Requirement of the cytosolic interaction between PATHOGENESIS-RELATED PROTEIN10 and LEUCINE-RICH REPEAT PROTEIN1 for cell death and defense signaling in pepper

Plants recruit innate immune receptors such as leucine-rich repeat (LRR) proteins to recognize pathogen attack and activate defense genes. Here, we identified the pepper (Capsicum annuum) pathogenesis-related protein10 (PR10) as a leucine-rich repeat protein1 (LRR1)-interacting partner. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the specific interaction between LRR1 and PR10 in planta. Avirulent Xanthomonas campestris pv vesicatoria infection induces PR10 expression associated with the hypersensitive cell death response. Transient expression of PR10 triggers hypersensitive cell death in pepper and Nicotiana benthamiana leaves, which is amplified by LRR1 coexpression as a positive regulator. LRR1 promotes the ribonuclease activity and phosphorylation of PR10, leading to enhanced cell death signaling. The LRR1-PR10 complex is formed in the cytoplasm, resulting in its secretion into the apoplastic space. Engineered nuclear confinement of both proteins revealed that the cytoplasmic localization of the PR10-LRR1 complex is essential for cell death-mediated defense signaling. PR10/LRR1 silencing in pepper compromises resistance to avirulent X. campestris pv vesicatoria infection. By contrast, PR10/LRR1 overexpression in Arabidopsis thaliana confers enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis. Together, these results suggest that the cytosolic LRR-PR10 complex is responsible for cell death-mediated defense signaling.     

Reduced expression of the tomato ethylene receptor gene LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv. vesicatoria

The hypersensitive response (HR) involves rapid death of cells at the site of pathogen infection and is thought to limit pathogen growth through the plant. Ethylene regulates senescence and developmental programmed cell death, but its role in hypersensitive cell death is less clear. Expression of two ethylene receptor genes, NR and LeETR4, is induced in tomato (Lycopersicon esculentum cv. Mill) leaves during an HR to Xanthomonas campestris pv. vesicatoria, with the greatest increase observed in LeETR4. LeETR4 antisense plants previously were shown to exhibit increased sensitivity to ethylene. These plants also exhibit greatly reduced induction of LeETR4 expression during infection and an accelerated HR at inoculum concentrations ranging from 10(5) to 10(7) CFU/ml. Increases in ethylene synthesis and pathogenesis-related gene expression are greater and more rapid in infected LeETR4 antisense plants, indicating an enhanced defense response. Populations of avirulent X. campestris pv. vesicatoria decrease more quickly and to a lower level in the transgenic plants, indicating a greater resistance to this pathogen. Because the ethylene action inhibitor 1-methylcyclopropene alleviates the enhanced HR phenotype in LeETR4 antisense plants, these changes in pathogen response are a result of increased ethylene sensitivity.     

The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene.

Resistance of tomato plants to the bacterial pathogen Pseudomonas syringae pv. tomato race 0 is controlled by the locus Pto. A bacterial avirulence gene was cloned by constructing a cosmid library from an avirulent P. syringae pv. tomato race, conjugating the recombinants into a strain of P. syringae pv. maculicola virulent on a tomato cultivar containing Pto, and screening for those clones that converted the normally virulent phenotype to avirulence. The cloned gene, designated avrPto, reduced multiplication of P. syringae pv. tomato transconjugants specifically on Pto tomato lines, as demonstrated by bacterial growth curve analyses. Analysis of F2 populations revealed cosegregation of resistance to P. syringae pv. tomato transconjugants carrying avrPto with resistance to P. syringae pv. tomato race 0. Surprisingly, mutation of avrPto in P. syringae pv. tomato race 0 does not eliminate the avirulent phenotype of race 0, suggesting that additional, as yet uncharacterized, avirulence genes and/or resistance genes may contribute to specificity in the avrPto-Pto interaction. Genetic analysis indicates that this resistance gene(s) would be tightly linked to Pto. Interestingly, P. syringae pv. glycinea transconjugants carrying avrPto elicit a typical hypersensitive resistant response in the soybean cultivar Centennial, suggesting conservation of Pto function between two crop plants, tomato and soybean.     

Plants expressing the Pto disease resistance gene confer resistance to recombinant PVX containing the avirulence gene AvrPto

Elicitation of hypersensitive cell death and induction of plant disease resistance by Pseudomonas syringae pv. tomato (Pst) is dependent on activity of the Pst Hrp secretion system and the gene-for-gene interaction between the tomato resistance gene Pto and the bacterial avirulence gene avrPto. AvrPto was expressed transiently in resistant or susceptible plant lines via a potato virus X (PVX) vector. We found that while PVX is normally virulent on tomato, a PVX derivative expressing avrPto was only capable of infecting plants lacking a functional Pto resistance pathway. Mutations in either the Pto or Prf genes allowed systemic spread of the recombinant virus. These results indicate that recognition of AvrPto by Pto in resistant plant lines triggers a plant defense response that can confer resistance to a viral as well as a bacterial pathogen.     

Mutational analysis of the Arabidopsis RPS2 disease resistance gene and the corresponding pseudomonas syringae avrRpt2 avirulence gene

Plants have evolved a large number of disease resistance genes that encode proteins containing conserved structural motifs that function to recognize pathogen signals and to initiate defense responses. The Arabidopsis RPS2 gene encodes a protein representative of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of plant resistance proteins. RPS2 specifically recognizes Pseudomonas syringae pv. tomato strains expressing the avrRpt2 gene and initiates defense responses to bacteria carrying avrRpt2, including a hypersensitive cell death response (HR). We present an in planta mutagenesis experiment that resulted in the isolation of a series of rps2 and avrRpt2 alleles that disrupt the RPS2-avrRpt2 gene-for-gene interaction. Seven novel avrRpt2 alleles incapable of eliciting an RPS2-dependent HR all encode proteins with lesions in the C-terminal portion of AvrRpt2 previously shown to be sufficient for RPS2 recognition. Ten novel rps2 alleles were characterized with mutations in the NBS and the LRR. Several of these alleles code for point mutations in motifs that are conserved among NBS-LRR resistance genes, including the third LRR, which suggests the importance of these motifs for resistance gene function.     

High-affinity binding site for ethylene-inducing xylanase elicitor on Nicotiana tabacum membranes

Challenge of Nicotiana tabacum cv Xanthi with the ethylene-inducing xylanase (EIX) from Trichoderma viride causes rapid induction of plant defense responses leading to hypersensitive necrosis. This phenomenon is cultivar-specific; no response is detected when N. tabacum cv Hicks is similarly treated. The responsiveness is determined in tobacco and tomato by a single dominant gene. EIX was labeled with fluorescein-isothiocyanate and incubated with cell suspension cultures, protoplasts or microsomal membranes. Binding of EIX to the microsomal membranes was found to be specific and saturable, with a dissociation constant of 6.2 nM. Using confocal laser microscopy, the EIX binding site was localized to the plasma membrane. Binding of EIX to its high-affinity site occurred in responsive species. These results demonstrate the existence of a high-affinity binding site for EIX on the plasma membrane of responsive cultivars. Chemical cross-linking of EIX to microsomal membranes from responding plants revealed a 66 kDa protein complex. This protein may function as the receptor that mediates the hypersensitive response induced by EIX binding.     

An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue

Cell viability or cell death is an important variable to monitor in many studies of host/pathogen interactions. However for studies that focus on events within the first few hours of the interaction, many of the viability assays currently being used are either too laborious and time consuming or measure the cell's temporary metabolic state rather than irreversible cell death. Evans blue has proven over the years to be a dependable stain for microscopic determination of cell death. We have used this stain to develop a spectrophotometric procedure that allows rapid, reproducible quantification of the stain retained by dead cells. This spectrophotometric procedure was used to compare plant/bacteria interactions involving either soybean/Pseudomonas syringae pv. glycinea or tobacco/P. syringae pv. syringae. Relative increases in cell death during these interactions in suspension cell systems were measured by both the spectrophotometric and microscopic technique and found to be similar. The spectrophotometric procedure was also adapted for leaf disc assays.Abbreviations HR hypersensitive response - SDS sodium dodecyl sulfate     

Molecular characterization of avirulence gene D from Pseudomonas syringae pv. tomato

Avirulence gene D, cloned from Pseudomonas syringae pv. tomato, caused P. s. pv. glycinea to elicit a hypersensitive defense response on certain cultivars of soybean. Nucleotide sequence data for a 5.6-kb HindIII fragment containing avrD disclosed five long open-reading frames (ORFs) occurring in tandem. The phenotype conferred by avrD was expressed in P. s. pv. glycinea solely by the first of these ORFs (933 bases) that encoded a protein of 34,115 daltons. Neither a signal peptide sequence nor significant regions of hydrophobicity were present that would indicate secretion of the protein or its membrane association. Hybridization analyses revealed that some but not all P. syringae pathovars contained DNA homologous to avrD. This included weak hybridization to all tested races of P. s. pv. glycinea, although none of them express the phenotype conferred by avrD. The avrD gene occurred on an indigenous 75-kb plasmid in several P. s. pv. tomato isolates.     

Systemic acquired tolerance to virulent bacterial pathogens in tomato

Recent studies on the interactions between plants and pathogenic microorganisms indicate that the processes of disease symptom development and pathogen growth can be uncoupled. Thus, in many instances, the symptoms associated with disease represent an active host response to the presence of a pathogen. These host responses are frequently mediated by phytohormones. For example, ethylene and salicylic acid (SA) mediate symptom development but do not influence bacterial growth in the interaction between tomato (Lycopersicon esculentum) and virulent Xanthomonas campestris pv vesicatoria (Xcv). It is not apparent why extensive tissue death is integral to a defense response if it does not have the effect of limiting pathogen proliferation. One possible function for this hormone-mediated response is to induce a systemic defense response. We therefore assessed the systemic responses of tomato to Xcv. SA- and ethylene-deficient transgenic lines were used to investigate the roles of these phytohormones in systemic signaling. Virulent and avirulent Xcv did induce a systemic response as evidenced by expression of defense-associated pathogenesis-related genes in an ethylene- and SA-dependent manner. This systemic response reduced cell death but not bacterial growth during subsequent challenge with virulent Xcv. This systemic acquired tolerance (SAT) consists of reduced tissue damage in response to secondary challenge with a virulent pathogen with no effect upon pathogen growth. SAT was associated with a rapid ethylene and pathogenesis-related gene induction upon challenge. SAT was also induced by infection with Pseudomonas syringae pv tomato. These data show that SAT resembles systemic acquired resistance without inhibition of pathogen growth.     

The avrRxo1 gene from the rice pathogen Xanthomonas oryzae pv. oryzicola confers a nonhost defense reaction on maize with resistance gene Rxo1

Maize lines that contain the single dominant gene Rxo1 exhibit a rapid hypersensitive response (HR) after infiltration with the rice bacterial streak pathogen Xanthomonas oryzae pv. oryzicola, but not with the rice bacterial blight pathogen X. oryzae pv. oryzae. The avirulence effector gene that corresponds to Rxo1, designated avrRxo1, was identified in an X. oryzae pv. oryzicola genomic library. When introduced into X. oryzae pv. oryzae, clones containing avrRxo1 induced an HR on maize with Rxo1, but not on maize without Rxo1. The avrRxo1 gene is 1,266 bp long and shows no significant homology to any database sequences. When expressed in an X. oryzae pv. oryzae hrpC mutant that is deficient in the type III secretion system, avrRxo1 did not elicit the HR, indicating that the avrRxo1-Rxo1 interaction is dependent on type III secretion. Transient expression of avrRxo1 in onion cells after biolistic delivery revealed that the protein product was associated with the plasma membrane. Transient expression in maize lines carrying Rxo1 resulted in cell death, suggesting that AvrRxo1 functions from inside maize cells to elicit Rxo1-dependent pathogen recognition.     

Vascular associated death1, a novel GRAM domain-containing protein, is a regulator of cell death and defense responses in vascular tissues

The hypersensitive response (HR) is a programmed cell death that is commonly associated with plant disease resistance. A novel lesion mimic mutant, vad1 (for vascular associated death1), that exhibits light conditional appearance of propagative HR-like lesions along the vascular system was identified. Lesion formation is associated with expression of defense genes, production of high levels of salicylic acid (SA), and increased resistance to virulent and avirulent strains of Pseudomonas syringae pv tomato. Analyses of the progeny from crosses between vad1 plants and either nahG transgenic plants, sid1, nonexpressor of PR1 (npr1), enhanced disease susceptibility1 (eds1), or non-race specific disease resistance1 (ndr1) mutants, revealed the vad1 cell death phenotype to be dependent on SA biosynthesis but NPR1 independent; in addition, both EDS1 and NDR1 are necessary for the proper timing and amplification of cell death as well as for increased resistance to Pseudomonas strains. VAD1 encodes a novel putative membrane-associated protein containing a GRAM domain, a lipid or protein binding signaling domain, and is expressed in response to pathogen infection at the vicinity of the hypersensitive lesions. VAD1 might thus represent a new potential function in cell death control associated with cells in the vicinity of vascular bundles.     

Overexpression of Pto activates defense responses and confers broad resistance.

The tomato disease resistance (R) gene Pto specifies race-specific resistance to the bacterial pathogen Pseudomonas syringae pv tomato carrying the avrPto gene. Pto encodes a serine/threonine protein kinase that is postulated to be activated by a physical interaction with the AvrPto protein. Here, we report that overexpression of Pto in tomato activates defense responses in the absence of the Pto-AvrPto interaction. Leaves of three transgenic tomato lines carrying the cauliflower mosaic virus 35S::Pto transgene exhibited microscopic cell death, salicylic acid accumulation, and increased expression of pathogenesis-related genes. Cell death in these plants was limited to palisade mesophyll cells and required light for induction. Mesophyll cells of 35S::Pto plants showed the accumulation of autofluorescent compounds, callose deposition, and lignification. When inoculated with P. s. tomato without avrPto, all three 35S::Pto lines displayed significant resistance and supported less bacterial growth than did nontransgenic lines. Similarly, the 35S::Pto lines also were more resistant to Xanthomonas campestris pv vesicatoria and Cladosporium fulvum. These results demonstrate that defense responses and general resistance can be activated by the overexpression of an R gene.