Syringolide 1 Triggers Ca2+ Influx,K+ Efflux,and Extracellular Alkalization in Soybean Cells Carrying the Disease-Resistance Gene Rpg4

Alleles of avirulence gene D (avrD) specify the production by bacteria of syringolides that elicit the hypersensitive response in soybean (Glycine max) plants carrying the disease-resistance gene Rpg4, but not rpg4 plants. Syringolide 1 caused extracellular alkalization, K+ efflux, and Ca2+ influx about 30 min after addition to suspension-cultured cells of two Rpg4 cultivars, Harosoy and Flambeau, but not in two rpg4 cultivars, Acme and Merit. All responses were sustained for at least 1.5 h and were inhibited by La3+, which blocks certain Ca2+ channels. These results suggest that syringolide 1 activates a Ca2+ influx-dependent signaling pathway only in Rpg4 soybean cells.     

Cloning of soybean genes induced during hypersensitive cell death caused by syringolide elicitor

Syringolide elicitors produced by bacteria expressing Pseudomonas syringae pv. glycinea avirulence gene D (avrD) induce hypersensitive cell death (HCD) only in soybean (Glycine max [L.] Merr.) plants carrying the Rpg4 disease resistance gene. Employing a differential display method, we isolated 13 gene fragments induced in cultured cells of a soybean cultivar Harosoy (Rpg4) treated with syringolides. Several genes for isolated fragments were induced by syringolides in an rpg4 cultivar Acme as well as in Harosoy; however, the genes for seven fragments designated as SIH (for syringolide-induced/HCD associated) were induced exclusively or strongly in Harosoy. cDNA clones for SIH genes were obtained from a cDNA library of Harosoy treated with syringolide. Several sequences are homologous to proteins associated with plant defense responses. The SIH genes did not respond to a non-specific -glucan elicitor, which induces phytoalexin accumulation but not HCD, suggesting that the induction of the SIH genes is specific for the syringolide–Harosoy interaction. HCD and the induction of SIH genes by syringolides were independent of H₂O₂. On the other hand, Ca²⁺ was required for HCD and the induction of some SIH genes. These results suggest that the induction of SIH genes by syringolides could be activated through the syringolide-specific signaling pathway and the SIH gene products may play an important role(s) in the processes of HCD induced by syringolides.Abbreviations AOS active oxygen species - CHS chalcone synthase - DPI diphenylene iodonium - HCD hypersensitive cell death - HR hypersensitive response - PAL phenylalanine ammonia lyase - SID syringolide-induced/defense associated - SIG syringolide-induced/general - SIH for syringolide-induced/HCD associated - XET xyloglucan endotransglycosylase     

The rpg4/ Rpg5 stem rust resistance locus in barley; resistance genes and cytoskeleton dynamic

Two closely linked resistance genes, rpg4 and Rpg5, conferring resistance to several races of Puccinia graminis, were cloned and characterized. The Rpg5 gene confers resistance to an isolate of Puccinia graminis f. sp. secalis (Pgs), while rpg4 confers resistance to Puccinia graminis f. sp. tritici (Pgt). Rpg5 is a novel gene containing nucleotide binding site-leucine rich repeat domains in combination with a serine threonine protein kinase domain. High-resolution mapping plus allele and recombinant sequencing identified the rpg4 gene, which encodes an actin depolymerizing factor-like protein (ADF2). Resistance against the Pgt races QCCJ, MCCF, TTKSK (aka Ug99) and RCRS requires both Rpg5 and rpg4, while Rpg5 alone confers resistance to Pgs isolate 92-MN-90. The dependency on the actin modifying protein ADF2 indicates cytoskeleton reorganization or redirection plays a role in pathogen-host interactions. Rpg5 may interact with ADF2 to activate or deactivate its function in the resistance response. Alternatively, Rpg5 could initiate signal transduction leading to resistance in response to detecting ADF2 protein modification. Pgt may redirect the actin cytoskeleton by inducing modifications of ADF2. The redirection of actin could possibly enable the pathogen to develop a haustoria-plant cell cytoskeleton interface for acquisition of nutrients.     

A specific, high-affinity binding site for the hepta-beta-glucoside elicitor exists in soybean membranes.

The presence of a specific binding site for a hepta-beta-glucoside elicitor of phytoalexin accumulation has been demonstrated in soybean microsomal membranes. A tyramine conjugate of the elicitor-active hepta-beta-glucoside was prepared and radiolabeled with 125I. The labeled hepta-beta-glucoside-tyramine conjugate was used as a ligand in binding assays with a total membrane fraction prepared from soybean roots. Binding of the radiolabeled hepta-beta-glucoside elicitor was saturable, reversible, and with an affinity (apparent Kd = 7.5 x 10(-10) M) comparable with the concentration of hepta-beta-glucoside required for biological activity. A single class of hepta-beta-glucoside binding sites was found. The binding site was inactivated by proteolysis and by heat treatment, suggesting that the binding site is a protein or glycoprotein. Competitive inhibition of binding of the radiolabeled hepta-beta-glucoside elicitor by a number of structurally related oligoglucosides demonstrated a direct correlation between the binding affinities and the elicitor activities of these oligoglucosides. Thus, the hepta-beta-glucoside-binding protein fulfills criteria expected of a bona fide receptor for the elicitor-active oligosaccharin.     

The Saccharomyces cerevisiae HCR1 gene encoding a homologue of the p35 subunit of human translation initiation factor 3 (eIF3) is a high copy suppressor of a temperature-sensitive mutation in the Rpg1p subunit of yeast eIF3.

The complex eukaryotic initiation factor 3 (eIF3) was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary complex, and aiding mRNA binding to 40 S ribosomal subunits. Recently, we described the identification of RPG1 (TIF32), the p110 subunit of the eIF3 core complex in yeast. In a screen for Saccharomyces cerevisiae multicopy suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified. When overexpressed, the 30-kDa gene product, named Hcr1p, was able to support, under restrictive conditions, growth of the rpg1-1 temperature-sensitive mutant, but not of a Rpg1p-depleted mutant. An hcr1 null mutant was viable, but showed slight reduction of growth when compared with the wild-type strain. Physical interaction between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation analysis. The combination of Deltahcr1 and rpg1-1 mutations resulted in a synthetic enhancement of the slow growth phenotype at a semipermissive temperature. In a computer search, a significant homology to the human p35 subunit of the eIF3 complex was found. We assume that the yeast Hcr1 protein participates in translation initiation likely as a protein associated with the eIF3 complex.     

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.     

The barley serine/threonine kinase gene Rpg1 providing resistance to stem rust belongs to a gene family with five other members encoding kinase domains

The barley (Hordeum vulgare L.) stem rust (Puccinia graminis f. sp. tritici) resistance gene Rpg1 encodes a serine/threonine protein kinase with two tandem kinase domains. The Rpg1 gene family was identified from the cv. Morex and consists of five additional members with divergent homology to Rpg1. All family members encode serine/threonine kinase-like proteins with at least one predicted catalytically active kinase domain. The five family members were sequenced from cDNA and genomic DNA and genetically mapped. The family member most closely related to Rpg1, ABC1037, is located on chromosome 1(7H) bin 01, very near (∼50 kb) but not co-segregating with Rpg1. Two others, ABC1036 and ABC1040, are closely related to each other and tightly linked on chromosome 7(5H) bin 07. ABC1041 mapped to chromosome 7(5H) bin 13, tightly linked to the rust resistance genes rpg4 and Rpg5 providing resistance to barley stem rust pathotype QCC and rye stem rust pathotype 92-MN-90, respectively, but segregated away in a high-resolution population. ABC1063 was localized to chromosome 4(4H) bin 6. An interesting Rpg1 allele that appears to be the result of unequal recombination between Rpg1 and ABC1037 was characterized. No known resistance loci cosegregated with any family members, however characterization of the Rpg1 family has provided insight into the evolution of this novel gene family and may present tools for understanding the functional domains of Rpg1. The genetic mapping, gene structures, and analysis of amino-acid sequences of the Rpg1 gene family members are presented.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Characterization of pPT23B, the Plasmid Involved in Syringolide Production by Pseudomonas syringae pv. tomato PT23

Avirulence gene D (avrD) in strain PT23 of Pseudomonas syringae pv. tomato (Pst) specifies the production of syringolides, which are elicitors of plant defense reactions. An 83-kb indigenous plasmid (pPT23B) that carries avrD has been mapped and characterized and a putative par region was identified. pPT23B contains a large amount of DNA that is repeated in other native plasmids in PT23. A putative mobile insertion element that occurs on plasmid pPT23A as well as on the chromosome was also identified in strain PT23. New broad-host-range expression vectors that functioned in Pst were constructed for overexpression of the cloned avrD gene and high-level production of the syringolides. Introduction of an avrD overexpression plasmid into PT23 or plasmid-cured strains led to identical syringolide peaks on HPLC with no new peaks observed. These results suggested that neither pPT23B nor other indigenous plasmids in Pst carry additional genes required for syringolide production or metabolism. Pst strains lacking pPT23B were not impaired in virulence on tomato plants.     

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.     

The P34 syringolide elicitor receptor interacts with a soybean photorespiration enzyme,NADH-dependent hydroxypyruvate reductase

The syringolide receptor P34 mediates avrD-Rpg4 gene-for-gene complementarity in soybean. However, the mechanism underlying P34 signal transmission after syringolide binding is unknown. In an effort to identify a second messenger for P34, soybean leaf proteins were run though a P34-affinity column. A 42-kDa protein which specifically bound to the column was identified as a putative plant NADH-dependent hydroxypyruvate reductase (HPR) by N-terminal peptide sequencing. HPR is an important enzyme involved in the plant photorespiration system. Screening of a soybean cDNA library yielded two distinct HPR clones that encoded proteins with 97% identity (P42-1 and P42-2). Surprisingly, only P42-2 displayed good binding with P34 in a yeast two-hybrid assay, indicating that P42-2, but not P42-1, is a potential second messenger for P34. Glycerate and its analogs, which are utilized in the photorespiration system, were tested for their inhibitory effect on syringolide-induced hypersensitive response (HR) to evaluate the biological significance of P42-2. Interestingly, the downstream products of HPR (glycerate and 3-phosphoglycerate) inhibited HR but the upstream compounds (hydroxypyruvate or serine) did not have a significant effect on HR. These results suggest that P42-2 is a primary target for a P34/syringolide complex and that P42-2 binding with the complex probably induces HR by inhibiting one or more HPR functions in soybean.     

High-affinity binding proteins for N-acetylchitooligosaccharide elicitor in the plasma membranes from wheat,barley and carrot cells: conserved presence and correlation with the responsiveness to the elicitor

Binding experiments as well as affinity labeling with an (125)I-labeled 2-(4-aminophenyl)ethylamino derivative of N-acetylchitooctaose revealed the presence of high-affinity binding sites/proteins for N-acetylchitooligosaccharide elicitor in the plasma membrane preparation from suspension-cultured carrot cells, barley cells and wheat leaves. Their binding specificity corresponded with the elicitor activity of N-acetylchitooligosaccharides and related sugars in these plant cells/tissues, and was similar to that reported for the binding site/protein previously reported for suspension-cultured rice cells. The molecular size of the binding proteins identified in carrot, barley and wheat was slightly smaller than that of rice. These plant cells were shown to respond to N-acetylchitooligosaccharides and generate reactive oxygen species, induced medium alkalinization, or previously shown to initiate lignification (wheat leaves, Barber et al. (1989) Physiol. Mol. Plant Pathol. 34: 3). No elicitor-binding protein nor the elicitor-induced cellular responses was detected for a cell line of tobacco BY-2 (BY-2T). On the other hand, another cell line of tobacco BY-2 (BY-2N) showed the presence of elicitor-binding protein and also elicitor-induced medium alkalinization. Thus, there was a good correlation between the existence of high-affinity binding proteins for the elicitor and elicitor-induced cellular responses among tested plant cells. These results indicated the wide distribution of N-acetylchitooligosaccharide elicitor-binding protein among various plants and added further support for the function of these plasma membrane proteins in the perception of the elicitor signal.     

Convergent evolution of disease resistance gene specificity in two flowering plant families

Plant disease resistance (R) genes that mediate recognition of the same pathogen determinant sometimes can be found in distantly related plant families. This observation implies that some R gene alleles may have been conserved throughout the diversification of land plants. To address this question, we have compared R genes from Glycine max (soybean), Rpg1-b, and Arabidopsis thaliana, RPM1, that mediate recognition of the same type III effector protein from Pseudomonas syringae, AvrB. RPM1 has been cloned previously, and here, we describe the isolation of Rpg1-b. Although RPM1 and Rpg1-b both belong to the coiled-coil nucleotide binding site (NBS) Leu-rich repeat (LRR) class of R genes, they share only limited sequence similarity outside the conserved domains characteristic of this class. Phylogenetic analyses of A. thaliana and legume NBS-LRR sequences demonstrate that Rpg1-b and RPM1 are not orthologous. We conclude that convergent evolution, rather than the conservation of an ancient specificity, is responsible for the generation of these AvrB-specific genes.     

Pseudomonas syringae effector avrB confers soybean cultivar-specific avirulence on Soybean mosaic virus adapted for transgene expression but effector avrPto does not

Soybean mosaic virus (SMV) was adapted for transgene expression in soybean and used to examine the function of avirulence genes avrB and avrPto of Pseudomonas syringae pvs. glycinea and tomato, respectively. A cloning site was introduced between the P1 and HC-Pro genes in 35S-driven infectious cDNAs of strains SMV-N and SMV-G7. Insertion of the uidA gene or the green fluorescent protein gene into either modified cDNA and bombardment into primary leaves resulted in systemic expression that reflected the pattern of viral movement into uninoculated leaves. Insertion of avrB blocked symptom development and detectable viral movement in cv. Harosoy, which carries the Rpg1-b resistance gene corresponding to avrB, but not in cvs. Keburi or Hurrelbrink, which lack Rpg1-b. In Keburi and Hurrelbrink, symptoms caused by SMV carrying avrB appeared more quickly and were more severe than those caused by the virus without avrB. Insertion of avrPto enhanced symptoms in Harosoy, Hurrelbrink, and Keburi. This result was unexpected because avrPto was reported to confer avirulence on P. syringae pv. glycinea inoculated to Harosoy. We inoculated Harosoy with P syringae pv. glycinea expressing avrPto, but observed no hypersensitive reaction, avrPto-dependent induction of pathogenesis-related protein la, or limitation of bacterial population growth. In Hurrelbrink, avrPto enhanced bacterial multiplication and exacerbated symptoms. Our results establish SMV as an expression vector for soybean. They demonstrate that resistance triggered by avrB is effective against SMV, and that avrB and avrPto have general virulence effects in soybean. The results also led to a reevaluation of the reported avirulence activity of avrPto in this plant.     

A High-Affinity Binding Site for the AVR9 Peptide Elicitor of Cladosporium fulvum Is Present on Plasma Membranes of Tomato and Other Solanaceous Plants

The race-specific Cladosporium fulvum peptide elicitor AVR9, which specifically induces a hypersensitive response in tomato genotypes carrying the Cf-9 resistance gene, was labeled with iodine-125 at the N-terminal tyrosine residue and used in binding studies. 125I-AVR9 showed specific, saturable, and reversible binding to plasma membranes isolated from leaves of tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) or from a near-isogenic genotype with the Cf-9 resistance gene (MM-Cf9). The dissociation constant was found to be 0.07 nM, and the receptor concentration was 0.8 pmol/mg microsomal protein. Binding was highly influenced by pH and the ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydrophobic interactions. Binding kinetics and binding capacity were similar for membranes of the MM-Cf0 and MM-Cf9 genotypes. In all solanaceous plant species tested, an AVR9 binding site was present, whereas in the nonsolanaceous species that were analyzed, such a binding site could not be identified. The ability of membranes isolated from different solanaceous plant species to bind AVR9 seems to correlate with the presence of members of the Cf-9 gene family, but whether this correlation is functional remains to be determined.     

Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley

Rpg1 is a stem rust resistance gene that has protected barley from severe losses for over 60 years in the US and Canada. It confers resistance to many, but not all, pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. A fast neutron induced deletion mutant, showing susceptibility to stem rust pathotype Pgt-MCC, was identified in barley cv. Morex, which carries Rpg1. Genetic and Rpg1 mRNA and protein expression level analyses showed that the mutation was a suppressor of Rpg1 and was designated Rpr1 (Required for P. graminis resistance). Genome-wide expression profiling, using the Affymetrix Barley1 GeneChip containing ∼22,840 probe sets, was conducted with Morex and the rpr1 mutant. Of the genes represented on the Barley1 microarray, 20 were up-regulated and 33 were down-regulated by greater than twofold in the mutant, while the Rpg1 mRNA level remained constant. Among the highly down-regulated genes (greater than fourfold), genomic PCR, RT-PCR and Southern analyses identified that three genes (Contig4901_s_at, HU03D17U_s_at, and Contig7061_s_at), were deleted in the rpr1 mutant. These three genes mapped to chromosome 4(4H) bin 5 and co-segregated with the rpr1-mediated susceptible phenotype. The loss of resistance was presumed to be due to a mutation in one or more of these genes. However, the possibility exists that there are other genes within the deletions, which are not represented on the Barley1 GeneChip. The Rpr1 gene was not required for Rpg5- and rpg4-mediated stem rust resistance, indicating that it shows specificity to the Rpg1-mediated resistance pathway.     

Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.     

Binding and structural characteristics of a soluble lactogen-binding protein from rabbit mammary-gland cytosol.

Specific receptors for prolactin (PRL) are known to be present on plasma membranes and intracellular membranes of mammary gland. We now report, however, the detection and characterization of a soluble lactogen-specific binding protein in high-speed (200,000 g) cytosolic preparations from pregnant- and non-pregnant-rabbit mammary gland. The binding protein was not detectable by poly(ethylene glycol) precipitation; instead, bound and free 125I-labelled human growth hormone (hGH; a potent lactogen) was separated using a mini-gel filtration technique. Specific binding of 125I-hGH reached an apparent equilibrium between 10 and 14 h at 21-23 degrees C. It was dependent on mammary-gland protein concentration and, partially, on Ca2+ or Mg2+ concentrations. Scatchard analysis revealed steep curvilinear plots, the high-affinity component having a KA of approximately 3 X 10(10) M-1. Gel filtration on calibrated Ultrogel AcA34 columns of 125I-hGH-cytosol complexes or of cytosol alone, followed by measurement of 125I-hGH binding in each eluted fraction, indicated that the binding protein had an Mr of 33,000-43,000. A specific binding protein of the same size was observed when 125I-ovine or -human PRL, but not 125I-bovine GH, was used as ligand. The apparent lactogenic specificity was confirmed by a lack of cross-reactivity of the binding protein with an anti-[GH receptor (rabbit liver)] monoclonal antibody. Polyacrylamide-gel electrophoresis of 125I-hGH covalently cross-linked to cytosol with disuccinimidyl suberate revealed binding proteins of Mr 35,000 (non-reduced) and 37,000 (reduced), results comparable with those obtained by gel filtration and indicating an absence of inter-subunit disulphide bonds. These studies have shown the presence of an apparently naturally soluble lactogen-binding protein in the cytosolic fraction of rabbit mammary gland. The relationship between this binding protein and the membrane PRL receptor is not yet known.     

AvrB mutants lose both virulence and avirulence activities on soybean and Arabidopsis

The Pseudomonas syringae pv. glycinea effector protein AvrB induces resistance responses in soybean varieties that contain the resistance gene Rpg1-b and Arabidopsis varieties that carry RPM1. In addition to this avirulence activity, AvrB also enhances bacterial virulence on soybean plants that lack Rpg1-b and induces a chlorotic phenotype on Arabidopsis plants that lack RPM1. We screened a library of avrB mutants for loss of avirulence on soybean and Arabidopsis, and assayed selected avirulence mutants for loss of virulence on both plants. All mutants screened were recognized similarly on both plant species. Nine single-site avrB mutations that affected avirulence localized to a solvent-accessible pocket in the protein structure. Seven of these mutated residues are absolutely conserved between AvrB and its nine homologues. Avirulence mutants generally lost virulence enhancement on susceptible soybean varieties and lost the ability to induce a chlorotic response on the rpm1 null Arabidopsis variety Mt-0. Three of four avirulence mutants tested failed to interact with RIN4, an Arabidopsis protein previously shown to be required for RPM1 function. Our results suggest that soybean and Arabidopsis recognize AvrB in the same manner, and that AvrB enzymatic activity is required for its function as an avirulence and virulence effector on two different plant species.     

Determining the GmRIN4 Requirements of the Soybean Disease Resistance Proteins Rpg1b and Rpg1r Using a Nicotiana glutinosa-Based Agroinfiltration System

Rpg1b and Rpg1r are soybean disease resistance (R) genes responsible for conferring resistance to Pseudomonas syringae strains expressing the effectors AvrB and AvrRpm1, respectively. The study of these cloned genes would be greatly facilitated by the availability of a suitable transient expression system. The commonly used Niciotiana benthamiana-based system is not suitable for studying Rpg1b and Rpg1r function, however, because expression of AvrB or AvrRpm1 alone induces a hypersensitive response (HR), indicating that N. benthamiana contains endogenous R genes that recognize these effectors. To identify a suitable alternative host for transient expression assays, we screened 13 species of Nicotiana along with 11 accessions of N. tabacum for lack of response to transient expression of AvrB and AvrRpm1. We found that N. glutinosa did not respond to either effector and was readily transformable as determined by transient expression of β-glucuronidase. Using this system, we determined that Rpg1b-mediated HR in N. glutinosa required co-expression of avrB and a soybean ortholog of the Arabidopsis RIN4 gene. All four soybean RIN4 orthologs tested worked in the assay. In contrast, Rpg1r did not require co-expression of a soybean RIN4 ortholog to recognize AvrRpm1, but recognition was suppressed by co-expression with AvrRpt2. These observations suggest that an endogenous RIN4 gene in N. glutinosa can substitute for the soybean RIN4 ortholog in the recognition of AvrRpm1 by Rpg1r.