Characterization of each aefR and mexT mutant in Pseudomonas syringae pv. tabaci 6605.

To investigate the mechanism of activation of the genes for resistance-nodulation-division (RND) family members MexE, MexF, and OprN for multidrug resistance (MDR), we mutagenized aefR and mexT, the potential regulators of mexEF/oprN transcription in Pseudomonas syringae pv. tabaci 6605 (Pta 6605). AefR is a member of the TetR transcription factors, and is known to be required for production of the quorum-sensing molecules, acyl homoserine lactones (AHL), in P. syringae. Furthermore, we found that AHL-synthesis-defective mutant strains in Pta 6605 showed enhanced expression of mexEF/oprN, and were highly tolerant to antimicrobial compounds such as chloramphenicol. MexT is a LysR-type transcription factor and is known to positively regulate transcription of mexEF/oprN in Pseudomonas aeruginosa. The ∆aefR mutant reduced the amount of growth in in vitro culture, caused the loss of AHL production, reduced the swarming motility, virulence and expression of psyI (AHL synthase) and psyR (AHL transcriptional regulator), and enhanced mexEF/oprN expression and tolerance to chloramphenicol, whereas the ∆mexT mutant retained the ability to produce AHL and did not show remarkable changes in in vitro growth, tolerance to antimicrobial compounds or virulence. Furthermore, unlike P. aeruginosa, the expression of mexEF/oprN is independent of MexT. These results indicate that (1) AefR is a regulator for the quorum-sensing system and MDR, and is required for swarming motility and virulence toward the host tobacco plant, and (2) MexT is not involved in the expression of mexEF/oprN in this bacterium.

Two flagellar stators and their roles in motility and virulence in Pseudomonas syringae pv. tabaci 6605.

The motor proteins around the flagellar basal body consist of two cytoplasmic membrane proteins, MotA and MotB, and function as a complex that acts as the stator to generate the torque that drives rotation. Genome analysis of several Pseudomonas syringae pathovars revealed that there are two sets of genes encoding motor proteins: motAB and motCD. Deduced amino acid sequences for MotA/B and MotC/D showed homologies to the H(+)-driven stator from Escherichia coli and Na(+)-driven stator from Vibrio alginolyticus, respectively. However, the swimming motility of P. syringae pv. tabaci (Pta) 6605 was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone but not by the sodium stator-specific inhibitor phenamil. To identify a gene encoding the stator protein required for motility, ∆motAB, ∆motCD, and ∆motABCD mutants were generated. The ∆motCD mutant had remarkably reduced and the ∆motABCD mutant completely abolished swimming motilities, whereas the ∆motAB mutant retained some degree of these abilities. The ∆motCD and ∆motABCD mutants did not produce N-acyl-homoserine lactones (AHLs), quorum-sensing molecules in this pathogen, and remarkably reduced the ability to cause disease in host tobacco leaves, as we previously observed in the ∆fliC mutant strain. These results strongly indicate that both stator pairs in Pta 6605 are proton-dependent and that MotCD is important for not only flagellar motility but also for production of AHLs and the ability to cause disease in host plants.

The siderophore pyoverdine of Pseudomonas syringae pv. tabaci 6605 is an intrinsic virulence factor in host tobacco infection.

To investigate the role of iron uptake mediated by the siderophore pyoverdine in the virulence of the plant pathogen Pseudomonas syringae pv. tabaci 6605, three predicted pyoverdine synthesis-related genes, pvdJ, pvdL, and fpvA, were mutated. The pvdJ, pvdL, and fpvA genes encode the pyoverdine side chain peptide synthetase III L-Thr-L-Ser component, the pyoverdine chromophore synthetase, and the TonB-dependent ferripyoverdine receptor, respectively. The Delta pvdJ and Delta pvdL mutants were unable to produce pyoverdine in mineral salts-glucose medium, which was used for the iron-depleted condition. Furthermore, the Delta pvdJ and Delta pvdL mutants showed lower abilities to produce tabtoxin, extracellular polysaccharide, and acyl homoserine lactones (AHLs), which are quorum-sensing molecules, and consequently had reduced virulence on host tobacco plants. In contrast, all of the mutants had accelerated swarming ability and increased biosurfactant production, suggesting that swarming motility and biosurfactant production might be negatively controlled by pyoverdine. Scanning electron micrographs of the surfaces of tobacco leaves inoculated with the mutant strains revealed only small amounts of extracellular polymeric matrix around these mutants, indicating disruption of the mature biofilm. Tolerance to antibiotics was drastically increased for the Delta pvdL mutant, as for the Delta psyI mutant, which is defective in AHL production. These results demonstrated that pyoverdine synthesis and the quorum-sensing system of Pseudomonas syringae pv. tabaci 6605 are indispensable for virulence in host tobacco infection and that AHL may negatively regulate tolerance to antibiotics.

RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens.

Colletotrichum higginsianum is a fungal pathogen that infects a wide variety of cruciferous plants, causing important crop losses. We have used map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum. This locus named RCH2 (for recognition of C. higginsianum) maps in an extensive cluster of disease-resistance loci known as MRC-J in the Arabidopsis ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 (resistance to Ralstonia solanacearum 1) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1-2, were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum. The screening identified an additional susceptible mutant (rps4-21) that has a 5-bp deletion in the neighboring gene, RPS4-Ws, which is a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). The rps4-21/rrs1-1 double mutant exhibited similar levels of susceptibility to C. higginsianum as the single mutants. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4. Thus, RPS4-Ws and RRS1-Ws function as a dual resistance gene system that prevents infection by three distinct pathogens.

Peroxisome biogenesis factor PEX13 is required for appressorium-mediated plant infection by the anthracnose fungus Colletotrichum orbiculare.

Peroxisomes are ubiquitous organelles of eukaryotic cells that fulfill a variety of biochemical functions, including beta-oxidation of fatty acids. Here, we report that an ortholog of the Saccharomyces cerevisiae peroxisome biogenesis gene PEX13 is required for pathogenicity of Colletotrichum orbiculare. CoPEX13 was identified by screening random insertional mutants for deficiency in fatty acid utilization. Targeted knockout mutants of CoPEX13 were unable to utilize fatty acids as a carbon source. Expression analysis using green fluorescent protein fused to the peroxisomal targeting signals PTS1 and PTS2 revealed that the import machinery for peroxisomal matrix proteins was impaired in copex13 mutants. Appressoria formed by the copex13 mutants were defective in both melanization and penetration ability on host plants, had thin cell walls, and lacked peroxisomes. Moreover, the concentration of intracellular glycerol was lower in copex13 appressoria than those of the wild type. These findings indicate that fatty acid oxidation in peroxisomes is required not only for appressorium melanization but also for cell wall biogenesis and metabolic processes involved in turgor generation, all of which are essential for appressorium penetration ability.

Amino acid sequence of bacterial microbe-associated molecular pattern flg22 is required for virulence.

Flagellin proteins derived from Pseudomonas syringae pv. tabaci 6605 and flg22Pa (QRLSTGSRINSAKDDAAGLQIA), one of the microbe-associated molecular patterns (MAMP) in bacterial flagellin, induce cell death and growth inhibition in Arabidopsis thaliana. To examine the importance of aspartic acid (D) at position 43 from the N-terminus of a flagellin in its elicitor activity, D43 was replaced with valine (V) and alanine (A) in P. syringae pv. tabaci flagellin and flg22Pta. The abilities of flagellins from P. syringae pv. tabaci D43V and D43A to induce cell death and growth inhibition were reduced, whereas the abilities of flg22PtaD43V and flg22PtaD43A were abolished. These results indicate that D43 is important for elicitor activity in P. syringae pv. tabaci. When tobacco plants were inoculated with each bacterium by the spray method, both P. syringae pv. tabaci D43V and D43A mutants had remarkably reduced ability to cause disease symptoms. Both mutants had reduced or no swimming and swarming motilities and adhesion ability. In P. syringae pv. tabaci D43V, little flagellin protein was detected and few flagella were observed by electron microscopy. These results indicate that mutant flagella are unstable and that flagellar motility is impaired. Thus, the amino acid residue required for MAMP activity is important for the intrinsic flagellar function.

Elicitin-responsive lectin-like receptor kinase genes in BY-2 cells.

The inhibition of elicitor-induced plant defense responses by the protein kinase inhibitors K252a and staurosporine indicates that defense responses require protein phosphorylation. We isolated a cDNA clone encoding Nicotiana tabacum lectin-like receptor protein kinase 1 (NtlecRK1), an elicitor-responsive gene; in tobacco bright yellow (BY-2) cells by a differential display method. NtlecRK forms a gene family with at least three members in tobacco. All three NtlecRK genes potentially encode the N-terminal legume lectin domain, transmembrane domain and C-terminal Ser/Thr-type protein kinase domain. Green fluorescent protein (GFP) fusion showed that the NtlecRK1 protein was located on the plasma membrane. In addition, NtlecRK1 and 3 were responsive to INF1 elicitin and the bacterial elicitor harpin. These results indicate that NtlecRKs are membrane-located protein kinases that are induced during defense responses in BY-2 cells.

Ultrastructural and Cytological Studies on Mycosphaerella pinodes Infection of the Model Legume Medicago truncatula.

Ascochyta (Mycosphaerella) blight on cultivated peas is primarily caused by infection through asexual spores (pycnospores) of Mycosphaerella pinodes (Berk. et Blox.) Vestergren [recently renamed Peyronellaea pinodes (Berk. & A. Bloxam) Aveskamp, Gruyter & Verkley]. Using a model pathosystem involving Medicago truncatula and Mycosphaerella pinodes strain OMP-1, we examined the histology and ultrastructure of early infection events and fungal development including penetration by appressoria, vegetative growth of infection hyphae, and host responses. On the susceptible ecotype R108-1, pycnospores germinated and grew over the surface of the epidermis, then formed an appressoria and penetrated the cuticle. Beneath the cuticle, the infection peg expanded into a hyphae that grew within the outer wall of the epidermis. Subsequently, the hyphae penetrated down within mesophyll cells and proliferated vigorously, eventually, forming asexual fruiting bodies (pycnidia). In contrast, successful penetration and subsequent growth of infection hyphae were considerably restricted in the ecotype Caliph. Detected by its reaction with cerium chloride (CeCl3) to generate electron-dense cerium perhydroxides in transmission electron micrographs, hydrogen peroxide (H2O2) accumulated in epidermal and mesophyll cells of Caliph challenged with pycnospores of M. pinodes. This intracellular localization was confirmed by energy-dispersive X-ray spectroscopy. Our observations thus indicate that the oxidative burst reaction leading to the generation of reactive oxygen species is associated with a local host defense response in Caliph, since no clear H2O2 accumulation was detectable in susceptible R108-1. Indeed, aberrant hyphae such as intrahyphal hyphae and dead hyphae, probably due to a local defense elicited by the fungus, were abundant in Caliph but not in R108-1. Our results on the cellular interactions between the fungus and host cells provide additional insights to understand foliar infection by M. pinodes on cultivated peas.

Leucine zipper motif in RRS1 is crucial for the regulation of Arabidopsis dual resistance protein complex RPS4/RRS1.

Arabidopsis thaliana leucine-rich repeat-containing (NLR) proteins RPS4 and RRS1, known as dual resistance proteins, confer resistance to multiple pathogen isolates, such as the bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum and the fungal pathogen Colletotrichum higginsianum. RPS4 is a typical Toll/interleukin 1 Receptor (TIR)-type NLR, whereas RRS1 is an atypical TIR-NLR that contains a leucine zipper (LZ) motif and a C-terminal WRKY domain. RPS4 and RRS1 are localised near each other in a head-to-head orientation. In this study, direct mutagenesis of the C-terminal LZ motif in RRS1 caused an autoimmune response and stunting in the mutant. Co-immunoprecipitation analysis indicated that full-length RPS4 and RRS1 are physically associated with one another. Furthermore, virus-induced gene silencing experiments showed that hypersensitive-like cell death triggered by RPS4/LZ motif-mutated RRS1 depends on EDS1. In conclusion, we suggest that the RRS1-LZ motif is crucial for the regulation of the RPS4/RRS1 complex.

Treatment of leptothrix cells with ultrapure water poses a threat to their viability.

The genus Leptothrix, a type of Fe/Mn-oxidizing bacteria, is characterized by its formation of an extracellular and microtubular sheath. Although almost all sheaths harvested from natural aquatic environments are hollow, a few chained bacterial cells are occasionally seen within some sheaths of young stage. We previously reported that sheaths of Leptothrix sp. strain OUMS1 cultured in artificial media became hollow with aging due to spontaneous autolysis within the sheaths. In this study, we investigated environmental conditions that lead the OUMS1 cells to die. Treatment of the cells with ultrapure water or acidic buffers (pH 6.0) caused autolysis of the cells. Under these conditions, the plasma membrane and cytoplasm of cells were drastically damaged, resulting in leakage of intracellular electrolytes and relaxation of genomic DNA. The autolysis was suppressed by the presence of Ca2+. The hydrolysis of peptidoglycan by the lysozyme treatment similarly caused autolysis of the cells and was suppressed also by the presence of Ca2+. However, it remains unclear whether the acidic pH-dependent autolysis is attributable to damage of peptidoglycan. It was observed that L. discophora strain SP-6 cells also underwent autolysis when suspended in ultrapure water; it is however, uncertain whether this phenomenon is common among other members of the genus Leptothrix.

Characterization of quorum sensing-controlled transcriptional regulator MarR and Rieske (2Fe-2S) cluster-containing protein (Orf5), which are involved in resistance to environmental stresses in Pseudomonas syringae pv. tabaci 6605.

Pseudomonas syringae pv. tabaci 6605 (Pta6605) produces acyl homoserine lactones (AHLs), quorum sensing (QS) molecules that are indispensable for virulence in host tobacco infection. Genome-wide transcriptional profiling of several QS-defective mutants revealed that the expression of the genes encoding the MarR family transcriptional regulator (MarR) and a Rieske 2Fe-2S cluster-containing protein (Orf5) located adjacent to psyI, a gene encoding AHL synthetase, are significantly repressed. Exogenous application of AHL recovered the expression of both marR and orf5 genes in the ΔpsyI mutant, indicating that AHL positively regulates the expression of these genes. To investigate the role of these genes in the virulence of Pta6605, ΔmarR and Δorf5 mutants were generated. Both mutants showed decreased swimming and swarming motilities, decreased survival ability under oxidative and nitrosative stresses and, consequently, reduced virulence on host tobacco plants. Transmission electron micrographs showed that the structure of the cell membranes of ΔmarR and Δorf5 mutants was severely damaged. Furthermore, not only the ratio of dead cells, but also the amount of flagella, extracellular DNA and protein released into the culture supernatant, was significantly increased in both mutants, indicating that the disruption of marR and orf5 genes might induce structural changes in the membrane and cell lysis. Because both mutants showed partly similar expression profiles, both gene products might be involved in the same regulatory cascades that are required for QS-dependent survival under environmentally stressed conditions.

The Colletotrichum lagenariu Ste12-like gene CST1 is essential for appressorium penetration.

Colletotrichum lagenarium is the causal agent of anthracnose of cucumber. This fungus produces a darkly melanized infection structure, appressoria, to penetrate the host leaves. The C. lagenarium CMK1 gene, a homologue of the Saccharomyces cerevisiae FUS3/KSS1 mitogen-activated protein (MAP) kinase genes, was shown to regulate conidial germination, appressorium formation, and invasive growth. In S. cerevisiae, Ste12p is known to be a transcriptional factor downstream of Fus3p/Kss1p MAP kinases. To evaluate the CMK1 MAP kinase pathway, we isolated the Ste12 homologue CST1 gene from C. lagenarium and characterized. The cst1delta strains were nonpathogenic on intact host leaves, but could form lesions when inoculated on wounded leaves. Conidia of the cst1delta strains could germinate and form melanized appressoria on both host leaf surface and artificial cellulose membrane, but could not produce infectious hyphae from appressoria, suggesting that CST1 is essential for appressorium penetration in C. lagenarium. In addition, matured appressoria of the cst1delta strains contained an extremely low level of lipid droplets compared with that of the wild-type strain. Lipid droplets were abundant in conidia of the cst1delta strains, but rapidly disappeared during appressorium formation. This misscheduled lipid degradation might be related to the failure of appressorium penetration in the cst1delta strain.

RCH1, a locus in Arabidopsis that confers resistance to the hemibiotrophic fungal pathogen Colletotrichum higginsianum.

When challenged with the crucifer pathogen Colletotrichum higginsianum, Arabidopsis thaliana ecotype Columbia (Col-0) was colonized by the fungus within 2 to 3 days, developing brown necrotic lesions surrounded by a yellow halo. Lesions spread from the inoculation site within 3 to 4 days, and subsequently continued to expand until they covered the entire leaf. Electron microscopy confirmed that C. higginsianum is a hemibiotroph on Arabidopsis, feeding initially on living cells as a biotroph before switching to a necrotrophic mode of growth. A collection of 37 ecotypes of Arabidopsis varied in their responses to infection by C. higginsianum. The ecotype Eil-0 was highly resistant, with symptoms limited to necrotic flecking and with only very limited fungal colonization. Analyses suggested that the hypersensitive response and reactive oxygen species may be important in this defense response. Expression analyses with cDNA microarrays indicated that the defense reaction depends primarily on the jasmonic acid- and ethylene-dependent signaling pathways and, to a lesser extent, on the salicylate-dependent pathway. Crosses between the Eil-0 and Col-0 ecotypes suggested that the resistance in Eil-0 was dominant and was conferred by a single locus, which we named RCH1. RCH1 is the first resistance locus to be identified from Arabidopsis against the hemibiotrophic fungus genus Colletotrichum.

cDNA cloning and characterization of tobacco ABC transporter: NtPDR1 is a novel elicitor-responsive gene.

We isolated an INF1 elicitin-inducible cDNA encoding a pleiotropic drug resistance (PDR)-type ATP-binding cassette (ABC) transporter homolog (NtPDR1) in suspension-cultured tobacco Bright Yellow-2 (BY-2) cells by application of differential display PCR. The NtPDR1 (Nicotiana tabacum PDR protein 1) gene also encodes a 162 kDa protein that includes two putative hydrophilic domains containing the ABC signature motif and two putative hydrophobic domains. Expression of the NtPDR1 gene was rapidly and strongly activated by treatment of BY-2 cells with INF1 elicitin. Further, treatment of BY-2 cells with flagellin, a bacterial proteinaceous hypersensitive reaction elicitor, or yeast extract, a general elicitor, also induced NtPDR1 gene expression. These results indicate that NtPDR1 may be involved in the general defense response in tobacco. This is the first report that microbial elicitors induce the expression of a plant ABC transporter gene.

Genomic structure of the NtPDR1 gene, harboring the two miniature inverted-repeat transposable elements, NtToya1 and NtStowaway101.

Here we report the genomic structure including the promoter sequence and coding region of NtPDR1 (Nicotiana tabacum Pleiotropic Drug Resistance 1), which is an elicitor-responsive gene encoding an ATP binding cassette (ABC) transporter that might be involved in the defense response in tobacco, as we reported recently. The NtPDR1 gene consists of 20 exons and 19 introns. Among the introns, the first and fifth are much larger than the others and harbor typical miniature inverted-repeat transposable elements (MITEs). One of the MITE elements in the first intron, termed NtToya1, belongs to the Toya family that was recently described in rice, while the other element in the fifth intron, termed NtStowaway101, shows high homology with the Stowaway elements of the IS630-Tc1-mariner family. Many of the genes we found to harbor Toya and Stowaway elements in Nicotiana species by BLAST search are also involved in stress responses or plant-pathogen interactions. The existence of putative cis-elements (a GCC box, three W boxes, and several JA-responsive elements) in the promoter region supports our previous finding that this gene is strongly inducible by elicitation and methyljasmonate, and that this ABC transporter might be essential for plant defense responses. Furthermore, Southern blot analysis and PCR amplification of the introns harboring the MITE-like elements from genomic DNA of three Nicotiana species suggests that NtPDR1 originated from N. sylvestris.

Occurrence of Free N-Glycans in Pea (Pisum sativum, L) Seedlings†.

Free N-glycans have been found in pea seedlings. These free N-glycans were coupled with 2-aminopyridine and purified by gel filtration, Con Α-Sepharose affinity chromatography, and size fractionation HPLC. These structures of pyridylaminated free N-glycans were analyzed by exomannosidase digestions and ion- spray tandem mass spectrometry. The structural analyses showed that the several oligomannose-type sugar chains having one GlcNAc residue at the reducing-end side occur in the seedlings, suggesting the endo-ß-N-acetylgIucosaminidase PS [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 228-232 (1996)] should be involved in the release of oligomannose-type N-glycans from the storage glycoproteins [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 1841-1850 (1996)] during the germination of pea seeds.

Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum.

Ralstonia solanacearum is a Gram-negative soil-borne bacterium that causes bacterial wilt disease in more than 200 plant species, including economically important Solanaceae species. In R. solanacearum, the hypersensitive response and pathogenicity (Hrp) type III secretion system is required for both the ability to induce the hypersensitive response (HR) in nonhost plants and pathogenicity in host plants. Recently, 72 effector genes, called rip (Ralstonia protein injected into plant cells), have been identified in R. solanacearum RS1000. RS1002, a spontaneous nalixidic acid-resistant derivative of RS1000, induced strong HR in the nonhost wild eggplant Solanum torvum in an Hrp-dependent manner. An Agrobacterium-mediated transient expression system revealed that Rip36, a putative Zn-dependent protease effector of R. solanacearum, induced HR in S. torvum. A mutation in the putative Zn-binding motif (E149A) completely abolished the ability to induce HR. In agreement with this result, the RS1002-derived Δrip36 and rip36E149A mutants lost the ability to induce HR in S. torvum. An E149A mutation had no effect on the translocation of Rip36 into plant cells. These results indicate that Rip36 is an avirulent factor that induces HR in S. torvum and that a putative Zn-dependent protease motif is essential for this activity.

Type IV pilin is glycosylated in Pseudomonas syringae pv. tabaci 6605 and is required for surface motility and virulence.

Type IV pilin (PilA) is a major constituent of pilus and is required for bacterial biofilm formation, surface motility and virulence. It is known that mature PilA is produced by cleavage of the short leader sequence of the pilin precursor, followed by methylation of N-terminal phenylalanine. The molecular mass of the PilA mature protein from the tobacco bacterial pathogen Pseudomonas syringae pv. tabaci 6605 (Pta 6605) has been predicted to be 12 329 Da from its deduced amino acid sequence. Previously, we have detected PilA as an approximately 13-kDa protein by immunoblot analysis with anti-PilA-specific antibody. In addition, we found the putative oligosaccharide-transferase gene tfpO downstream of pilA. These findings suggest that PilA in Pta 6605 is glycosylated. The defective mutant of tfpO (ΔtfpO) shows reductions in pilin molecular mass, surface motility and virulence towards host tobacco plants. Thus, pilin glycan plays important roles in bacterial motility and virulence. The genetic region around pilA was compared among P. syringae pathovars. The tfpO gene exists in some strains of pathovars tabaci, syringae, lachrymans, mori, actinidiae, maculicola and P. savastanoi pv. savastanoi. However, some strains of pathovars tabaci, syringae, glycinea, tomato, aesculi and oryzae do not possess tfpO, and the existence of tfpO is independent of the classification of pathovars/strains in P. syringae. Interestingly, the PilA amino acid sequences in tfpO-possessing strains show higher homology with each other than with tfpO-nonpossessing strains. These results suggest that tfpO and pilA might co-evolve in certain specific bacterial strains.

Degeneration of hrpZ gene in Pseudomonas syringae pv. tabaci to evade tobacco defence: an arms race between tobacco and its bacterial pathogen.

The HrpZ harpin of Pseudomonas syringae is known to induce a hypersensitive response (HR) in some plants. In P. syringae pv. tabaci (Pta), the harpin gene hrpZ has been spontaneously disrupted by an internal deletion in its open reading frame and a frame shift. The loss of the ability of the recombinant harpin polypeptide of Pta to induce HR despite the high sensitivity of tobacco plants to harpin led us to investigate the meaning of the disrupted hrpZ gene in the virulence of Pta 6605. The hrpZ gene from P. syringae pv. pisi was introduced into wild-type (WT) Pta. The hrpZ-complemented Pta secreted harpin into the culture medium, but failed to cause disease symptoms by both infiltration and spray inoculation. Inoculation with the hrpZ-complemented Pta induced defence responses in tobacco plants, whereas the defence responses of tobacco plants were not prominent on inoculation with WT Pta. These results indicate that an ancestor of Pta might have disrupted hrpZ by an internal deletion to evade plant defences and confer the ability to cause disease in tobacco plants.

A dual resistance gene system prevents infection by three distinct pathogens.

Colletotrichum higginsianum causes typical anthracnose lesions on the leaves, petioles, and stems of cruciferous plants. Inoculation of Arabidopsis thaliana ecotype Columbia leaves with C. higginsianum results in fungal growth and disease symptoms reminiscent of those induced in other cruciferous plants. We performed map-based cloning and natural variation analysis of 19 A. thaliana ecotypes to identify a dominant resistance locus against C. higginsianum. We found that the A. thaliana RCH2 (for recognition of C. higginsianum) locus encodes two NB-LRR proteins, both of which are required for resistance to C. higginsianum in the A. thaliana ecotype Ws-0. Both proteins are well-characterized R proteins involved in resistance against bacterial pathogens; RRS1 (resistance to Ralstonia solanacearum 1) confers resistance to strain Rs1000 of R. solanacearum and RPS4 to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). Furthermore, we found that both RRS1-Ws and RPS4-Ws genes are required for resistance to Pst-avrRps4 and to Rs1002 R. solanacearum. We therefore demonstrate that a pair of neighboring genes, RRS1-Ws and RPS4-Ws, function cooperatively as a dual R-gene system against at least three distinct pathogens.