Bs2 Gene Research Articles

Interfamily transfer of Bs2 from pepper to cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is the principal source of calories among root and tuber crops in tropical countries. Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) is the most prevalent bacterial disease in cassava. Genome and mutagenesis analysis of Xam strains has led to the identification of an effector similar to avrBs2 from Xanthomonas euvesicatoria among a core set effectors contributing to Xam virulence. Previous studies have demonstrated that transgenic tomato plants expressing Bs2, the AvrBs2 cognate resistance protein from pepper, are resistant to Xanthomonas euvesicatoria. According to avrBs2 wide distribution and important contribution to Xam virulence, we aimed at overexpressing Bs2 in cassava plants as a strategy to control CBB. The susceptible cultivar 60444 was transformed with the Bs2 gene from pepper and transgenic cassava plants that functionally express Bs2 were regenerated. Our results show that overexpression of Bs2 in a highly susceptible cultivar leads to reactive oxygen species production. However, the overexpression of Bs2 neither leads to an HR in cassava nor reduces Xam growth on in vitro plants. These results suggest that BS2 activates defense-signaling pathways in cassava such as ROS production, although this is not sufficient to restrict Xam growth.

The Genetic Transformation of Sweet Orange (Citrus sinensis L. Osbeck) for Enhanced Resistance to Citrus Canker.

Developing disease resistance is one of the most important components of any plant breeding program. Citrus traditional breeding methods (bud sport selection, crossbreeding, and other breeding channels) are a laborious task and often hampered by long juvenility, a high degree of heterozygosity, polyembryony, self-incompatibility, and abortion of reproductive organs. An interesting alternative to the classical breeding approach is the use of genetic transformation, which provides the means for adding a single agronomic trait to a plant without otherwise altering its phenotype. Agrobacterium tumefaciens-mediated transformation has been carried out with numerous hybrids and citrus species. This technique allowed us to introduce the Bs2 gene in Citrus, as well as to increase citrus canker resistance in transgenic Bs2 gene-expressing lines.

Transgenic Expression of EFR and Bs2 Genes for Field Management of Bacterial Wilt and Bacterial Spot of Tomato.

Field trials were conducted at two locations in Florida to evaluate transgenic tomato expressing the ELONGATION FACTOR TU RECEPTOR (EFR) gene from Arabidopsis thaliana, the Bs2 gene from pepper, or both Bs2 and EFR (Bs2/EFR) for managing bacterial wilt caused by Ralstonia solanacearum and bacterial spot caused by Xanthomonas perforans. Expression of EFR or Bs2/EFR in the susceptible genotype Fla. 8000 significantly reduced bacterial wilt incidence (50 to 100%) and increased total yield (57 to 114%) relative to lines expressing only Bs2 or the nontransformed Fla. 8000 control, although the marketable yield was not significantly affected. Following harvest, surviving symptomatic and nonsymptomatic plants were assessed for colonization by R. solanacearum. There were no significant differences in the population at the lower stem. Interestingly, in the middle stem, no bacteria could be recovered from EFR or Bs2/EFR lines but viable bacterial populations were recovered from Bs2 and nontransformed control lines at 102 to 105 CFU/g of stem tissue. In growth-chamber experiments, the EFR transgenic tomato lines were found to be effective against seven different R. solanacearum strains isolated from the southeastern United States, indicating utility across the southeastern United States. In all of the bacterial spot trials, EFR and Bs2/EFR lines had significantly reduced disease severity (22 to 98%) compared with the Fla. 8000 control. The marketable and total yield of Bs2/EFR were significantly higher (43 to 170%) than Fla. 8000 control in three of four field trials. These results demonstrate for the first time the potential of using the EFR gene for field management of bacterial wilt and bacterial spot diseases of tomato.

Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease.

Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

Good, the Bad, and the Ugly: What the Future Could Hold for Bs2 Tomatoes

Bs2 tomatoes are transgenic tomatoes that have been engineered to contain the Bs2 gene from pepper. As such, they are considered a genetically modified (GM) food, or a genetically modified organism (GMO). Numerous trials conducted by University of Florida researchers have shown the benefits of these cultivars for bacterial spot disease management, and growers and industry members recognize the potential for Bs2 tomatoes to make Florida tomato production more sustainable. This 4-page fact sheet discusses the benefits that might be realized by the adoption of Bs2 tomato varieties, and the challenges standing in the way of their commercial production. Written by S. F. Hutton, J. W. Scott, J. B. Jones, R. E. Stall, G. E. Vallad, B. J. Staskawicz, and D. M. Horvath , and published by the UF Department of Horticultural Sciences, April 2015. HS1259/HS1259: The Good, the Bad, and the Ugly: What the Future Could Hold for Bs2 Tomatoes (ufl.edu)

Transgenic resistance confers effective field level control of bacterial spot disease in tomato.

We investigated whether lines of transgenic tomato (Solanum lycopersicum) expressing the Bs2 resistance gene from pepper, a close relative of tomato, demonstrate improved resistance to bacterial spot disease caused by Xanthomonas species in replicated multi-year field trials under commercial type growing conditions. We report that the presence of the Bs2 gene in the highly susceptible VF 36 background reduced disease to extremely low levels, and VF 36-Bs2 plants displayed the lowest disease severity amongst all tomato varieties tested, including commercial and breeding lines with host resistance. Yields of marketable fruit from transgenic lines were typically 2.5 times that of the non-transformed parent line, but varied between 1.5 and 11.5 fold depending on weather conditions and disease pressure. Trials were conducted without application of any copper-based bactericides, presently in wide use despite negative impacts on the environment. This is the first demonstration of effective field resistance in a transgenic genotype based on a plant R gene and provides an opportunity for control of a devastating pathogen while eliminating ineffective copper pesticides.

Transient expression of pepper Bs2 gene in Citrus limon as an approach to evaluate its utility for management of citrus canker disease

The pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains containing the avrBs2 avirulence gene in susceptible pepper and tomato. The avrBs2 gene is highly conserved in the Xanthomonas genus and when bacteria lack this gene their growth in a susceptible host is diminished, indicating that the avrBs2 gene product could confer an adaptive advantage to the pathogen. The avrBs2 of Xanthomonas citri subsp. citri (Xcc), cause of citrus canker, shares 96% homology with avrBs2 of Xcv. To evaluate if Bs2 could recognize avrBs2 of Xcc in citrus plants and thereby activate plant defence mechanisms to increase resistance to canker, transient expression experiments were conducted using Agrobacterium tumefaciens in lemon plants subsequently challenged with wildtype Xcc. The results showed that transient expression of Bs2 reduced canker formation in lemon and induced plant defence mechanisms, as shown by callose deposition and PR‐1 expression. Moreover, when an avrBs2 mutant of Xcc was used, no decrease in disease symptoms was observed. This work shows that the Bs2 gene from Solanaceae is functional in lemon, a member of the Rutaceae family. Therefore, Bs2 is a potential candidate gene for stable expression in transgenic citrus plants in order to improve resistance to canker disease.

Development of gene-based markers for the Bs2 bacterial spot resistance gene for marker-assisted selection in pepper (Capsicum spp.)

The Bs2 gene of pepper (Capsicum spp.) confers resistance to the most commonly detected races of Xanthomonas campestris pv vesicatora (Xcv). Using information from a published database, we developed gene-based markers for selection of Bs2 in pepper. High polymorphism was detected in exon 3, intron 2 and 3’-UTR of the Bs2 gene. The three regions were sequenced and numerous SNPs and In/Dels were identified between resistant and susceptible inbred lines. Seven SNPs were selected to develop SNP markers using the tetra-primer amplification refractory mutation system-PCR methodology (Tetra-primer ARMS-PCR). One SNP (G/C) was used to develop a molecular marker that can perfectly identify the presence of the bacterial spot resistance. The gene-based markers were further validated to test association with resistance in 80 lines and cultivars. The markers successfully identified a bell pepper commercial cultivar 62-2 that harbors the Bs2 gene. The successful development of the markers in this study provides a useful tool for MAS to screen the Bs2 gene in pepper.

Near-Isogenic Lines for Genes Conferring Hypersensitive Resistance to Bacterial Spot in Chili Pepper

In order to develop chili pepper bacterial spot resistant cultivars and near-isogenic lines (NILs) to prompt the molecular mapping of the resistance gene, we have run backcross breeding program since 1994. Two resistance genes against Xanthomonas axonopodis pv. vesicatoria Bs2 from Fla. XVR 3-25 and Bs3 from our breeding line 25-11-3-2, were introduced into a land race, Chilseongcho (abbreviated to Chilseong hereafter) with good fruit guality. We report here the testing of <TEX>$BC_4F_3\;to\;BC_4F_5$</TEX>. We found that <TEX>$BC_4F_5$</TEX> lines of the crosses were homozygous with respect to the respective genes of introduction. The lines, in which Bs2 gene was introduced, were hypersensitively resistant to both race 1 and race 3 of X. axonopodis pv. vesicatoria, whereas, those in which Bs3 was introduced were resistant to race 1.

Molecular Genetic Evidence for the Role of SGT1 in the Intramolecular Complementation of Bs2 Protein Activity in Nicotiana benthamiana

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Co-immunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Co-immunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.

Temperature Sensitivity of the Hypersensitive Response of Bell Pepper to Xanthomonas axonopodis pv. vesicatoria

ABSTRACT When bacterial spot-resistant pepper plants carrying resistance gene Bs2 and infiltrated with incompatible strains of Xanthomonas axonopodis pv. vesicatoria carrying a functional avrBs2 gene (races P1 and P3) were incubated at 32 degrees C, they exhibited an electrolyte leakage and bacterial multiplication pattern in planta similar to that obtained with a compatible strain (race P4) carrying a nonfunctional avrBs2 gene. They also developed disease-like symptoms. Pretreatment of incompatible bacteria at 32 degrees C before infiltration caused a delay in electrolyte leakage less pronounced than that caused by exposing plants to 32 degrees C. Also, plants had to be exposed to 32 degrees C for an hour prior to inoculation to increase symptom expression. These data suggest that the Bs2 gene is temperature sensitive. In other experiments, the avrBs1-Bs1 interaction appeared to be the most heat tolerant and thus the least likely to revert to compatible, whereas the avrBs3-Bs3 interaction had an intermediate sensitivity to elevated temperatures.

Xanthomonas resistance in Hungarian 4 spice pepper varieties

With a view to further enhance the reputation of Hungarian spice pepper it was necessary to improve resistance to the bacterium Xanthomonas campestris pv.vesicatoria, the most dangerous pathogen of pepper varieties. From among the familiar resistance genes in Hungary only the gene Bs-2 could provide sufficient protection against the aggressiveness spectrum of the bacterium species X.c.pv. vesicatoria. The first results of the resistance breeding are the spice pepper varieties Kaldom and Kalorez. In addition to the Bs-2 gene attempts are also being made at building in a gds gene into pepper, a gene creating a general defense system, a different strategy towards Xanthomonas campestris pv. vesicatoria.&#x0D;

DEVELOPMENT OF DNA MARKERS LINKED TO BACTERIAL LEAF SPOT RESISTANCE OF CHILLI

Bacterial leaf spot disease (Xanthomonas campestris pv. vesicatoria; Xcv) causes severe damage to chilli (Capsicum annuum L.) and has been a major yield constraint. An introduced chilli accession 3-25-27 derived from Florida XVR 3-25 has a dominant gene, Bs2, which confers resistance to Xcv races 1, 2 and 3. To identify RAPD markers linked to Bs2, bulked segregant analysis (BSA) and random amplified polymorphic DNA (RAPD) analysis were performed. Isogenic DNA pools were made from 10 resistant and 10 susceptible plants in a BC 1 F 1 population derived from crosses between 3-25-27 and Early California Wonder. Two RAPD markers, OPD05 and OPF10, were identified from a total of 460 10-basepair primers. From the linkage analysis it was found that two markers were located on either side of the Bs2 gene. OPD05 was located 5.3 cM away from Bs2 gene and OPF10 was located 4.9 cM from the Bs2 on the opposite side to OPD05. For practical use, identified RAPD markers were converted to sequence characterized amplified regions (SCARs). Primer sequences with various lengths were designed and examined for their ability to detect a resistance marker. SCF10, originating from the RAPD marker OPF10, was amplified using a 24-basepair primer set, and it showed polymorphism between resistant and susceptible individuals. The initial SCARs developed from OPD05, however, showed monomorphism between resistant and susceptible plants. After trials with various primer combinations, it was possible to develop a polymorphic SCD05 marker. These markers enable the identification of the Bs2 gene on segregating progenies for marker-assisted selection.

Bacterial Spot Resistance, Yield, and Quality of Bell and Specialty Peppers

Bacterial spot epidemics, caused by Xanthomonas campestris pv. vesicatoria (Xcv), are still considered serious risks for commercial pepper (Capsicum annuum) growers in a number of eastern, southern and midwestern states. Newly released bell pepper cultivars with the Bs2 gene for resistance to Xcv races 1, 2, and 3 were compared in 2000 under bacterial spot-free and severe (natural) bacterial spot epidemic conditions in central and eastern Kentucky where similar trials had been conducted from 1995 to 1997. In addition to the replicated bell pepper trials, 49 hot and specialty pepper cultivars were grown for observation in single plots at the same two locations. As in previous trials, there were economically important differences in resistance and marketable yields among bell pepper cultivars having the Bs2 gene; some resistant cultivars were as susceptible as susceptible checks. Others were highly resistant in spite of the presence of Xcv races 3 and 6 in the eastern Kentucky trial. Only a few were highly resistant with excellent fruit quality. With a few notable exceptions, most of the hot and specialty cultivars were very susceptible to bacterial spot. Two of the three new jalapeño cultivars carrying Bs2 were highly resistant to bacterial spot and high yielding under severe epidemic conditions.

Construction of a yeast artificial chromosome library of pepper (Capsicum annuum L.) and identification of clones from the Bs2 resistance locus

A yeast artificial chromosome (YAC) library was constructed using high-molecular-weight DNA isolated from pepper (Capsicum annuum L.) leaf protoplasts. Insert DNA was prepared by partial digestion using EcoRI and subjected to electrophoretic fractionation before in-gel ligation to the pJS97/98 YAC vector. Prior to transformation of yeast spheroplasts, ligation products were subjected to a second electrophoretic size selection. The library consists of about 19 000 clones with an average insert size of 500 kb, thus representing approximately three haploid genome equivalents. Three PCR-based markers tightly linked to the pepper Bs2 resistance gene were used to assess the utility of this library for positional cloning. Three YAC clones containing pepper genomic DNA from the Bs2 resistance locus were isolated from the library. The clones ranged in size from 270 kb to 1.2 Mb and should prove useful for the cloning of the Bs2 gene.

High-resolution genetic and physical mapping of the region containing the Bs2 resistance gene of pepper

The Bs2 resistance gene of pepper confers resistance against the bacterial pathogen Xanthomonas campestris pv. vesicatoria. As a first step toward isolation of the Bs2 gene, molecular markers tightly linked to the gene were identified by randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis of near-isogenic lines. Markers flanking the locus were identified and a high-resolution linkage map of the region was developed. One AFLP marker, A2, was found to cosegregate with the locus, while two others, F1 and B3, flank the locus and are within 0.6 cM. Physical mapping of the A2 and F1 markers indicates that these markers may be within 150 kb of each other. Together, these results indicate that the Bs2 region may be cloned either by chromosome walker or landing. The linked markers were also used to characterize gamma-irradiation-induced mutants at the Bs2 locus.

Races of Xanthomonas campestris pv. vesicatoria overcoming the gene Bs2 for bacterial spot resistance in pepper, prevalent on Capsicum chinense in Barbados and Grenada and weakly pathogenic on bell pepper and tomato in the field

A total of 404 isolates of Xanthomonas campestris pv. vesicatoria, obtained from Capsicum chinense cv. West Indian Red grown in Barbados and Grenada, were differentiated into pathogenic races, and of these, 96 were tested also for selected taxonomic group phenotypes. The response of C. chinense to infection by several X. campestris pv. vesicatoria races and the contribution of races isolated from this cultivar to severity of bacterial spot of bell pepper and tomato were also investigated. P4T2, P5T2 and P6T2 were the predominant races of X. campestris pv. vesicatoria isolated from C. chinense grown in Grenada, whereas nine races (T1, P4, P6, P0T2, P1T2, P4T1, P4T2, P6T1 and P6T2) were isolated in Barbados. Race P4T2 comprised 46.0 and 71.4% of the isolates from Barbados and Grenada, respectively. The 96 isolates, all of which overcame resistance conferred by the gene Bs2, shared taxonomic group B strain characteristics, including the presence of the β‐protein band, positive amylolytic activity and inability to oxidize cis‐aconitate. The C. chinense cv. West Indian Red was susceptible only to races of X. campestris pv. vesicatoria that can overcome Bs2 gene resistance. Of six such races identified in Barbados, only P4T1, P4T2 and P6T1 affected bacterial spot‐susceptible bell pepper or tomato in the field, and they amounted to only 1.5–2.1% of each sample of isolates from these plant species. Moreover, they were confined to the smallest bacterial spot lesions. Bell pepper was most severely affected by combinations of races T1 with P3T2 and T2 with P0T1, and tomato by race T1 only and combinations of races P0T1 with P0T2 and P1T1 with P1T0, all of which prevailed in the field despite selection against them by C. chinense cv. West Indian Red.

Comparison of Resistant Cultivars for Management of Bacterial Spot in Peppers

Bacterial spot epidemics, caused by Xanthomonas campestris pv. vesicatoria (Doidge) Dye, continue to plague bell pepper (Capsicum annuum L.) growers in a number of southern and midwestern states. A 3-year study designed to compare cultivars and breeding lines under induced bacterial spot epidemic and bacterial spot-free conditions began soon after the first release of cultivars having the Bs2 gene for resistance to races 1 to 3 of the pathogen. Bacterial spot epidemics were created by transplanting `Merlin' plants (inoculated with races 1 to 3) into plots of each test cultivar at an isolated location in eastern Kentucky. Plots of the same trial entries at a second location were kept free of bacterial spot for 2 of the 3 years of trials; however, a moderate natural epidemic occurred at this location in 1996. Bacterial spot resistance had the greatest impact on yields and returns per acre in the inoculated trials. Cultivars with only Bs1 or a combination of Bs1 and Bs3 were highly susceptible in the inoculated trials. There were statistically significant and economically important differences in resistance among cultivars and breeding lines having the Bs2 gene; some were nearly as susceptible as susceptible checks. Although many Bs2-gene cultivars showed satisfactory levels of resistance, only a few were highly resistant, horticulturally acceptable, and comparable in yields to the best susceptible hybrids in a bacterial spot-free environment.

A developmentally regulated gene of trypanosomes encodes a homolog of rat protein-disulfide isomerase and phosphoinositol-phospholipase C

We have isolated and characterized a developmentally regulated gene in Trypanosoma brucei, arbitrarily termed BS2. BS2 mRNA is substantially more abundant in bloodstream-form trypanosomes than in procyclic culture forms. Its nucleotide sequence reveals a single contiguous open-reading frame of 497 codons and is predicted to encode a protein of approximately 55.5 kilodaltons. A search of the NBRF protein data base revealed that within the predicted amino acid sequence are two of the evolutionarily conserved redox sites typified by thioredoxin of bacteria. Of this family of proteins, the recently sequenced rat genes encoding protein-disulfide isomerase (PDI) and form I phosphoinositide-specific phospholipase C (PIPLC) showed homology extending over the length of all three proteins (i.e., between BS2, PDI, and PIPLC). Although this homology includes the acidic C-terminus characteristic of proteins localized to the lumen of the endoplasmic reticulum, the BS2 product is predicted to possess multiple sites for N-linked glycosylation while PDI and PIPLC have none. Possible roles of the BS2 gene product in trypanosome physiology are discussed.