs2:Cya fusion protein results in detectable increases of cAMP levels in plant cells as early as 3 h after inoculation. Adenylate cyclase activity was shown to be type III secretion-dependent as the Xanthomonas hrp mutations, hrcV or hrpF, failed to produce detectable levels of cAMP in infected pepper plants. Furthermore, the N-terminal secretion and translocation signals of AvrBs2 were shown to be required for activity of the fusion protein in the plant. A single genomic copy of the avrBs2:cya fusion gene expressed under the control of the wild-type avrBs2 promoter was used to compare the effect of a susceptible and resistant plant interaction on the kinetics of effector protein delivery. Implications of these results and additional applications of this reporter construct are discussed.

Molecular Genetic Evidence for the Role of SGT1 in the Intramolecular Complementation of Bs2 Protein Activity in Nicotiana benthamiana

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens–mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2–mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Coimmunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Coimmunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1–containing protein complex that is required for the expression of bacterial disease resistance.