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In tomato plants, resistance to bacterial speck disease is mediated by a phosphorylation cascade, which is triggered by the specific recognition between the plant serine/threonine protein kinase Pto and the bacterial AvrPto protein. In the present study, we investigated in vitro biochemical properties of Pto, which appears to function as an intracellular receptor for the AvrPto signal molecule. Pto and its downstream effector Pti1, which is also a serine/threonine protein kinase, were expressed in Escherichia coli as maltose-binding protein and glutathione S-transferase fusion proteins, respectively. The two kinases each autophosphorylated at multiple sites as determined by phosphopeptide mapping. In addition, Pto and Pti1 autophosphorylation occurred via an intramolecular mechanism, as their specific activity was not affected by their molar concentration in the assay. Moreover, an active glutathione S-transferase-Pto fusion failed to phosphorylate an inactive maltose-binding protein-Pto(K69Q) fusion excluding an intermolecular mechanism of phosphorylation for Pto. Pti1 phosphorylation by Pto was also characterized and found to occur with a Km of 4.1 microM at sites similar to those autophosphorylated by Pti1. Pto and the product of the recessive allele pto phosphorylated Pti1 at similar sites, as observed by phosphopeptide mapping. This suggests that the inability of the kinase pto to confer resistance to bacterial speck disease in tomato is not caused by altered recognition specificity for Pti1 phosphorylation sites.

Resistance to bacterial speck disease in tomato occurs when the Pto kinase in the plant responds to expression of the avirulence gene avrPto in the Pseudomonas pathogen. Transient expression of an avrPto transgene in plant cells containing Pto elicited a defense response. In the yeast two-hybrid system, the Pto kinase physically interacted with AvrPto. Alterations of AvrPto or Pto that disrupted the interaction in yeast also abolished disease resistance in plants. The physical interaction of AvrPto and Pto provides an explanation of gene-for-gene specificity in bacterial speck disease resistance.

The Pto gene encodes a serine/threonine kinase that confers resistance in tomato to Pseudomonas syringae pv. tomato strains that express the avirulence gene avrPto . Partial characterization of the Pto signal transduction pathway and the availability of transgenic tomato lines (± Pto ) make this an ideal system for exploring the molecular basis of disease resistance. In this paper, we test two transgenic tomato cell suspension cultures (± Pto ) for production of H 2 O 2 following independent challenge with two strains of P. syringae pv. tomato (± avrPto ). Only when Pto and avrPto are present in the corresponding organisms are two distinct phases of the oxidative burst seen, a rapid first burst followed by a slower and more prolonged second burst. In the remaining three plant–pathogen interactions, we observe either no burst or only a first burst, indicating that the second burst is correlated with disease resistance. Further support for this observation comes from the finding that both resistant and susceptible tomato lines produce the critical second oxidative burst when challenged with P. syringae pv. tabaci , a nonhost pathogen that elicits a hypersensitive response on both tomato lines. The Pto kinase is not required, however, for the oxidative burst initiated by non-specific elicitors such as oligogalacturonides or osmotic stress. A model describing a possible role for the Pto kinase in the overall scheme of oxidative burst signaling is proposed.

It has been 90 years since the first confirmation of the genetic basis of plant disease resistance.1 Since then resistance (R) genes have been identified in all major economic plant species and have been incorporated into numerous elite cultivars.2–6 This intensive effort in the discovery and utilization of plant R genes has dramatically increased and stabilized food production throughout the world and ranks as one of the major achievements of modern agriculture.

Resistance to bacterial speck in tomato is governed by a gene-for-gene interaction in which a single resistance locus (Pto) in the plant responds to the expression of a specific avirulence gene (avrPto) in the pathogen. Disease susceptibility results if either Pto or avrPto are lacking from the corresponding organisms. Leaves of tomato cultivars that contain the Pto locus also exhibit a hypersensitive-like response upon exposure to an organophosphorous insecticide, fenthion. Recently, the Pto gene was isolated by a map-based cloning approach and was shown to be a member of a clustered multigene family with similarity to various protein-serine/threonine kinases. Another member of this family, termed Fen, was found to confer sensitivity to fenthion. The Pto protein shares 80% identity (87% similarity) with Fen. Here, Pto and Fen are shown to be functional protein kinases that probably participate in the same signal transduction pathway.

The original concept behind map-based or positional cloning was to find a DNA marker linked to a gene of interest, and then to ‘walk’ to the gene via overlapping clones (e.g. cosmids or YACs). While chromosome walking is straightforward in organisms with small genomes, it is difficult to apply in most plant species, which typically have large, complex genomes. The strategy of chromosome walking is based on the assumption that it is difficult and time consuming to find DNA markers that are physically close to a gene of interest. Recent technological developments invalidate this assumption for many species. As a result, the mapping paradigm has now changed such that one first isolates one or more DNA marker(s) at a physical distance from the targeted gene that is less than the average insert size of the genomic library being used for clone isolation. The DNA marker is then used to screen the library and isolate (or ‘land’ on) the clone containing the gene, without any need for chromosome walking and its associated problems. Chromosome landing, together with the technology that has made it possible, is likely to become the main strategy by which map-based cloning is applied to isolate both major genes and genes underlying quantitative traits in plant species.

A map-based cloning technique for crop plants is being developed using tomato as a model system. The target gene jointless is a recessive mutation that completely suppresses the formation of flower and fruit pedicel abscission zones. Previously, the jointless locus was mapped to a 3 cM interval between the two molecular markers TG523 and RPD158. Physical mapping of the jointless region by pulsed-field gel electrophoresis demonstrated that TG523 and RPD158 reside on a 600 kb SmaI fragment. In this study, TG523 was used as a probe to screen a tomato yeast artificial chromosome (YAC) library. Six tomato YAC (TY) clones were isolated, ranging from 220 to 380 kb in size. Genetic mapping of YAC ends demonstrated that this set of overlapping YACs encompasses the jointless locus. Two YAC ends, TY159L (L indicates left end) and TY143R (R indicates right end), cosegregate with the jointless locus. Only one of the six YACs (TY142) contained single-copy DNA sequences at both ends that could be mapped. The two ends of TY142 were mapped to either side of the jointless locus, indicating that TY142 contains a contiguous 285 kb tomato DNA fragment that probably includes the jointless locus. Physical mapping of the TY142 clone revealed that TY159L and TY143R reside on a 55 kb SalI fragment. Southern blot hybridization analysis of the DNAs of tomato lines nearly isogenic for the jointless mutation has allowed localization of the target locus to a region of less than 50 kb within the TY142 clone.

Tomato (Lycopersicon esculentum) cultivars that conatin the Pto bacterial resistance locus also exhibit sensitivity to fenthion, an organophosphorous insecticide. Resistance to Pseudomonas syringae pv. tomato (Pst) encoded by the Pto gene and sensitivity to fenthion cosegregate in large F2 populations and were apparently introgressed together into tomato from the wild species Lycopersion pimpinellifolium. In order to isolate the genes responsible for these two phenotypes and to study their molecular basis, a multistep map-based cloning project was initiated. A closely linked RFLP marker was used to isolate a yeast artificial chromosome (YAC) clone that spanned the Pto region. Transcribed sequences within the Pto region were identified by isolating cDNA clones that hybridized to the YAC clone. Transformation of candidate cDNA clones into a Pst-susceptible, fenthion-insensitive tomato line succeeded in identifying a cDNA conferring Pst resistance and a separate cDNA conferring sensitivity to fenthion. The cDNA clones represent members of a clustered gene family and encode putative serine-threonine protein kinases. The role of these kinases in recognizing a signal from Pst or the fenthion molecule and in activating the plant response is currently being investigated.

Yeast artificial chromosome (YAC) libraries are becoming widely used for the construction of long-range physical maps and for map-based cloning [ 1–6]. In plants, YAC libraries are now available for Arabidopsis, barley, carrot, maize, rice and tomato [7–14]. Recently, map-based cloning projects using YAC libraries have been successful in isolating several plant genes that were known only by their phenotype [15–18]. Detailed analysis of individual YACs is likely to provide new insights into the way repeated sequences and transcribed regions are organized in plant genomes.

The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

Resistance to Pseudomonas syringae pv. tomato (Pst) in tomato is conferred by a single dominant locus, Pto. We are attempting to isolate the Pto locus using a multi-step positional cloning strategy. First, using near-isogenic lines (NILs) we identified RFLP and RAPD markers that are tightly linked to the locus (4). Second, we developed and screened a large F2 population (251 plants) that segregated for Pst resistance. This population was used to create a high resolution (0.2 cM) linkage map that allowed the precise placement of Pto in relation to molecular markers (6). One marker, TG538, was found to cosegregate with Pto. Third, we screened a tomato YAC library (5) with TG538 and identified five YACs ranging from 400 to 600 kb that carry sequences derived from the Pto region. Mapping the ends of the cloned tomato DNA from the smallest YAC (PTY538-1) showed that this YAC encompasses the Pto locus. Fourth, PTY538-1 was used to identify cDNA clones from a library made from Pst-induced leaf tissue. Two cDNA clones CD106 and CD127-1, were isolated by this method. Linkage mapping of these clones showed that CD106 maps 0.4 cM from the Pto locus, whereas CD127 cosegregates with Pto. Once we have identified a full-length clone of CD127 we will introduce it (and the corresponding genomic sequence) into tomato plants in an effort to transform a Pst-susceptible line to resistance. The eventual isolation of Pto should open up many avenues of research into the molecular mechanism of plant disease resistance.

We have constructed a yeast artificial chromosome (YAC) library of tomato for chromosome walking that contains the equivalent of three haploid genomes (22,000 clones). The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato. Approximately 11,000 YACs have been screened with RFLP markers that cosegregate with Tm-2a and Pto - loci conferring resistance to tobacco mosaic virus and Pseudomonas syringae pv. tomato, respectively. Five YACs were identified that hybridized to the markers and are therefore starting points for chromosome walks to these genes. A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato chromosomes, TGRII, an interspersed repeat, and TGRIII, a repeat that occurs primarily at centromeric sites. Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences. Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome. The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis. Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.

An approach to isolating DNA sequences that are linked to important plant genes is described. The strategy is based upon a recent modification of the polymerase chain reaction in which synthetic primers are used to amplify random sequences from genomic DNA. This technique, used in conjunction with near-isogenic lines (which differ only by the presence or absence of the target gene and a small region of surrounding DNA), leads to the rapid identification of sequences linked to the gene of interest. The feasibility of this method has been demonstrated by analyzing a pair of tomato near-isogenic lines that differ for a region on chromosome 5 that contains a gene (Pto) conferring resistance to Pseudomonas syringae pv. tomato. One hundred forty-four random primers were screened on these lines, and seven amplified products were identified that were present in one but not the other line. Of four products that were further investigated, three were confirmed by segregation analysis to be tightly linked to the Pto gene. Linked sequences identified by this method are useful for detecting the presence of the target gene in plant populations (e.g., in plant breeding) and, if very tightly linked, as starting points for a chromosome walk to isolate the gene. Since near-isogenic lines are a typical product of plant breeding and classical genetic studies, this method is applicable to a wide variety of species.

Previous research on the automotive industry points to the difficulty manufacturers and suppliers face in managing their supply chains. Both groups report challenges in managing the numerous supply change interfaces between companies due particularly to differing inter-company relationship models. They also report significant differences between how well they perform Supply Chain Management (SCM) tasks and how important these activities are to their companies' future success. They also discuss the major risks companies face in sourcing globally, which when combined with increasing product and manufacturing complexity leads to conflicts in business and organisational goals.

PLANT BIOLOGY. For the article “Suppression of pathogen-inducible NO synthase (iNOS) activity in tomato increases susceptibility to Pseudomonas syringae ,” by Meena R. Chandok, Sophia K. Ekengren, Gregory B. Martin, and Daniel F. Klessig, which appeared in issue 21, May 25, 2004, of Proc. Natl. Acad. Sci. USA (101, 8239–8244; first published May 14, 2004; …

Tomato is a well-established model organism for studying many biological processes including resistance and susceptibility to pathogens and the development and ripening of fleshy fruits. The availability of the complete Arabidopsis genome sequence will expedite map-based cloning in tomato on the basis of chromosomal synteny between the two species, and will facilitate the functional analysis of tomato genes.

The avrPto gene of Pseudomonas syringae pv tomato triggers race-specific resistance in tomato plants carrying Pto, a resistance gene encoding a protein kinase. When introduced into P. s. tabaci, avrPto triggers resistance in tobacco W38 plants that carry the corresponding R gene. The AvrPto protein is believed to be secreted into host cells through the bacterial type III secretion pathway, where it activates disease resistance in tomato by interacting with Pto. We report here the identification of two distinct regions in AvrPto that determine the recognition specificity of this protein in tomato and tobacco. Point mutations in the central region disrupted the avirulence activity in tomato but not in tobacco. Conversely, point mutations in the C-terminal region abolished the avirulence in tobacco but not in tomato. We further report that AvrPto was localized to the plasma membrane of plant cells. Disrupting the membrane association by mutating a putative myristoylation motif of AvrPto abolished the avirulence activity in both tomato and tobacco. These findings demonstrate that AvrPto is recognized differently by the R genes in tomato and tobacco and that the recognition of AvrPto probably is associated with the plasma membrane.

Leaves of tomato cultivars that contain the Pto bacterial resistance locus develop small necrotic lesions within 24 hr after exposure to fenthion, an organophosphorous insecticide. Recently, the Pto gene was isolated and shown to be a putative serine/threonine protein kinase. Pto is one member of a multigene family that is clustered within a 400-kb region on chromosome 5. Here, we report that another member of this gene family, termed Fen, is responsible for the sensitivity to fenthion. Fen was isolated by map-based cloning using closely linked DNA markers to identify a yeast artificial chromosome clone that spanned the Pto region. After transformation with the Fen gene under control of the cauliflower mosaic virus (CaMV) 35S promoter, tomato plants that are normally insensitive to fenthion rapidly developed extensive necrotic lesions upon exposure to fenthion. Two related insecticides, fensulfothion and fenitrothion, also elicited necrotic lesions specifically on Fen-transformed plants. Transgenic tomato plants harboring integrated copies of the Pto gene under control of the CaMV 35S promoter displayed sensitivity to fenthion but to a lesser extent than did wild-type fenthion-sensitive plants. The Fen protein shares 80% identity (87% similarity) with Pto but does not confer resistance to Pseudomonas syringae pv tomato. These results suggest that Pto and Fen participate in the same signal transduction pathway.

Bean germplasm collections in northern Malawi revealed the existence of diverse landraces, which have probably been maintained by the local farmers as heterogeneous mixtures since the precolonial introduction of Phaseolus vulgaris into eastern Africa. The various seed types comprising these mixtures are known by an array of local names, reflecting farmer perceptions of seed color and pattern, eating quality, plant structure, origin, and other characteristics. A principal components analysis of morphological, phenological, and agronomic metrical traits for 375 lines randomly selected from 15 landraces revealed a clinal pattern, with the northern and southern areas forming the extremes. Genetic distances, based on the first six PCs, indicated greater between-area variability than within-area variability. Bean landrace diversity in Malawi is likely the result of a complex interplay among forces that generate variability such as outcrossing and human and environmental selection.

Bean (Phaseolus vulgaris) landraces in northern Malawi were found to be heterogeneous for many qualitative and quantitative traits. An earlier 21- character evaluation of 25 lines in 15 landraces found significant differences in all characters among lines and among landraces. The present study has concentrated on two factors that are likely to be involved in the generation and maintenance of this heterogeneity: seed- handling practices and natural outcrossing. Seed- handling practices during harvest, storage, and marketing at the village, local market, and national levels were found to result in physical and usually nonselective mixing of many seed types. Experiments determined that outcrossing does occur at a low level among beans in Malawi and can result in the generation of many new seed phenotypes. A scenario was developed integrating seed- handling practices and outcrossing that lays a foundation for understanding the generation and maintenance of variability in these bean landraces. The process whereby landrace heterogeneity arises and the ways it is maintained, if understood, should assist plant breeders in the development of improved germplasm for subsistence farmers.

We isolated the ntrC gene from Bradyrhizobium japonicum, the endosymbiont of soybean (Glycine max), and examined its role in regulating nitrogen assimilation. Two independent ntrC mutants were constructed by gene replacement techniques. One mutant was unable to produce NtrC protein, while the other constitutively produced a stable, truncated NtrC protein. Both ntrC mutants were unable to utilize potassium nitrate as a sole nitrogen source. In contrast to wild-type B. japonicum, the NtrC null mutant lacked glnII transcripts in aerobic, nitrogen-starved cultures. However, the truncated-NtrC mutant expressed glnII in both nitrogen-starved and nitrogen-excess cultures. Both mutants expressed glnII under oxygen-limited culture conditions and in symbiotic cells. These results suggest that nitrogen assimilation in B. japonicum is regulated in response to both nitrogen limitation and oxygen limitation and that separate regulatory networks exist in free-living and symbiotic cells.

Tomato MAPK kinase SlMKK2 is a key protein regulating immunity-associated programmed cell death (PCD) in plants. We examined the role of the N-terminal MAPK-docking site (or D-site) of SlMKK2 in PCD elicitation. In vivo assays revealed that SlMKK2 interacted with the downstream MAPK SlMPK3 independent of PCD elicitation and two conserved leucines in the D-site were required for both interaction with SlMPK3 and PCD elicitation. These results demonstrate that two leucines in the D-site of SlMKK2 play a critical role in regulation of signal transfer to the downstream MAPK by regulating their physical interaction.

Bradyrhizobium japonicum induces the formation of nitrogen-fixing symbiotic root nodules on soybean plants. The B. japonicum genome encodes two isoforms of glutamine synthetase (GS). One form, GSI, encoded by the gene glnA, is similar in structure and activity to the enzyme found in all other bacteria. The second form, GSII, encoded by glnII, is structurally related to the eucaryotic enzyme. Genetic analyses indicate that glnA or glnII alone is sufficient to provide glutamine prototrophy, whereas the double mutation glnA glnII produces glutamine auxotrophy. The glnA gene is transcribed from a single promoter that has a structure most similar to that of the bacterial consensus promoter. The level of transcription of glnA is not specifically affected by nitrogen limitation of growth. The glnII gene is also transcribed from a single promoter; however, this promoter has structural features characteristic of promoters controlled by the nitrogen regulation system. In contrast to glnA, physiological studies indicate that glnII transcription is regulated in response to nitrogen source availability. Under aerobic growth conditions, expression of glnII is induced when growth is limited by nitrogen source depletion as expected for regulation by the nitrogen regulation system.

Bean production practices common to the small East African country of Malawi are typical of practices followed generally throughout the temperate highlands of East Africa, where beans (Phaseolus vulgaris L.) are widely grown. The similarity extends to the widespread use of mixtures of seed and plant types as “varieties.”

The glnB gene from Bradyrhizobium japonicum, the endosymbiont of soybeans (Glycine max), was isolated and sequenced, and its expression was examined under various culture conditions and in soybean nodules. The B. japonicum glnB gene encodes a 12,237-dalton polypeptide that is highly homologous to the glnB gene products from Klebsiella pneumoniae and Escherichia coli. The gene is located directly upstream from glnA (encoding glutamine synthetase), a linkage not observed in enteric bacteria. The glnB gene from B. japonicum is expressed from tandem promoters, which are differentially regulated in response to the nitrogen status of the medium. Expression from the downstream promoter involves the B. japonicum ntrC gene product (NtrC) in both free-living and symbiotic cells. Thus, glnB, a putative nitrogen-regulatory gene in B. japonicum, is itself Ntr regulated, and NtrC is active in B. japonicum cells in their symbiotic state.

Background The plant plasma membrane is a key battleground in the war between plants and their pathogens. Plants detect the presence of pathogens at the plasma membrane using sensor proteins, many of which are targeted to this lipophilic locale by way of fatty acid modifications. Pathogens secrete effector proteins into the plant cell to suppress the plant’s defense mechanisms. These effectors are able to access and interfere with the surveillance machinery at the plant plasma membrane by hijacking the host’s fatty acylation apparatus. Despite the important involvement of protein fatty acylation in both plant immunity and pathogen virulence mechanisms, relatively little is known about the role of this modification during plant-pathogen interactions. This dearth in our understanding is due largely to the lack of methods to monitor protein fatty acid modifications in the plant cell. Results We describe a rapid method to detect two major forms of fatty acylation, N-myristoylation and S-acylation, of candidate proteins using alkyne fatty acid analogs coupled with click chemistry. We applied our approach to confirm and decisively demonstrate that the archetypal pattern recognition receptor FLS2, the well-characterized pathogen effector AvrPto, and one of the best-studied intracellular resistance proteins, Pto, all undergo plant-mediated fatty acylation. In addition to providing a means to readily determine fatty acylation, particularly myristoylation, of candidate proteins, this method is amenable to a variety of expression systems. We demonstrate this using both Arabidopsis protoplasts and stable transgenic Arabidopsis plants and we leverage Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves as a means for high-throughput evaluation of candidate proteins. Conclusions Protein fatty acylation is a targeting tactic employed by both plants and their pathogens. The metabolic labeling approach leveraging alkyne fatty acid analogs and click chemistry described here has the potential to provide mechanistic details of the molecular tactics used at the host plasma membrane in the battle between plants and pathogens. Electronic supplementary material The online version of this article (doi:10.1186/s13007-016-0138-2) contains supplementary material, which is available to authorized users.

Background: Effector proteins are translocated into host cells by plant-pathogens to undermine pattern-triggeredimmunity (PTI), the plant response to microbe-associated molecular patterns that interferes with the infection process. Individual effectors are found in variable repertoires where some constituents target the same pathways.The effector protein AvrPto from Pseudomonas syringae has a core domain (CD) and C-terminal domain (CTD) that each promotes bacterial growth and virulence in tomato. The individual contributions of each domain and whether they act redundantly is unknown. Results: We use RNA-Seq to elucidate the contribution of the CD and CTD to the suppression of PTI in tomato leaves 6 h after inoculation. Unexpectedly, each domain alters transcript levels of essentially the same genes but to a different degree. This difference, when quantified, reveals that although targeting the same host genes, the two domains act synergistically. AvrPto has a relatively greater effect on genes whose expression is suppressed during PTI, and the effect on these genes appears to be diminished by saturation. Conclusions: RNA-Seq profiles can be used to observe relative contributions of effector subdomains to PTI suppression. Our analysis shows the CD and CTD multiplicatively affect the same gene transcript levels with a greater relative impact on genes whose expression is suppressed during PTI. The higher degree of up-regulation versus down-regulation during PTI is plausibly an evolutionary adaptation against effectors that target immune signaling, Facultad de Ciencias Naturales y Museo, Instituto de Fisiología Vegetal