Fine mapping and analysis of a candidate gene in tomato accession PI128216 conferring hypersensitive resistance to bacterial spot race T3

Bacterial spot caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X. gardneri is one of the most destructive diseases in tomatoes (Solanum lycopersicum L.) growing in tropical and subtropical regions. Exploring resistance genes from diverse germplasm and incorporating them into cultivated varieties are critical for controlling this disease. The S. pimpinellifolium accession PI128216 was reported to carry the Rx4 gene on chromosome 11 conferring hypersensitivity and field resistance to race T3. To facilitate the use of marker-assisted selection in breeding and map-based cloning of the gene, an F₂ population derived from a cross between the susceptible variety OH88119 and the resistant accession PI128216 was created for fine mapping of the Rx4 gene. Using 18 markers developed through various approaches, we mapped the gene to a 45.1-kb region between two markers pcc17 and pcc14 on chromosome 11. A NBS-LRR class of resistance gene was identified as the candidate for the Rx4 gene based on annotation results from the International Tomato Annotation Group. Comparison of the genomic DNA sequences of the Rx4 alleles in PI128216 and OH88119 revealed a 6-bp insertion/deletion (InDel) and eight SNPs. The InDel marker was successfully used to distinguish resistance and susceptibility in 12 tomato lines. These results will facilitate cloning the Rx4 gene and provide a useful tool for marker-assisted selection of this gene in tomato breeding programs.     

An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins

Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1(D481V), which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.     

Identification and expression profiling of tomato genes differentially regulated during a resistance response to Xanthomonas campestris pv. vesicatoria

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. Plants of the tomato line Hawaii 7981 are resistant to race T3 of X. campestris pv. vesicatoria expressing the type III effector protein AvrXv3 and develop a typical hypersensitive response upon bacterial challenge. A combination of suppression subtractive hybridization and microarray analysis identified a large set of cDNAs that are induced or repressed during the resistance response of Hawaii 7981 plants to X. campestris pv. vesicatoria T3 bacteria. Sequence analysis of the isolated cDNAs revealed that they correspond to 426 nonredundant genes, which were designated as XRE (Xanthomonas-regulated) genes and were classified into more than 20 functional classes. The largest functional groups contain genes involved in defense, stress responses, protein synthesis, signaling, and photosynthesis. Analysis of XRE expression kinetics during the tomato resistance response to X. campestris pv. vesicatoria T3 revealed six clusters of genes with coordinate expression. In addition, by using isogenic X. campestris pv. vesicatoria T2 strains differing only by the avrXv3 avirulence gene, we found that 77% of the identified XRE genes were directly modulated by expression of the AvrXv3 effector protein. Interestingly, 64% of the XRE genes were also induced in tomato during an incompatible interaction with an avirulent strain of Pseudomonas syringae pv. tomato. The identification and expression analysis of X. campestris pv. vesicatoria T3-modulated genes, which may be involved in the control or in the execution of plant defense responses, set the stage for the dissection of signaling and cellular responses activated in tomato plants during the onset of spot disease resistance.     

Xv4-vrxv4: a new gene-for-gene interaction identified between Xanthomonas campestris pv. vesicatoria race T3 and wild tomato relative Lycopersicon pennellii

Strains of tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria elicit a hypersensitive response (HR) in leaves of Lycopersicon pennellii LA716. Genetic segregation of the resistance exhibited ratios near 3:1 in F2 populations, which confirmed that a single dominant gene controlled the inheritance of this trait. With the aid of a collection of introgression lines, restriction fragment length polymorphism, and cleaved amplified polymorphic sequence markers, the resistance locus was located on chromosome 3 between TG599 and TG134. An avirulence gene named avrXv4 was also isolated by mobilizing a total of 600 clones from a genomic DNA library of the T3 strain 91-118 into the X. campestris pv. vesicatoria strain ME90, virulent on L. pennellii. One cosmid clone, pXcvT3-60 (29-kb insert), induced HR in resistant plants. The avirulent phenotype of pXcvT3-60 was confirmed by comparing growth rates in planta and electrolyte leakages among transconjugants carrying a mutated or intact clone with the wild-type T3 strain 91-118. A 1.9-kb DNA fragment contained within a 6.8-kb active subclone was sequenced and was determined to carry an open reading frame of 1,077 bp. The predicted AvrXv4 protein exhibits high similarity to members of an emerging new family of bacterial proteins from plant and mammalian pathogens comprising AvrRxv, AvrBsT, YopJ, YopP, AvrA, and YL40.     

Use of virus-induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize

Plant disease resistance proteins (R-proteins) detect specific pathogen-derived molecules, triggering a defence response often including a rapid localized cell death at the point of pathogen penetration called the hypersensitive response (HR). The maize Rp1-D21 gene encodes a protein that triggers a spontaneous HR causing spots on leaves in the absence of any pathogen. Previously, we used fine mapping and functional analysis in a Nicotiana benthamiana transient expression system to identify and characterize a number of genes associated with variation in Rp1-D21-induced HR. Here we describe a system for characterizing genes mediating HR, using virus-induced gene silencing (VIGS) in a maize line carrying Rp1-D21. We assess the roles of 12 candidate genes. Three of these genes, SGT1, RAR1, and HSP90, are required for HR induced by a number of R-proteins across several plant–pathogen systems. We confirmed that maize HSP90 was required for full Rp1-D21-induced HR. However, suppression of SGT1 expression unexpectedly increased the severity of Rp1-D21-induced HR while suppression of RAR1 expression had no measurable effect. We confirmed the effects on HR of two genes we had previously validated in the N. benthamiana system, hydroxycinnamoyltransferase and caffeoyl CoA O-methyltransferase. We further showed the suppression the expression of two previously uncharacterized, candidate genes, IQ calmodulin binding protein (IQM3) and vacuolar protein sorting protein 37, suppressed Rp1-D21-induced HR. This approach is an efficient way to characterize the roles of genes modulating the hypersensitive defence response and other dominant lesion phenotypes in maize.     

Identification of QTL associated with resistance to bacterial spot race T4 in tomato

Bacterial spot of tomato (Solanum lycopersicum L.), caused by several Xanthomonas sp., is a serious but difficult disease to control by chemical means. Development of resistance has been hindered by emergence of races virulent to tomato, by the quantitative inheritance of resistance, and by a low correlation between seedling assays and resistance in the field. Resistance to multiple races, including race T4, has been described in the S. lycopersicum var. cerasiformae accession PI 114490. We used molecular markers to identify associations with quantitative trait loci (QTL) in an elite inbred backcross (IBC) population derived from OH 9242, PI 114490 and Fla. 7600, a breeding line with tomato accession Hawaii 7998 (H7998) in its pedigree. Race T4 resistance has also been described in the advanced breeding lines Fla. 8233, Fla. 8517, and Fla. 8326, and a selective genotyping approach was used to identify introgressions associated with resistance in segregating progeny derived from crosses with these lines. In the IBC population, loci on chromosomes 11 and 3, respectively, explained as much as 29.4 and 4.8% of resistance variation. Both these loci were also confirmed by selective genotyping: PI 114490 and H7998 alleles on chromosome 11 each provided resistance. The PI 114490 allele on chromosome 3 was confirmed in the Fla. 8517 population, and an allele of undetermined descent was confirmed at this locus in the Fla. 8326 population. A chromosome 12 allele was associated with susceptibility in the Fla. 8517 population. Additional loci contributing minor effects were also implicated in the IBC population or by selective genotyping. Selection for the major QTL in a marker-directed phenotyping approach should significantly improve the efficiency of breeding for resistance to bacterial spot race T4, although as yet undetected QTL would be necessary to carry out strict marker assisted selection.     

Structural and functional analysis of SGT1 reveals that its interaction with HSP90 is required for the accumulation of Rx, an R protein involved in plant immunity

SGT1 (for suppressor of G2 allele of skp1) and RAR1 (for required for Mla12 resistance) are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90 (for heat shock protein90). In plants, SGT1, RAR1, and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. Here, we present structural and functional characterization of plant SGT1 proteins. Random mutagenesis of Arabidopsis thaliana SGT1b revealed that its CS (for CHORD-SGT1) and SGS (for SGT1 specific) domains are essential for disease resistance. NMR-based interaction surface mapping and mutational analyses of the CS domain showed that the CHORD II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain. Functional analysis of the CS mutations indicated that the interaction between SGT1 and HSP90 is required for the accumulation of Rx, a potato (Solanum tuberosum) R protein. Biochemical reconstitution experiments suggest that RAR1 may function to enhance the SGT1-HSP90 interaction by promoting ternary complex formation.     

Excess copper in soil as a factor affecting bacterial spots caused by <Emphasis Type="Italic">Xanthomonas vesicatoria</Emphasis> in tomato plants; bio-interaction between two stress factors and their influence on plants

The investigations presented in this paper focus on studying the influence of excess soil copper levels in young tomato plants and their relationship to bacterial spot severity caused by Xanthomonas vesicatoria race T3. The plants were grown under greenhouse conditions in pots containing soil artificially contaminated with copper in concentrations of 250 and 400 mg kg⁻¹. Both stress factors were applied individually and in combination. Soil copper levels did influence bacterial spot severity. The copper doses applied restricted bacterial spot growth. Strongest responses were observed when the pathogen was infiltrated into seeds. Deformations of plant, as well as increased plastid synthesis inhibition occurred when the plants were exposed to copper and X. vesicatoria. The applicability of the investigation is related to the fight against bacterial spot caused by X. vesicatoria in tomatoes grown on copper-polluted soils. In this case, the remedial action should be carried out very carefully in order to avoid additional damage to plants.     

The role of RAR1 in Agrobacterium-mediated plant transformation

RAR1 is identified as a critical protein involved in plant innate immunity. We investigated the role of RAR1 in Agrobacterium-mediated plant transformation based on the previous findings that accessory proteins associated with the E3 ligase complex such as SGT1, which tightly interacts with RAR1, play a role in the transformation process. RAR1 gene silencing in Nicotiana benthamiana and Arabidopsis rar1 mutant analysis suggested that RAR1 is required for early stages of Agrobacterium-mediated plant transformation. This finding further illustrates that RAR1, along with SGT1, that serve as a HSP90 co-chaperone is important for Agrobacterium-mediated plant transformation.     

Structural and functional analysis of SGT1-HSP90 core complex required for innate immunity in plants

SGT1 (Suppressor of G2 allele of skp1), a co-chaperone of HSP90 (Heat-shock protein 90), is required for innate immunity in plants and animals. Unveiling the cross talks between SGT1 and other co-chaperones such as p23, AHA1 (Activator of HSP90 ATPase 1) or RAR1 (Required for Mla12 resistance) is an important step towards understanding the HSP90 machinery. Nuclear magnetic resonance spectroscopy and mutational analyses of HSP90 revealed the nature of its binding with the CS domain of SGT1. Although CS is structurally similar to p23, these domains were found to non-competitively bind to various regions of HSP90; yet, unexpectedly, full-length SGT1 could displace p23 from HSP90. RAR1 partly shares the same binding site with HSP90 as the CS domain, whereas AHA1 does not. This analysis allowed us to build a structural model of the HSP90–SGT1 complex and to obtain a compensatory mutant pair between both partners that is able to restore virus resistance in vivo through Rx (Resistance to potato virus X) immune sensor stabilization.     

Molecular chaperons and co-chaperons,Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana

Ralstonia solanacearum is the causal agent of bacterial wilt disease. To better understand the molecular mechanisms involved in interaction between Nicotiana benthamiana and R. solanacearum, we focused on Hsp90, RAR1 and SGT1. Appearances of wilt symptom were significantly suppressed in Hsp90, RAR1 and SGT1-silenced plants compared with control plants. In RAR1-silenced plants, population of R. solanacearum increased in a similar manner to control plants. In contrast, multiplication of R. solanacearum was significantly suppressed in Hsp90 and SGT1-silenced plants. In addition, expression of PR genes were increased in Hsp90 and SGT1-silenced plants challenged with R. solanacearum. Therefore, RAR1 might be required for disease development or suppression of disease tolerance. These results also suggested that Hsp90 and/or SGT1 might play an important role in suppression of plant defenses leading to disease susceptibility and disease development.     

SGT1 positively regulates the process of plant cell death during both compatible and incompatible plant–pathogen interactions

SGT1 (suppressor of G2 allele of Skp1), an interactor of SCF (Skp1‐Cullin‐F‐box) ubiquitin ligase complexes that mediate protein degradation, plays an important role at both G1–S and G2–M cell cycle transitions in yeast, and is highly conserved throughout eukaryotes. Plant SGT1 is required for both resistance (R) gene‐mediated disease resistance and nonhost resistance to certain pathogens. Using virus‐induced gene silencing (VIGS) in Nicotiana benthamiana, we demonstrate that SGT1 positively regulates the process of cell death during both host and nonhost interactions with various pathovars of Pseudomonas syringae. Silencing of NbSGT1 in N. benthamiana plants delays the induction of hypersensitive response (HR)‐mediated cell death against nonhost pathogens and the development of disease‐associated cell death caused by the host pathogen P. syringae pv. tabaci. Our results further demonstrate that NbSGT1 is required for Erwinia carotovora‐ and Sclerotinia sclerotiorum‐induced disease‐associated cell death. Overexpression of NbSGT1 in N. benthamiana accelerates the development of HR during R gene‐mediated disease resistance and nonhost resistance. Our data also indicate that SGT1 is required for pathogen‐induced cell death, but is not always necessary for the restriction of bacterial multiplication in planta. Therefore, we conclude that SGT1 is an essential component affecting the process of cell death during both compatible and incompatible plant–pathogen interactions.     

Rice Pti1a negatively regulates RAR1-dependent defense responses

Tomato (Solanum lycopersicum) Pto encodes a protein kinase that confers resistance to bacterial speck disease. A second protein kinase, Pti1, physically interacts with Pto and is involved in Pto-mediated defense signaling. Pti1-related sequences are highly conserved among diverse plant species, including rice (Oryza sativa), but their functions are largely unknown. Here, we report the identification of a null mutant for the Pti1 homolog in rice and the functional characterization of Os Pti1a. The rice pti1a mutant was characterized by spontaneous necrotic lesions on leaves, which was accompanied by a series of defense responses and resistance against a compatible race of Magnaporthe grisea. Overexpression of Pti1a in rice reduced resistance against an incompatible race of the fungus recognized by a resistance (R) protein, Pish. Plants overexpressing Pti1a were also more susceptible to a compatible race of the bacterial pathogen Xanthomonas oryzae pv oryzae. These results suggest that Os Pti1a negatively regulates defense signaling for both R gene-mediated and basal resistance. We also demonstrated that repression of the rice RAR1 gene suppressed defense responses induced in the pti1a mutant, indicating that Pti1a negatively regulates RAR1-dependent defense responses. Expression of a tomato Pti1 cDNA in the rice pti1a mutant suppressed the mutant phenotypes. This contrasts strikingly with the previous finding that Sl Pti1 enhances Pto-mediated hypersensitive response (HR) induction when expressed in tobacco (Nicotiana tabacum), suggesting that the molecular switch controlling HR downstream of pathogen recognition has evolved differently in rice and tomato.     

Fine genetic mapping of RXopJ4, a bacterial spot disease resistance locus from Solanum pennellii LA716

The RXopJ4 resistance locus from the wild accession Solanum pennellii (Sp) LA716 confers resistance to bacterial spot disease of tomato (S. lycopersicum, Sl) caused by Xanthomonas perforans (Xp). RXopJ4 resistance depends on recognition of the pathogen type III effector protein XopJ4. We used a collection of Sp introgression lines (ILs) to narrow the RXopJ4 locus to a 4.2-Mb segment on the long arm of chromosome 6, encompassed by the ILs 6-2 and 6-2-2. We then adapted or developed a collection of 14 molecular markers to map on a segregating F₂ population from a cross between the susceptible parent Sl FL8000 and the resistant parent RXopJ4 8000 OC₇. In the F₂ population, a 190-kb segment between the markers J350 and J352 cosegregated with resistance. This fine mapping will enable both the identification of candidate genes and the detection of resistant plants using cosegregating markers. The RXopJ4 resistance gene(s), in combination with other recently characterized genes and a quantitative trait locus (QTL) for bacterial spot disease resistance, will likely be an effective tool for the development of durable resistance in cultivated tomato.     

Evaluation of tomato wild species for resistance to race T1 nad T3 of Xanthomonas vesicatoria

Thirty six tomato wild species accessions of the subgenera Eulycopersicon and Eriopersicon of the genus Lycopersicon were inoculated with race T1 and T3 of Xanthomonas vesicatoria by vaccum infiltration method. Degree of diseases was evaluated by scale of 0 to 4. It was established that some accessions showed low degree of disease to race T1 and others to race T3. LA 2623 indicated very low degree of disease to race T1 and was immune in inoculation with T3. LA 386 and LA 1297 manifested hypersensitive reaction to both races and PI 127826 to race T3 only. The accessions possessing low degree of disease or hypersensitive reaction to race T1 and race T3 are new promising sources of resistance to Xanthomonas vesicatoria.     

Identification of a pathogenicity locus in Xanthomonas campestris pv. vesicatoria

Summary Mutants of a tomato strain ofXanthomonas campestris pv.vesicatoria (XCV), causal agent of bacterial spot of tomato and pepper, were produced using the transposon Tn5 carried in the suicide plasmid pGS9. One prototrophic mutant, M461, was isolated which caused no visible reaction on tomato or pepper, but maintained the wild-type ability to induce a hypersensitive reaction (HR) on tobacco. This mutant showed similar growth characteristics to the wild-type in culture, but growth in planta was reduced. A genomic library of wild-type XCV was constructed in the broad host range cosmid vector pLAFR3. Clone p6AD4 restored pathogenicity to M461 on tomato and the ability to induce a HR on pepper. This clone contained ca. 22 kb of XCV DNA. The insertion in M461 was in a site corresponding to a 1.1 kbEcoRI fragment of p6AD4.     

Occurrence of Pseudomonas syringae pv. Tomato race 1 in Italy on Pto Gene-Bearing Tomato Plants

Bacterial speck symptoms on leaves of the tomato cultivar Erminia Fl (Petoseed), heterozygous for the Pto resistant gene, were observed in June 1995 in Northern Italy. Using individual-lesion isolation, 8 bacterial isolates were obtained from the affected leaves, which were identified as Pseudomonas syringae pv. tomato by biochemical, physiological, nutritional and pathogenicity tests. When the differential cultivar Ontario 7710, homozygous for the Pto gene, was inoculated with the bacterial isolates, 4 of them provoked the typical bacterial speck symptoms and therefore belonged to race 1. This is the first occurrence of P. syringae pv. tomato race 1 in Italy and the first time this race has been isolated on a Pto gene-bearing tomato cultivar grown in open field.