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Principal component analysis (PCA) with 36,621 polymorphic genome-anchored single nucleotide polymorphisms (SNPs) identified collectively for Capsicum annuum and Capsicum baccatum was used to characterize population structure and species domestication of these two important incompatible cultivated pepper species. Estimated mean nucleotide diversity (p) and Tajima's D across various chromosomes revealed biased distribution toward negative values on all chromosomes (except for chromosome 4) in cultivated C. baccatum, indicating a population bottleneck during domestication of C. baccatum. In contrast, C. annuum chromosomes showed positive p and Tajima's D on all chromosomes except chromosome 8, which may be because of domestication at multiple sites contributing to wider genetic diversity. For C. baccatum, 13,129 SNPs were available, with minor allele frequency (MAF) ≥0.05; PCA of the SNPs revealed 283 C. baccatum accessions grouped into 3 distinct clusters, for strong population structure. The fixation index (FST) between domesticated C. annuum and C. baccatum was 0.78, which indicates genome-wide divergence.We conducted extensive linkage disequilibrium (LD) analysis of C. baccatum var. pendulum cultivars on all adjacent SNP pairs within a chromosome to identify regions of high and low LD interspersed with a genome-wide average LD block size of 99.1 kb. We characterized 1742 haplotypes containing 4420 SNPs (range 9-2 SNPs per haplotype). Genome-wide association study (GWAS) of peduncle length, a trait that differentiates wild and domesticated C. baccatum types, revealed 36 significantly associated genome-wide SNPs. Population structure, identity by state (IBS) and LD patterns across the genome will be of potential use for future GWAS of economically important traits in C. baccatum peppers. [ABSTRACT FROM AUTHOR]

Most agricultural traits are controlled by quantitative trait loci (QTLs); however, there are fewstudies on QTLmapping of horticultural traits in pepper (Capsicumspp.) due to the lack of high-densitymolecular maps and the sequence information. In this study, an ultra-high-density map and 120 recombinant inbred lines (RILs) derived from a cross between C. annuum 'Perennial' and C. annuum 'Dempsey' were used for QTL mapping of horticultural traits. Parental lines and RILs were resequenced at 18× and 1× coverage, respectively. Using a sliding window approach, an ultra-high-density bin map containing 2,578 bins was constructed. The total map length of the map was 1,372 cM, and the average interval between binswas 0.53 cM. A total of 86 significant QTLs controlling 17 horticultural traitswere detected. Among these, 32 QTLs controlling 13 traits were major QTLs. Our research shows that the construction of bin maps using low-coverage sequence is a powerful method for QTL mapping, and that the short intervals between bins are helpful for fine-mapping of QTLs. Furthermore, bin maps can be used to improve the quality of reference genomes by elucidating the genetic order of unordered regions and anchoring unassigned scaffolds to linkage groups. [ABSTRACT FROM AUTHOR]

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Cryptochromes are flavoproteins that play a central role in the circadian oscillations of all living organisms except archaea. Cryptochromes are clustered into three subfamilies: plant-type cryptochromes, animal-type cryptochromes and cryptochrome-DASH proteins. These subfamilies are composed of photolyase/cryptochrome superfamily with 6–4 photolyase and cyclobutane pyrimidine dimer photolyase. Cryptochromes have conserved domain architectures with two distinct domains, an N-terminal photolyase-related domain and a C-terminal domain. Although the molecular function and domain architecture of cryptochromes are conserved, their molecular mechanisms differ between plants and animals. Thus, cryptochromes are one of the best candidates for comparative and evolutionary studies. Here, we have developed a Web-based platform for comparative and evolutionary studies of cryptochromes, dbCRY (http://www.dbcryptochrome.org/). A pipeline built upon the consensus domain profile was applied to 1438 genomes and identified 1309 genes. To support comparative and evolutionary genomics studies, the Web interface provides diverse functions such as (i) browsing by species, (ii) protein domain analysis, (iii) multiple sequence alignment, (iv) homology search and (v) extended analysis opportunities through the implementation of ‘Favorite Browser’ powered by the Comparative Fungal Genomics Platform 2.0 (CFGP 2.0; http://cfgp.snu.ac.kr/). dbCRY would serve as a standardized and systematic solution for cryptochrome genomics studies. [ABSTRACT FROM AUTHOR]

Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the iso-lation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Ss4C. These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach. [ABSTRACT FROM AUTHOR]

Background: Among the Solanaceae plants, the pepper genome is three times larger than that of tomato. Although the gene repertoire and gene order of both species are well conserved, the cause of the genome-size difference is not known. To determine the causes for the expansion of pepper euchromatic regions, we compared the pepper genome to that of tomato. Results: For sequence-level analysis, we generated 35.6 Mb of pepper genomic sequences from euchromatin enriched 1,245 pepper BAC clones. The comparative analysis of orthologous gene-rich regions between both species revealed insertion of transposons exclusively in the pepper sequences, maintaining the gene order and content. The most common type of the transposon found was the LTR retrotransposon. Phylogenetic comparison of the LTR retrotransposons revealed that two groups of Ty3/Gypsy-like elements (Tat and Athila) were overly accumulated in the pepper genome. The FISH analysis of the pepper Tat elements showed a random distribution in heterochromatic and euchromatic regions, whereas the tomato Tat elements showed heterochromatinpreferential accumulation. Conclusions: Compared to tomato pepper euchromatin doubled its size by differential accumulation of a specific group of Ty3/Gypsy-like elements. Our results could provide an insight on the mechanism of genome evolution in the Solanaceae family. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are inevitably generated in aerobic organisms as by-products of normal metabolism or as the result of defense and development. ROS readily oxidize methionine (Met) residues in proteins/peptides to form Met-R-sulfoxide or Met-S-sulfoxide, causing inactivation or malfunction of the proteins. A pepper (Capsicum annuum) methionine sulfoxide reductase B2 gene (CaMsrB2) was isolated, and its roles in plant defense were studied. CaMsrB2 was down-regulated upon inoculation with either incompatible or compatible pathogens. The down-regulation, however, was restored to the original expression levels only in a compatible interaction. Gain-of-function studies using tomato (Solanum lycopersicum) plants transformed with CaMsrB2 resulted in enhanced resistance to Phytophthora capsici and Phytophthora infestans. Inversely, loss-of-function studies of CaMsrB2 using virus-induced gene silencing in pepper plants (cv Early Calwonder-30R) resulted in accelerated cell death from an incompatible bacterial pathogen, Xanthomonas axonopodis pv vesicatoria (Xav) race 1, and enhanced susceptibility to a compatible bacterial pathogen, virulent X. axonopodis pv vesicatoria race 3. Measurement of ROS levels in CaMsrB2-silenced pepper plants revealed that suppression of CaMsrB2 increased the production of ROS, which in turn resulted in the acceleration of cell death via accumulation of ROS. In contrast, the CaMsrB2-transgenic tomato plants showed reduced production of hydrogen peroxide. Taken together, our results suggest that the plant MsrBs have novel functions in active defense against pathogens via the regulation of cell redox status. [ABSTRACT FROM AUTHOR]

Trichomes are present on nearly all land plants and protect plants against insect herbivores, drought and UV radiation. The trichome-bearing phenotype is conferred by the dominant allele of the pepper trichome locus 1 ( Ptl1) in Capsicum annuum, Mexican ‘Criollo de Morelos-334’ (CM334). A genetic analysis using simple sequence repeats from pepper cDNA identified the HpmsE031 marker as tightly linked to Ptl1 in 653 individuals of an F2 population derived from a cross between CM334 and Chilsungcho varieties. A bacterial artificial chromosome (BAC) library from CM334 covering 12× of the genome was screened using the HpmsE031 SSR marker as a probe and three BAC clones were identified. The Ptl1 region was covered by one 80 kb BAC clone, TT1B7. Fluorescence in situ hybridization (FISH) confirmed that TT1B7 localized to pepper chromosome 10. One co-dominant marker, Tco, and one dominant marker, Tsca, were successfully developed from the TT1B7 BAC sequence. Tco mapped 0.33 cM up from Ptl1 and Tsca mapped 0.75 cM down from Ptl1. Analysis of the BAC sequence predicts the presence of 14 open reading frames including 60S ribosomal protein L21-like protein ( Solanum demissum), protein kinase 2 ( Nicotiana tabacum), hypothetical proteins, and unnamed protein products. These results will provide not only useful information for map-based cloning of Ptl1 in Capsicum but also the starting points for analysis of R-gene cluster inked with Ptl1. [ABSTRACT FROM AUTHOR]

•Plant receptor-like kinases belong to a large gene family. The Capsicum annuum receptor-like kinase 1 ( CaRLK1) gene encodes a transmembrane protein with a cytoplasmic kinase domain and an extracellular domain. •The CaRLK1 extracellular domain (ECD)–green fluorescent protein (GFP) fusion protein was targeted to the plasma membrane, and the kinase domain of the CaRLK1 protein exhibited autophosphorylation activity. CaRLK1 transcripts were more strongly induced in treatment with Xag8ra than in treatment with Xag8-13. Furthermore, infection with incompatible Xanthomonas campestris pv. vesicatoria race 3 induced expression of CaRLK1 more strongly than in the compatible interaction. •Cell death caused by both a disease-forming and an HR-inducing pathogen was delayed in the CaRLK1-transgenic plants. Ectopic expression of CaRLK1 also induced transcripts of the lesion stimulating disease ( LSD) gene, a negative regulator of cell death. Respiratory burst oxidase homolog ( RBOH) genes were up-regulated in the transgenic plants compared with the wild type, as the concentration of the superoxide anion was increased. In contrast, the concentration of H2O2 did not differ between the transgenic and wild-type plants. •These results support the theory that the suppression of plant cell death by CaRLK1 is associated with consistent production of the superoxide anion and induction of the RBOH genes and the LSD gene, but not with the concentration of H2O2. Thus, CaRLK1 may be a receptor of an as yet unidentified pathogen molecular pattern and may function as a negative regulator of plant cell death. [ABSTRACT FROM AUTHOR]

It has been suggested that serotonin biosynthesis is regulated by tryptophan decarboxylase (TDC) in plants. To determine if TDC plays a rate-limiting role in serotonin biosynthesis, two TDC genes, PepTDC1 and PepTDC2, were cloned from pepper ( Capsicum annuum L.) fruits infected with anthracnose fungus and their expression was then examined in various organs, including fruit that had been treated with the fungus or various chemicals. PepTDC1 expression was highly induced in pepper fruits after treatment with fungus and ethylene, while PepTDC2 was constitutively expressed at low levels in all pepper tissues. Additionally, predominant induction of PepTDC1 mRNA and TDC enzyme activity was detected in the unripe-green fruit, but not in the ripe-red fruit upon pathogen infection. Higher expression of TDC in unripe-green fruit was closely associated with increased levels of tryptamine, serotonin, and serotonin derivatives. However, unlike the enhanced serotonin synthesis, tryptophan levels responded unchanged when challenged with the pathogen in both the unripe-green fruit and the ripe-red fruit. Expression of two key tryptophan biosynthetic genes, anthranilate synthase ( ASα) and tryptophan synthase ( TSβ), remained unchanged in response to treatment. Also, anthranilate synthase enzyme activity remained steady regardless of pathogen infection. Taken together, these results suggest that the synthesis of serotonin was regulated by the induction of TDC without a simultaneous increase in tryptophan levels in pepper fruits. [ABSTRACT FROM AUTHOR]

• Induction of cell death is an important component of plant defense against pathogens. There have been many reports on the role of phytohormones in pathogen-induced cell death, but jasmonic acid (JA) has not been implicated as a regulator of the response. Here, we report the function of NbHB1, Nicotiana benthamiana homeobox1, in pathogen-induced cell death in connection with JA signaling. • Involvement of NbHB1 in cell death was analysed by gain- and loss-of-function studies using Agrobacterium-mediated transient overexpression and virus-induced gene silencing, respectively. Expression of NbHB1 following pathogen inoculations and various treatments was monitored by reverse transcription polymerase chain reaction. • Transcript levels of NbHB1 were upregulated by infection with virulent and avirulent bacterial pathogens. Ectopic expression of NbHB1 accelerated cell death following treatment with darkness, methyl jasmonate, or pathogen inoculation. Conversely, when NbHB1 was silenced, pathogen-induced cell death was delayed. NbHB1-induced cell death was also delayed by silencing of NbCOI1, indicating a requirement for JA-mediated signaling. Overexpression of the domain-deleted proteins of NbHB1 revealed that the homeodomain, leucine zipper, and part of the variable N-terminal region were necessary for NbHB1 functionality. • These results strongly suggest the role of NbHB1 in pathogen-induced plant cell death via the JA-mediated signaling pathway. [ABSTRACT FROM AUTHOR]

The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD3645.1, suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb11 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34-amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2. [ABSTRACT FROM AUTHOR]

The article highlights a study which examined the characteristics of a tomato alpha-dioxygenase1 (alpha-DOXI) promoter. Based on reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the promoter is found to be root-specific. The promoter could be a useful tool for foreign gene expression in plant root.

Background: Tandemly repeated DNA, also called as satellite DNA, is a common feature of eukaryotic genomes. Satellite repeats can expand and contract dramatically, which may cause genome size variation among genetically-related species. However, the origin and expansion mechanism are not clear yet and needed to be elucidated. Results: FISH analysis revealed that the satellite repeat showing homology with intergenic spacer (IGS) of rDNA present in the tomato genome. By comparing the sequences representing distinct stages in the divergence of rDNA repeat with those of canonical rDNA arrays, the molecular mechanism of the evolution of satellite repeat is described. Comprehensive sequence analysis and phylogenetic analysis demonstrated that a long terminal repeat retrotransposon was interrupted into each copy of the 18S rDNA and polymerized by recombination rather than transposition via an RNA intermediate. The repeat was expanded through doubling the number of IGS into the 25S rRNA gene, and also greatly increasing the copy number of type I subrepeat in the IGS of 25-18S rDNA by segmental duplication. Homogenization to a single type of subrepeat in the satellite repeat was achieved as the result of amplifying copy number of the type I subrepeat but eliminating neighboring sequences including the type II subrepeat and rRNA coding sequence from the array. FISH analysis revealed that the satellite repeats are commonly present in closely-related Solanum species, but vary in their distribution and abundance among species. Conclusion: These results represent that the dynamic satellite repeats were originated from intergenic spacer of rDNA unit in the tomato genome. This result could serve as an example towards understanding the initiation and the expansion of the satellite repeats in complex eukaryotic genome. [ABSTRACT FROM AUTHOR]

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F2 individuals from a cross between Capsicum annuum ‘CM334’ and C. annuum ‘Chilsungcho’. This linkage map consisted of 202 restriction fragment length polymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F2:3 originated from a cross between resistant Mexican landrace C. annuum ‘CM334’ and susceptible Korean landrace C. annuum ‘Chilsungcho’ using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum ‘CM334’ was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici. [ABSTRACT FROM AUTHOR]

Abstract  The Arabidopsis bypass1 mutant (bps1) exhibits defective shoot and root growth that is associated with constitutive production of a root-derived carotenoid-related signal (Van Norman et al., Curr Biol 14:1739–1746, 2004). Since the identity of the signal and the function of BPS1 are still unknown, we investigated effects of BPS1 depletion in Nicotiana benthamiana to elucidate BPS1 function in plant growth and development. The predicted protein of NbBPS1, a BPS1 homolog of N. benthamiana, contains a central transmembrane domain, and a NbBPS1:GFP fusion protein was mainly associated with the endoplasmic reticulum. Virus-induced gene silencing (VIGS) of NbBPS1 resulted in pleiotrophic phenotypes, including growth retardation and abnormal leaf development. At the cellular level, the plants exhibited hyperproliferation of the cambial cells and defective xylem differentiation during stem vascular development. Hyperactivity of the cambium was associated with an elevated auxin and cytokinin response. In contrast, the leaves had reduced numbers of cells with increased cell size and elevated endoreduplication. Cell death in NbBPS1 VIGS leaves started with vacuole collapse, followed by degeneration of the organelles. Interestingly, these phenotypes were mainly caused by silencing of NbBPS1 in the aerial parts of the plants, different from the case of the Arabidopsis bps1 mutant. These results suggest that NbBPS1 plays a role in the control of cell division and differentiation in the cambium of N. benthamiana, and BPS homologs may have a diverse function in different tissues and in different species. [ABSTRACT FROM AUTHOR]

• Plants respond to pathogens by regulating a network of signaling pathways that fine-tune transcriptional activation of defense-related genes. • The aim of this study was to determine the role of Capsicum annuum WRKY zinc finger-domain transcription factor 1 (CaWRKY1) in defense. In previous studies, CaWRKY1 was found to be rapidly induced in C. annuum (chili pepper) leaves by incompatible and compatible pathogen inoculations, but the complexity of the network of the WRKY family prevented the function of CaWRKY1 in defense from being elucidated. • Virus-induced gene silencing of CaWRKY1 in chili pepper leaves resulted in decreased growth of Xanthomonas axonopodis pv. vesicatoria race 1. CaWRKY1-overexpressing transgenic plants showed accelerated hypersensitive cell death in response to infection with tobacco mosaic virus and Pseudomonas syringe pv. tabaci. Lower levels of pathogenesis-related gene induction were observed in CaWRKY1-overexpressing transgenic plants following salicylic acid (SA) treatments. • This work suggests that the newly characterized CaWRKY1, which is strongly induced by pathogen infections and the signal molecule SA, acts as a regulator to turn off systemic acquired resistance once the pathogen challenge has diminished and to prevent spurious activation of defense responses at suboptimal concentrations of SA. [ABSTRACT FROM AUTHOR]

Using cDNA microarray analysis, we isolated a cDNA clone, CaATL1 ( Capsicum annuum L. Bukang AT-hook-Like gene 1), from a chili pepper plant incompatibly interacting with bacterial pathogens. The deduced amino acid sequence has a potential nuclear localization sequence and an AT-hook DNA binding motif which can bind AT-rich sequence elements. Expression of CaATL1 was specifically induced in host- and non-host-resistant responses against bacterial and viral pathogens in pepper plants. In addition, CaATL1 transcripts also increased following salicylic acid and ethephone treatment but were only mildly induced by methyl-jasmonate treatment. CaATL1::smGFP (soluble-modified green fluorescent protein) fusion protein localized to nuclei in tobacco BY2 protoplasts. The C-terminal region of the CaATL1 protein fused to the LexA DNA binding domain was able to activate reporter gene expression in yeast. To analyse further the role of the CaATL1 in pathogen defence response, we generated CaATL1-over-expressing transgenic tomato plants. These transgenic plants showed enhanced disease resistance against bacterial and oomycete pathogens. Taken together, these results provide the first evidence of a role for a plant AT-hook motif-containing transcription factor in pathogen defence response. [ABSTRACT FROM AUTHOR]

The coordinated expression of the genes involved in respiration in the photosynthetic cyanobacterium Synechocystis sp. FCC 6803 during the early period of glucose (Gic) treatment is poorly understood. When photoautotrophically grown cells were supplemented with 10 mm Gic in the light or after a dark adaptation period of 14 h, significant increases in the respiratory activity, as determined by NAD(P)H turnover, respiratory 02 uptake rate, and cytosolic alkalization, were observed. At the same time, the transcript levels of 18 genes coding for enzymes associated with respiration increased with differential induction kinetics; these genes were classified into three groups based on their half-rising times. Transcript levels of the four genes gpi, zwf, pdhB, and atpB started to increase along with a net increase in NAD(P)H, while the onset of net NAD(P)H consumption coincided with an increase in those of the genes tktA, ppc, pdhD, icd, ndhD2, ndbA, ctaDl, cydA, and atpE. In contrast, the expression of the atpl/G/D/A/C genes coding for AlP synthase subunits was the slowest among respiratory genes and their expression started to accumulate only after the establishment of cytosolic alkalization. These differential effects of Gic on the transcript levels of respiratory genes were not observed by inactivation of the genes encoding the Glc transporter or glucokinase. In addition, several Glc analogs could not mimic the effects of Glc. Our findings suggest that genes encoding some enzymes involved in central carbon metabolism and oxidative phosphorylation are coordinately regulated at the transcriptional level during the switch of nutritional mode. [ABSTRACT FROM AUTHOR]

The CCR4-associated factor 1 (CAF1) protein belongs to the CCR4-NOT complex, which is an evolutionary conserved protein complex and plays an important role in the control of transcription and mRNA decay in yeast and mammals. To investigate the function of CAF1 in plants, we performed gain- and loss-of-function studies by overexpression of the pepper CAF1 ( CaCAF1) in tomato and virus-induced gene silencing (VIGS) of the gene in pepper plants. Overexpression of CaCAF1 in tomato resulted in significant growth enhancement, with increasing leaf thickness, and enlarged cell size by more than twofold when compared with the control plants. A transmission electron microscopic analysis revealed that the CaCAF1-transgenic tomato plants had thicker cell walls and cuticle layers than the control plants. In addition to developmental changes, overexpression of CaCAF1 in tomato plants resulted in enhanced resistance against the oomycete pathogen Phytophthora infestans. Additionally, microarray, northern and real-time polymerase chain reaction analyses of CaCAF1-transgenic tomato plants revealed that multiple genes were constitutively upregulated, including genes involved in polyamine biosynthesis, defence reactions and cell-wall organogenesis. In contrast, VIGS of CaCAF1 in pepper plants caused significant growth retardation and enhanced susceptibility to the pepper bacterial spot pathogen Xanthomonas axonopodis pv. vesicatoria. Our results suggest roles for plant CAF1 in normal growth and development, as well as in defence against pathogens. [ABSTRACT FROM AUTHOR]

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F2 individuals from an interspecific cross of TF68 × Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding. [ABSTRACT FROM AUTHOR]

Agrobacterium radiobacter K84. used worldwide to biocontrol crown gall disease caused by Agrobacterium tumefaciens, produces an antiagrobacterial compound called agrocin 84. We report the nucleotide sequence of pAgK84. a 44.42-kb plasmid coding for production of this disubstituted adenine nucleotide antibiotic. pAgK84 encodes 36 ORFs, 17 of which (agn) code for synthesis of or immunity to agrocin 84. Two genes, agnB2 and agnA, encode aminoacyl tRNA synthetase homologues. We have shown that the toxic moiety of agrocin 84 inhibits cellular leucyl-tRNA synthetases and AgnB2, which confers immunity to the antibiotic, is a resistant form of this enzyme. AgnA, a truncated homologue of asparaginyl tRNA synthetase could catalyze the phosphoramidate bond between a precursor of the methyl pentanamide side group and the nucleotide. We propose previously undescribed chemistry, catalyzed by AgnB1, to generate the precursor necessary for this phosphoramidate linkage. AgnC7 is related to ribonucleotide reductases and could generate the 3′-deoxyarabinose moiety of the nucleoside. Bioinformatics suggest that agnC3, agnc4, and agnC6 contribute to maturation of the methyl pentanamide, whereas agnC2 may produce the glucofuranose side group bound to the adenine ring. AgnG is related to bacterial exporters. An agnG mutant accumulated agrocin 84 intracellularly but did not export the antibiotic. pAgK84 is transmissible and encodes genes for conjugative DNA processing but lacks a type IV secretion system. suggesting that pAgK84 transfers by mobilization. By sequence analysis, the deletion engineered into pAgK1026 removed the oriT and essential tra genes, confirming the enhanced environmental safety of this modified form of pAgK84. [ABSTRACT FROM AUTHOR]

Pepper (Capsicum annuum) serotonin N-hydroxycinnamoyltransferase (SHT) catalyzes the synthesis of N-hydroxycinnamic acid amides of serotonin, including feruloylserotonin and p-coumaroylserotonin. To elucidate the domain or the key amino acid that determines the amine substrate specificity, we isolated a tyramine N-hydroxycinnamoyltransferase (THT) gene from pepper. Purified recombinant THT protein catalyzed the synthesis of N-hydroxycinnamic acid amides of tyramine, including feruloyltyramine and p-coumaroyltyramine, but did not accept serotonin as a substrate. Both the SHT and THT mRNAs were found to be expressed constitutively in all pepper organs. Pepper SHT and THT, which have primary sequences that are 78% identical, were used as models to investigate the structural determinants responsible for their distinct substrate specificities and other enzymatic properties. A series of chimeric genes was constructed by reciprocal exchange of DNA segments between the SHT and THT cDNAs. Functional characterization of the recombinant chimeric proteins revealed that the amino acid residues 129 to 165 of SHT and the corresponding residues 125 to 160 in THT are critical structural determinants for amine substrate specificity. Several amino acids are strongly implicated in the determination of amine substrate specificity, in which glycine-158 is involved in catalysis and amine substrate binding and tyrosine-149 plays a pivotal role in controlling amine substrate specificity between serotonin and tyramine in SHT. Furthermore, the indisputable role of tyrosine is corroborated by the THT-F145Y mutant that uses serotonin as the acyl acceptor. The results from the chimeras and the kinetic measurements will direct the creation of additional novel N-hydroxycinnamoyltransferases from the various N-hydroxycinnamoyltransferases found in nature. [ABSTRACT FROM AUTHOR]

To understand better the defence responses of plants to pathogen attack, we challenged hot pepper plants with bacterial pathogens and identified transcription factor-encoding genes whose expression patterns were altered during the subsequent hypersensitive response. One of these genes,CaPIF1(Capsicum annuum Pathogen-Induced Factor 1), was characterized further. This gene encodes a plant-specific EPF-type protein that contains two Cys2/His2 zinc fingers.CaPIF1expression was rapidly and specifically induced when pepper plants were challenged with bacterial pathogens to which they are resistant. In contrast, challenge with a pathogen to which the plants are susceptible only generated weakCaPIF1expression.CaPIF1expression was also strongly induced in pepper leaves by the exogenous application of ethephon, an ethylene-releasing compound, and salicylic acid, whereas methyl jasmonate had only moderate effects. CaPIF1 localized to the nuclei of onion epidermis when expressed as a CaPIF1–smGFP fusion protein. Transgenic tobacco plants over-expressingCaPIF1driven by the CaMV 35S promoter showed increased resistance to challenge with a tobacco-specific pathogen or non-host bacterial pathogens. These plants also showed constitutive up-regulation of multiple defence-related genes. Moreover, virus-induced silencing of theCaPIF1orthologue inNicotiana benthamianaenhanced susceptibility to the same host or non-host bacterial pathogens. These observations provide evidence that an EPF-type Cys2/His2 zinc-finger protein plays a crucial role in the activation of the pathogen defence response in plants. [ABSTRACT FROM AUTHOR]

Large-scale single-pass sequencing of cDNA libraries and microarray analysis have proven to be useful tools for discovering new genes and studying gene expression. As a first step in elucidating the defense mechanisms in hot pepper plants, a total of 8,525 expressed sequence tags (ESTs) were generated and analyzed in silico. The cDNA microarray analysis identified 613 hot pepper genes that were transcriptionally responsive to the non-host soybean pustule pathogen Xanthomonas axonopodis pv. glycines (Xag). Several functional types of genes, including those involved in cell wall modification/biosynthesis, transport, signaling pathways and divergent defense reactions, were induced at the early stage of Xag infiltration. In contrast, genes encoding proteins that are involved in photosynthesis, carbohydrate metabolism and the synthesis of chloroplast biogenetic proteins were down-regulated at the late stage of Xag infiltration. These expression profiles share common features with the expression profiles elicited by other stresses, such as fungal challenge, wounding, cold, drought and high salinity. However, we also identified several novel transcription factors that may be specifically involved in the defense reaction of the hot pepper. We also found that the defense reaction of the hot pepper may involve the deactivation of gibberellin. Furthermore, many genes encoding proteins with unknown function were identified. Functional analysis of these genes may broaden our understanding of non-host resistance. This study is the first report of large-scale sequencing and non-host defense transcriptome analysis of the hot pepper plant species. (The sequence data in this paper have been submitted to the dbEST and GenBank database under the codes 10227604–10236595 and BM059564–BM068555, respectively. Additional information is available at http://plant.pdrc.re.kr/ks200201/pepper.html). [ABSTRACT FROM AUTHOR]

To understand the function of metallothioneins (MTs) in plants, we introduced the Nicotiana gilltinosa MT gene into tobacco (N. tabacum) plants via an Agrobacterium mediated transformation. Full-length MT cDNA was fused between the cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (nos) terminator of the pMBP1 binary vector in sense orientation. Tobacco leaf discs which were cocultivated with Agrobacterium carrying the chimeric MT gene, formed kanamycin-resistant shoots on medium containing kanamycin. The kanamycin-resistant shoots were subsequently rooted on medium containing 200 μM CdSO4• Approximately 30% of individual transgenic plants developed normally. Nontransgenic plants promptly underwent leaf chlorosis, and their growth and development were inhibited on MS medium containing 50 μM CdSO4• Genomic Southern blot analysis showed that the MT gene was stably integrated into the nuclear genome of transgenic tobacco plants. The expression level of MT transcripts was analyzed by RNA gel blot analysis. Selfpollinated seeds obtained from transgenic tobacco plants showing cadmium tolerance were germinated on a medium containing 100 μM CdSO4• PCR analysis from sensitive and stably resistant T2 seedlings for cadmium sulfate confirmed a high correlation between the phenotypic expression of the MT gene and the transgenic genotype, indicating that the MT gene is inherited in the next generation. [ABSTRACT FROM AUTHOR]

To understand the coordinated functions of the different classes of defense-related genes expressed in plant disease resistance, the expression patterns of pathogenesis related (PR) protein genes and genes involved in antioxidation and the production of secondary metabolites were examined. The expression patterns of the respective defense-related genes were monitored following TMV infection or salicylic acid treatment. Northern blot analyses showed that PR genes such as PR-l, β-l,3-glucanase and chitinase were strongly induced in tobacco leaves upon TMV infection or salicylic acid treatment. 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and phenylalanine ammonialyase (PAL), involved in isoprenoid and phenylpropanoid biosynthesis, respectivel y, were mildly induced at the late stage of normal hypersensitive response (HR) or after salicylic acid treatment when compared with the PR-gene expressions. However, in acute HR, they were strongly expressed at the early stage. Interestingly, the expression of the antioxidative genes, anionic peroxidase and ascorbate peroxidase, were inversely expressed following TMV infection and salicylic acid treatment. Differential expression of 3 groups of genes involved in plant defense responses are discussed in relation to different signal transduction pathways. [ABSTRACT FROM AUTHOR]