Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3

Bacterial spot disease of tomato and pepper caused by Xanthomonas campestris pv vesicatoria is prevented by resistance genes in the plant that match genes for avirulence in the bacterium. Based on DNA homology to the avirulence gene avrBs3, which induces the resistance response on pepper, we have isolated another avirulence gene from X. c. vesicatoria, designated avrBs3-2. This gene differs in specificity from avrBs3 in inducing the hypersensitive response on tomato but not on pepper. Sequence analysis of the avrBs3-2 gene revealed a high degree of conservation: the 3480 by open reading frame contains an internal region of 17.5 nearly identical 102 bp repeat units that differ in their order from those present in the avrBs3 gene. The coding region is 97% identical to avrBs3 and expresses constitutively a 122 kDa protein, thus representing a natural allele of this gene. The previously isolated 1.7 kb avrBsP gene from X. c. vesicatoria is 100% identical to the corresponding avrBs3-2 sequence, indicating that these genes might be identical. Interestingly, derivatives of avrBs3-2 lacking the C-terminal region and part of the repetitive region are still able to confer incompatibility in tomato. The avrBs3-2 gene is compared with the sequence of avrBs3 derivatives generated by deletion of repeat units that also have avirulence activity on tomato. Both genes, avrBs3 and avrBs3-2, are flanked by a 62 by long inverted repeat, which prompts speculations about the origin of the members of the avrBs3 gene family.     

Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

Summary The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6–3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%–100% homology with each other.     

A gene from Xanthomonas campestris pv. vesicatoria that determines avirulence in tomato is related to avrBs3.

Strains of Xanthomonas campestris pv. vesicatoria that were avirulent in tomato leaves but virulent in pepper leaves were identified. A cloned gene, avrBsP, from one of the strains, Xv 87-7, converted a virulent strain in tomato to avirulent in tomato. A 1.7-kb subclone containing the avirulence gene cross-hybridized with the avirulence gene, which determines race 1 within the pepper group of strains (avrBs3). However, the two avirulence genes differ in their biological activity. The base sequences of the two avirulence genes were almost identical through the 1.7-kb segment of avrBsP, with significant differences only in some bases in the repeat region.     

Analysis of pathogenicity mutants of a bacteriocin producing Xanthomonas perforans

Since the initial discovery of Xanthomonas perforans on tomato in 1991, it has completely displaced Xanthomonas euvesicatoria as the bacterial spot of tomato pathogen in Florida. Previous research has shown that X. perforans produces at least three different bacteriocin-like compounds (BcnA, BcnB, BcnC) antagonistic toward X. euvesicatoria strains. In this study pathogenicity-attenuated, bacteriocin-producing mutants of X. perforans were created to determine their potential as biological control agents for control of X. euvesicatoria. Several candidate genes were chosen based on previous studies in which mutant phenotypes exhibited reduced virulence in either X. perforans (OpgH_Xcv) or the closely related X. euvesicatoria strain 85-10 (hpaB, hpaC, xopA, xopD, avrBs2 and gumD). Each candidate gene in X. perforans was amplified and PCR-assisted deletion mutagenesis was performed in the wild-type (wt) X. perforans strain to create potential attenuation mutants. Each mutant was tested for growth rate, disease severity and antagonism toward X. euvesicatoria strains. Three mutants, XopA, opgH, and gumD were significantly less pathogenic than the wild-type strain with the opgH mutant reaching significantly lower internal populations than all other mutants except hpaC. The opgH-strain was the most affected in its ability to grow internally in plant tissue while inhibiting X. euvesicatoria populations equal to or more than the other mutant strains. This mutant strain could potentially be used as part of an effective biological control strategy.     

Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism

A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum. The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a nonhost plant. In this study we present the complementation analysis and nucleotide sequence of a 4772 by region of this hrp gene cluster. Three complete open reading frames (ORFs) are predicted within this region. The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38607, 73 990 and 21959 dalton, respectively. HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues. A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants. None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported. Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y. enterocolitica, FlbF of Caulobacter crescentus, F1hA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv. vesicatoria. These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins. Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide. The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81 % similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P. solanacearum and X. campestris. An efflux of plant electrolytes was found to be associated with the interactions between P. solanacearum and both tomato and tobacco leaves. This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.     

High-resolution genetic mapping of the pepper resistancelocus Bs3 governing recognition of the Xanthomonascampestris pv vesicatora AvrBs3 protein

The pepper (Capsicum annuum) Bs3 gene confers resistance to Xanthomonas campestris pv vesicatoria strains expressing the avirulence protein AvrBs3. Using amplified fragment length polymorphism (AFLP) and bulked DNA templates from resistant and susceptible plants we identified markers linked to Bs3 and defined a 2.1-cM interval containing the target gene. Bs3-linked AFLP fragments were cloned and conformity of isolated PCR products with the desired markers was determined by hybridisation to membrane-bound AFLP reactions. AFLP markers flanking the target gene were converted into locus-specific PCR-based markers. These markers were employed for the analysis of 790 plants segregating for Bs3, resulting in a linkage map with a genetic resolution of 0.13 cM. Mapping of Bs3-linked markers in tomato placed them to a syntenic interval on tomato chromosome 2. Received: 15 October 1999 / Accepted: 29 November 1999     

对增强辣椒疫霉菌抗性的研究

病原物诱导型启动子能精确控制抗病基因在侵染位点的表达,是抗病基因工程的有效工具。prp1-1是来自马铃薯谷胱甘肽巯基转移酶基因启动子的一个273bp的片段,能够快速准确地启动被侵染位点抗病基因的表达;Rs-AFP2是具有对致病性丝状真菌的广谱抗性。该研究构建prp1-1调控Rs-AFP2基因表达的载体,经农杆菌介导转化法导入辣椒。逆转录PCR检测发现,转基因辣椒只在受到疫霉菌孢子侵染时,才由prp1-1启动Rs-AFP2基因的转录。用疫霉菌孢子灌根接种转基因辣椒T1代植株,35株T1代辣椒中有29株表现出明显的疫霉菌抗性。另将23株T1代辣椒种于人工气候箱,发现其形态和发育特征与相同条件下的非转基因植株无明显区别。研究表明,prp1-1调控Rs-AFP2的诱导表达达到了增强辣椒疫霉菌抗性的目的,而且避免了负面效应的发生。     

Genetic mapping and functional analysis of the tomato Bs4 locus governing recognition of the Xanthomonas campestris pv. vesicatoria AvrBs4 protein

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.). Analysis of 17 different Lycopersicon accessions with avrBs4-expressing X. campestris pv. vesicatoria strains identified 15 resistant and two susceptible tomato genotypes. Genetic analysis revealed that AvrBs4 recognition in tomato is governed by a single locus, designated Bs4 (bacterial spot resistance locus no. 4). Amplified fragment length polymorphism and bulked DNA templates from resistant and susceptible plants were used to define a 2.6-cM interval containing the Bs4 locus. A standard tomato mapping population was employed to localize Bs4-linked markers on the short arm of chromosome 5. Investigation of X. campestris pv. vesicatoria hrp mutant strains revealed that AvrBs4 secretion and avirulence activity are hrp dependent. Agrobacterium-based delivery of the avrBs4 gene into tomato triggered a plant response that phenotypically resembled the hypersensitive response induced by avrBs4-expressing X. campestris pv. vesicatoria strains, suggesting symplastic perception of the avirulence protein. Mutations in the avrBs4 C-terminal nuclear localization signals (NLSs) showed that NLSs are dispensable for Bs4-mediated recognition. Our data suggest that tomato Bs4 and pepper Bs3 employ different recognition modes for detection of the highly homologous X. campestris pv. vesicatoria avirulence proteins AvrBs4 and AvrBs3.     

Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf. In this paper we present evidence for the induction of avr9 gene expression in C. fulvum grown in vitro under conditions of nitrogen limitation. Only growth medium with very low amounts of nitrogen (nitrate, ammonium, glutamate or glutamine) induced the expression of avr9. Limitation of other macronutrients or the addition of plant factors did not induce the expression of avr9. The induced expression of avr9 is possibly mediated by a positive-acting nitrogen regulatory protein, homologous to the Neurospora crassa NIT2 protein, which induces the expression of many genes under conditions of nitrogen limitation. The avr9 promoter contains several putative NIT2 binding sites. The expression of avr9 during the infection process was explored cytologically using transformants of C. fulvum carrying an avr9 promoter--glucuronidase reporter gene fusion. The possibility that expression of avr9 in C. fulvum growing in planta is caused by nitrogen limitation in the apoplast of the tomato leaf is discussed.     

The Cladosporium fulvum resistance gene Cf-ECP3 is part of the Orion cluster on the short arm of tomato Chromosome 1

The resistance of tomato to the pathogenic fungus Cladosporiumfulvum complies with the gene-for-gene relationship. Race specificresistance is based on Cf-gene mediated recognition ofsecreted avirulence products, resulting in a hypersensitive response (HR).Besides the avirulence gene products, C. fulvum secretes anumber of extra cellular proteins (ECPs) into the apoplast. Two L.esculentum accessions have previously been identified that reactedwith a HR upon injection with purified ECP3. The corresponding resistance genedesignated Cf-ECP3 was mapped by using an F₂population composed of 192 plants from the cross of susceptible MoneyMaker toresistant L. esculentum G1.1153.Cf-ECP3 inherited monogenically, cosegragated with theChromosome 1 Cleaved Amplified Polymorphic Sequence (CAPS) marker CT116 and wasmapped accurately at Orion, a locus harbouring Cf-ECP2 inother genotypes. RFLP anaysis with a Cf-9 probe furtherdemonstrated cosegregation of Cf-ECP3 with anHcr9 (Homologue of Cladosporiumfulvumresistance gene Cf-9) indicating that this gene is likelyan Hcr9. Thus in addition to the Milky Way locusharbouringthe Cf-4, Cf-4A andCf-9 resistance genes targeted against AVR4, AVR4A andAVR9, Orion is another complex locus on the short arm of Chromosome 1 thatharbours at least two functional Cf-genes,Cf-ECP2 and Cf-ECP3, targeted againstthe fungal excreted proteins ECP2 and ECP3.     

Characterization of pACK4, a mobilizable plasmid from Staphylococcus simulans biovar staphylolyticus

Staphylococcus simulans biovar staphylolyticus, the lysostaphin-producing organism, contains five plasmids designated pACK1–pACK5. pACK4 was found to be relaxable and to share sequence similarity with a number of well-characterized mobilizable plasmids from other staphylococci. All mobilizable staphylococcal plasmids characterized to date mediate resistance to various antibiotics, but pACK4 is unique because it contains no recognizable antibiotic resistance genes. pACK4 was found to contain an origin of transfer (oriT) region that shares inverted repeat regions and the same nic site as several other mobilizable staphylococcal plasmids. The presence of this conserved oriT region suggested that pACK4 might be mobilized in the presence of a conjugative plasmid. Filter mating studies revealed that pACK4 was mobilized by the conjugative plasmid pGO1. In addition, pACK4 was found to be virtually identical to the recently described plasmid pVGA from Staphylococcus aureus, except that pVGA contains an additional region (vgaA) that confers resistance to pleuromutilin, streptogramin A, and lincosamide. The high sequence similarity among pACK4, pVGA, and several previously described mobilizable staphylococcal plasmids suggests a common origin for these plasmids.     

A new insertion element, ISH26, from Halobacterium halobium

Summary A new halobacterial insertion element, ISH26, is described which occurs in the genome of Halobacterium halobium NRC817 in at least seven copies. A copy of ISH26 was isolated from the bacterio-opsin gene (bop) of the Bop mutant M140 of strain H. halobium R1 and characterized in more detail. It shows typical structural features of a transposable element with 16 pb terminal inverted repeat sequences and an 11 bp duplication of the target site. Two partially overlapping open reading frames (ORFs) are contained in the sequence of ISH26 on one strand. The terminal 16 bp inverted repeat of ISH26 is almost identical to the first 16 bp of the terminal inverted repeat of the ISH2 insertion element. The remaining sequences of ISH26 and ISH2 are entirely different. Two size variants of ISH26, 1,384 bp and 1,705 bp in size, are found in the H. halobium genome. The larger one (ISH26-1) contains an imperfect duplication of 321 bp at one end of ISH26.     

Induced defence responses and protective effects on tomato against Xanthomonas vesicatoria by an aqueous extract from Solanum lycocarpum infected with Crinipellis perniciosa

This study investigated the induced defence responses and protective effects on susceptible tomato (Lycopersicon esculentum Mill.) against Xanthomonas vesicatoria (Doidge) by a heat-treated aqueous extract (VLA) from dry necrotic tissue of ‘Lobeira’ (Solanum lycocarpum St. Hil.) branches infected with the fungus Crinipellis perniciosa (Stahel) compared with acibenzolar-S-methyl (ASM), a commercial inducer of resistance. Plantlets were sprayed with VLA and ASM and challenged 4 days later with a virulent strain of X. vesicatoria, under greenhouse conditions. The disease severity, fresh weight of shoots, the activities of phenol peroxidase (POX), polyphenol oxidase (PPO), chitinase (CHI), phenylalanine ammonia-lyase (PAL), lignin deposition, and soluble phenolic contents were evaluated in the leaf tissues. Reduction of the bacterial spot severity was observed in plantlets treated with VLA which conferred 63% of the ASM protection. This protective effect and lesion reduction promoted by VLA were probably associated particularly with POX and PAL activities, lignin deposition on leaf tissues and, to a less extent, CHI activity.     

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.     

番茄SlGRAS4基因特征分析和耐热功能鉴定

GRAS转录因子是调控植物生长发育和非生物胁迫响应的重要转录因子之一,而目前还没有GRAS调控高温胁迫的研究。为了深入研究番茄SlGRAS4生物功能,以耐热番茄LA2093为试验材料,分析番茄SlGRAS4基因结构、启动子序列及进化关系,利用qRT-PCR检测SlGRAS4在不同胁迫和不同激素处理下的表达水平,利用VIGS验证SlGRAS4基因耐热功能。结果表明:(1)生物信息学分析显示,SlGRAS4蛋白长度为666 aa,分子量为75 737.72 Da,理论等电点为6.31,含有GRAS转录因子家族典型的结构域,主要集中在C末端的277～657 aa之间;在SlGRAS4启动子区域发现脱落酸(ABA)和水杨酸(SA)响应元件;SlGRAS4与烟草NtGRAS1蛋白亲缘关系最近,推测SlGRAS4可能与其同源基因具有相似的生物功能。(2)在高温、低温、盐和干旱胁迫处理12 h时番茄SlGRAS4基因表达量升至最高,分别增加到对照的8.86、4.86、55.38和7.63倍;在ABA和SA激素处理8 h时SlGRAS4基因的表达量达到峰值,分别达到对照的120.72和3.55倍,说明SlGRAS4可能参与了多种非生物胁迫响应和激素信号传导。(3)沉默SlGRAS4基因番茄植株(VSlGRAS4)在高温胁迫下较对照植株(Ve)更容易萎蔫,且F_v/F_m与SOD、POD活性显著降低,REL和H_2O_2含量显著升高,说明在高温胁迫下沉默SlGRAS4使番茄植株细胞膜氧化损伤加重,光合能力降低,活性氧(ROS)清除酶活性减弱。(4)qRT-PCR分析显示,VSlGRAS4植株中高温信号应答关键基因HsfA1b、ROS信号应答基因ZAT10和ZAT12以及ROS清除酶编码基因CuZnSOD、FeSOD、APX1、APX2、CAT的表达水平均显著低于Ve植株,表明SlGRAS4转录因子可以通过调控高温和ROS信号转导来影响番茄的耐热性。研究认为,高温、低温、干旱、盐、ABA和SA均可显著诱导番茄SlGRAS4基因的表达,沉默SlGRAS4基因番茄植株的耐热性显著降低,证明番茄SlGRAS4基因具有耐热功能,为进一步解析SlGRAS4参与番茄耐热调控的分子机制奠定基础。     

Molecular analysis of theBg-rbg transposable element system ofZea mays L.

Summary The two components of theBg-rbg transposable element system of maize have been cloned. TheBg element, isolated from the mutable allelewx-m32 :: Bg is inserted in the intron of theWaxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp.Bg has 5 by terminal inverted repeats, and generates upon insertion an 8 by direct duplication of the target sequence. Both ends of theBg element contain a 76 by direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCG_kC ^G is here repeated several times in direct or inverse orientation. Therbg element was isolated from the mutable alleleo2m(r) where it is located in the promoter region of theOpaque-2 (O2) gene.rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of theBg element, and is also flanked by an 8 by direct duplication at the target site. LikeBg, rbg carries the 76 by direct repeats. Restriction enzyme analysis reveals that, compared toBg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between therbg element and the autonomousBg element.     

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12

Summary Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate(pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product).Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.Abbreviations pCMB p-chloromercuribenzoate - DM Davis and Mingioli - Ap ampicillin - NTG N-methyl-N-nitro-N-nitrosoguanidine - EMS ethylmethane sulfonate - Str streptomycin - Tet tetracycline - Ac l-azetidine-2-carboxylic acid - DHP 3, 4-dehydro-d,l-proline - MTT 3-(4,5-dimethyl-2)2,5-diphenyl tetrazolium bromide - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - Kan kanamycin - Spc spectinomycin