During rice blast fungal attack, plant xylanase inhibitor proteins (XIPs) that inhibit fungal xylanase activity are believed to act as a defensive barrier against fungal pathogens. To understand the role of XIPs in rice, a xylanase inhibitor was cloned from rice. The expression of this gene was examined at the transcriptional/translational levels during compatible and incompatible interactions, and the biochemical activity of this protein was also examined.
Sequence alignment revealed that the deduced amino acid sequence of OsCLP shares a high degree of similarity with that of other plant TAXI-type XIPs. However, recombinant OsCLP did not display inhibitory activity against endo-1,4-β-xylanase enzymes from Aureobasidium pullulans (A. pullulans) or Trichoderma viride (T. viride). Instead, an in-gel activity assay revealed strong chitinase activity. The transcription and translation of OsCLP were highly induced when rice was exposed to pathogens in an incompatible interaction. In addition, exogenous treatment with OsCLP affected the growth of the basidiomycete fungus Rhizoctonia solani through degradation of the hyphal cell wall. These data suggest that OsCLP, which has chitinase activity, may play an important role in plant defenses against pathogens.
Taken together, our results demonstrate that OsCLP may have antifungal activity. This protein may directly inhibit pathogen growth by degrading fungal cell wall components through chitinase activity.
Antifungal activity; Chitinase; Oryza sativa; Xylanase inhibitor
The coffee berry borer, Hypothenemus hampei, reproduces and feeds exclusively on the mature endosperm of the coffee seed, which has a cell wall composed mainly of a heterogeneous mixture of hemicellulose polysaccharides, including arabinoxylans. Xylanases are digestive enzymes responsible for the degradation of xylan based polymers, hydrolyzing them into smaller molecules that are easier to assimilate by insects. We report the cloning, expression and enzymatic characterization of a xylanase gene that was identified in the digestive tract of the coffee berry borer.
The complete DNA sequence encoding a H. hampei xylanase (HhXyl) was obtained using a genome walking technique in a cDNA library derived from the borer digestive tract. The XIP-I gene was amplified from wheat (Triticum aestivum variety Soisson). A Pichia pastoris expression system was used to express the recombinant form of these enzymes. The xylanase activity and XIP-I inhibitory activity was quantified by the 3,5-dinitrosalicylic (DNS). The biological effects of XIP-I on borer individuals were evaluated by providing an artificial diet enriched with the recombinant XIP-I protein to the insects.
The borer xylanase sequence contains a 951 bp open reading frame that is predicted to encode a 317-amino acid protein, with an estimated molecular weight of 34.92 kDa and a pI of 4.84. Bioinformatic analysis revealed that HhXyl exhibits high sequence homology with endo-β-D-xylanases of Streptomyces bingchenggensis from glycosyl hydrolase 10 (GH10). The recombinant xylanase showed maximal activity at pH 5.5 and 37°C. XIP-I expressed as a recombinant protein inhibited HhXyl activity in vitro and caused individual H. hampei mortality in bioassays when included as a supplement in artificial diets.
A xylanase from the digestive tract of the coffee berry borer was identified and functionally characterized. A xylanase inhibitor protein, XIP-I, from wheat was shown to be a potent inhibitor of this xylanase, suggesting that its deployment has potential as a strategy to control coffee berry borer colonization of coffee plants.
Leaf rust, which is caused by the fungus Hemileia vastatrix (Pucciniales), is a devastating disease that affects coffee plants (Coffea arabica L.). Disadvantages that are associated with currently developed phytoprotection approaches have recently led to the search for alternative strategies. These include genetic manipulations that constitutively activate disease resistance signaling pathways. However, molecular actors of such pathways still remain unknown in C. arabica. In this study, we have isolated and characterized the coffee NDR1 gene, whose Arabidopsis ortholog is a well-known master regulator of the hypersensitive response that is dependent on coiled-coil type R-proteins.
Two highly homologous cDNAs coding for putative NDR1 proteins were identified and cloned from leaves of coffee plants. One of the candidate coding sequences was then expressed in the Arabidopsis knock-out null mutant ndr1-1. Upon a challenge with a specific strain of the bacterium Pseudomonas syringae (DC3000::AvrRpt2), analysis of both macroscopic symptoms and in planta microbial growth showed that the coffee cDNA was able to restore the resistance phenotype in the mutant genetic background. Thus, the cDNA was dubbed CaNDR1a (standing for Coffea arabica Non-race specific Disease Resistance 1a). Finally, biochemical and microscopy data were obtained that strongly suggest the mechanistic conservation of the NDR1-driven function within coffee and Arabidopsis plants. Using a transient expression system, it was indeed shown that the CaNDR1a protein, like its Arabidopsis counterpart, is localized to the plasma membrane, where it is possibly tethered by means of a GPI anchor.
Our data provide molecular and genetic evidence for the identification of a novel functional NDR1 homolog in plants. As a key regulator initiating hypersensitive signalling pathways, CaNDR1 gene(s) might be target(s) of choice for manipulating the coffee innate immune system and achieving broad spectrum resistance to pathogens. Given the potential conservation of NDR1-dependent defense mechanisms between Arabidopsis and coffee plants, our work also suggests new ways to isolate the as-yet-unidentified R-gene(s) responsible for resistance to H. vastatrix.
Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to spread across the southeast and midsouth regions of the United States, necessitating the use of fungicides by producers. Our objective in this research was to identify ASR proteins expressed early during infection for the development of immunodiagnostic assays. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (for Phakopsora
extracellular protein). Two highly expressed protein family members, PHEP 107 and PHEP 369, were selected as ideal immunodiagnostic targets for antibody development, after we detected PHEPs in plants as early as 3 days postinfection (dpi). Monoclonal antibodies (MAbs; 2E8E5-1 and 3G6H7-3) generated against recombinant PHEP 369 were tested for sensitivity against the recombinant protein and extracts from ASR-infected plants and for specificity against a set of common soybean pathogens. These antibodies should prove applicable in immunodiagnostic assays to detect infected soybeans and to identify ASR spores from sentinel surveillance plots.
Phakopsora pachyrhizi is an obligate fungal pathogen causing Asian soybean rust (ASR). A dual approach was taken to examine the molecular and biochemical processes occurring during the development of appressoria, specialized infection structures by which P. pachyrhizi invades a host plant. Suppression subtractive hybridization (SSH) was utilized to generate a cDNA library enriched for transcripts expressed during appressoria formation. Two-dimensional gel electrophoresis and mass spectroscopy analysis were used to generate a partial proteome of proteins present during appressoria formation.
Sequence analysis of 1133 expressed sequence tags (ESTs) revealed 238 non-redundant ESTs, of which 53% had putative identities assigned. Twenty-nine of the non-redundant ESTs were found to be specific to the appressoria-enriched cDNA library, and did not occur in a previously constructed germinated urediniospore cDNA library. Analysis of proteins against a custom database of the appressoria-enriched ESTs plus Basidiomycota EST sequences available from NCBI revealed 256 proteins. Fifty-nine of these proteins were not previously identified in a partial proteome of P. pachyrhizi germinated urediniospores. Genes and proteins identified fell into functional categories of metabolism, cell cycle and DNA processing, protein fate, cellular transport, cellular communication and signal transduction, and cell rescue. However, 38% of ESTs and 24% of proteins matched only to hypothetical proteins of unknown function, or showed no similarity to sequences in the current NCBI database. Three novel Phakopsora genes were identified from the cDNA library along with six potentially rust-specific genes. Protein analysis revealed eight proteins of unknown function, which possessed classic secretion signals. Two of the extracellular proteins are reported as potential effector proteins.
Several genes and proteins were identified that are expressed in P. pachyrhizi during appressoria formation. Understanding the role that these genes and proteins play in the molecular and biochemical processes in the infection process may provide insight for developing targeted control measures and novel methods of disease management.
Most disease-resistance (R) genes in plants encode NBS-LRR proteins and belong to one of the largest and most variable gene families among plant genomes. However, the specific evolutionary routes of NBS-LRR encoding genes remain elusive. Recently in coffee tree (Coffea arabica), a region spanning the SH3 locus that confers resistance to coffee leaf rust, one of the most serious coffee diseases, was identified and characterized. Using comparative sequence analysis, the purpose of the present study was to gain insight into the genomic organization and evolution of the SH3 locus.
Sequence analysis of the SH3 region in three coffee genomes, Ea and Ca subgenomes from the allotetraploid C. arabica and Cc genome from the diploid C. canephora, revealed the presence of 5, 3 and 4 R genes in Ea, Ca, and Cc genomes, respectively. All these R-gene sequences appeared to be members of a CC-NBS-LRR (CNL) gene family that was only found at the SH3 locus in C. arabica. Furthermore, while homologs were found in several dicot species, comparative genomic analysis failed to find any CNL R-gene in the orthologous regions of other eudicot species. The orthology relationship among the SH3-CNL copies in the three analyzed genomes was determined and the duplication/deletion events that shaped the SH3 locus were traced back. Gene conversion events were detected between paralogs in all three genomes and also between the two sub-genomes of C. arabica. Significant positive selection was detected in the solvent-exposed residues of the SH3-CNL copies.
The ancestral SH3-CNL copy was inserted in the SH3 locus after the divergence between Solanales and Rubiales lineages. Moreover, the origin of most of the SH3-CNL copies predates the divergence between Coffea species. The SH3-CNL family appeared to evolve following the birth-and-death model, since duplications and deletions were inferred in the evolution of the SH3 locus. Gene conversion between paralog members, inter-subgenome sequence exchanges and positive selection appear to be the major forces acting on the evolution of SH3-CNL in coffee trees.
Coffee is one of the world's most important crops; it is consumed worldwide and plays a significant role in the economy of producing countries. Coffea arabica and C. canephora are responsible for 70 and 30% of commercial production, respectively. C. arabica is an allotetraploid from a recent hybridization of the diploid species, C. canephora and C. eugenioides. C. arabica has lower genetic diversity and results in a higher quality beverage than C. canephora. Research initiatives have been launched to produce genomic and transcriptomic data about Coffea spp. as a strategy to improve breeding efficiency.
Assembling the expressed sequence tags (ESTs) of C. arabica and C. canephora produced by the Brazilian Coffee Genome Project and the Nestlé-Cornell Consortium revealed 32,007 clusters of C. arabica and 16,665 clusters of C. canephora. We detected different GC3 profiles between these species that are related to their genome structure and mating system. BLAST analysis revealed similarities between coffee and grape (Vitis vinifera) genes. Using KA/KS analysis, we identified coffee genes under purifying and positive selection. Protein domain and gene ontology analyses suggested differences between Coffea spp. data, mainly in relation to complex sugar synthases and nucleotide binding proteins. OrthoMCL was used to identify specific and prevalent coffee protein families when compared to five other plant species. Among the interesting families annotated are new cystatins, glycine-rich proteins and RALF-like peptides. Hierarchical clustering was used to independently group C. arabica and C. canephora expression clusters according to expression data extracted from EST libraries, resulting in the identification of differentially expressed genes. Based on these results, we emphasize gene annotation and discuss plant defenses, abiotic stress and cup quality-related functional categories.
We present the first comprehensive genome-wide transcript profile study of C. arabica and C. canephora, which can be freely assessed by the scientific community at http://www.lge.ibi.unicamp.br/coffea. Our data reveal the presence of species-specific/prevalent genes in coffee that may help to explain particular characteristics of these two crops. The identification of differentially expressed transcripts offers a starting point for the correlation between gene expression profiles and Coffea spp. developmental traits, providing valuable insights for coffee breeding and biotechnology, especially concerning sugar metabolism and stress tolerance.
Background and Aims
Galactomannans act as storage reserves for the seeds in some plants, such as guar (Cyamopsis tetragonoloba) and coffee (Coffea arabica and Coffea canephora). In coffee, the galactomannans can represent up to 25 % of the mass of the mature green coffee grain, and they exert a significant influence on the production of different types of coffee products. The objective of the current work was to isolate and characterize cDNA encoding proteins responsible for galactomannan synthesis in coffee and to study the expression of the corresponding transcripts in the developing coffee grain from C. arabica and C. canephora, which potentially exhibit slight galactomannan variations. Comparative gene expression analysis was also carried out for several other tissues of C. arabica and C. canephora.
cDNA banks, RACE-PCR and genome walking were used to generate full-length cDNA for two putative coffee mannan synthases (ManS) and two galactomannan galactosyl transferases (GMGT). Gene-specific probe-primer sets were then generated and used to carry out comparative expression analysis of the corresponding genes in different coffee tissues using quantitative RT-PCR
Two of the putative galactomannan biosynthetic genes, ManS1 and GMGT1, were demonstrated to have very high expression in the developing coffee grain of both Coffea species during endosperm development, consistent with our proposal that these two genes are responsible for the production of the majority of the galactomannans found in the grain. In contrast, the expression data presented indicates that the ManS2 gene product is probably involved in the synthesis of the galactomannans found in green tissue.
The identification of genes implicated in galactomannan synthesis in coffee are presented. The data obtained will enable more detailed studies on the biosynthesis of this important component of coffee grain and contribute to a better understanding of some functional differences between grain from C. arabica and C. canephora.
Coffea; galactomannans; mannan synthase; galactomannan galactosyl transferase; coffee grain
Two experiments were carried out to evaluate the effects of Pratylenchus brachyurus and P. coffeae on Coffea arabica. The first experiment was conducted in a greenhouse to determine the effects of Pratylenchus brachyurus and P. coffeae on seedlings of Coffea arabica cv. Mundo Novo. Both Pratylenchus spp. reduced the growth of coffee seedlings. Higher contents of soluble sugars were detected in the leaves of infected plants. The reproduction rate of P. brachyurus was very low on cv. Mundo Novo, indicating an intolerance to this nematode. In a second experiment, C. arabica cultivars Mundo Novo and Catuaf both were intolerant hosts of P. brachyurus.
caffeine; carbohydrates; chlorophyll; coffee; intolerance; Pratylenchus brachyurus; Pratylenchus coffeae; protein susceptibility
Asian soybean rust (ASR), caused by Phakopsora pachyrhizi Syd., has the potential to become a serious threat to soybean, Glycine max L. Merr., production in the USA. A novel rust resistance gene, Rpp?(Hyuuga), from the Japanese soybean cultivar Hyuuga has been identified and mapped to soybean chromosome 6 (Gm06). Our objectives were to fine-map the Rpp?(Hyuuga) gene and develop a high-throughput single nucleotide polymorphism (SNP) assay to detect this ASR resistance gene. The integration of recombination events from two different soybean populations and the ASR reaction data indicates that the Rpp?(Hyuuga) locus is located in a region of approximately 371 kb between STS70887 and STS70923 on chromosome Gm06. A set of 32 ancestral genotypes which is predicted to contain 95% of the alleles present in current elite North American breeding populations and the sources of the previously reported ASR resistance genes (Rpp1, Rpp2, Rpp3, Rpp4, Rpp5, and rpp5) were genotyped with five SNP markers. We developed a SimpleProbe assay based on melting curve analysis for SNP06-44058 which is tighly linked to the Rpp?(Hyuuga) gene. This SNP assay can differentiate plants/lines that are homozygous/homogeneous or heterozygous/heterogeneous for the resistant and susceptible alleles at the Rpp?(Hyuuga) locus.
Microsatellite markers, also known as SSRs (Simple Sequence Repeats), have proved to be excellent tools for identifying variety and determining genetic relationships. A set of 127 SSR markers was used to analyze genetic similarity in twenty five Coffea arabica varieties. These were composed of nineteen commercially important Brazilians and six interspecific hybrids of Coffea arabica, Coffea canephora and Coffealiberica. The set used comprised 52 newly developed SSR markers derived from microsatellite enriched libraries, 56 designed on the basis of coffee SSR sequences available from public databases, 6 already published, and 13 universal chloroplast microsatellite markers. Only 22 were polymorphic, these detecting 2-7 alleles per marker, an average of 2.5. Based on the banding patterns generated by polymorphic SSR loci, the set of twenty-five coffee varieties were clustered into two main groups, one composed of only Brazilian varieties, and the other of interspecific hybrids, with a few Brazilians. Color mutants could not be separated. Clustering was in accordance with material genealogy thereby revealing high similarity.
SSR; coffee; genetic similarity; molecular marker
The montane rainforests of SW Ethiopia are the primary centre of diversity of Coffea arabica and the origin of all Arabica coffee cultivated worldwide. This wild gene pool is potentially threatened by forest fragmentation and degradation, and by introgressive hybridization with locally improved coffee varieties. We genotyped 703 coffee shrubs from unmanaged and managed coffee populations, using 24 microsatellite loci. Additionally, we genotyped 90 individuals representing 23 Ethiopian cultivars resistant to coffee berry disease (CBD). We determined population genetic diversity, genetic structure, and admixture of cultivar alleles in the in situ gene pool. We found strong genetic differentiation between managed and unmanaged coffee populations, but without significant differences in within-population genetic diversity. The widespread planting of coffee seedlings including CBD-resistant cultivars most likely offsets losses of genetic variation attributable to genetic drift and inbreeding. Mixing cultivars with original coffee genotypes, however, leaves ample opportunity for hybridization and replacement of the original coffee gene pool, which already shows signs of admixture. In situ conservation of the wild gene pool of C. arabica must therefore focus on limiting coffee production in the remaining wild populations, as intensification threatens the genetic integrity of the gene pool by exposing wild genotypes to cultivars.
admixture; Afromontane rainforest; coffee; crop wild relative; ecosystem services; genetic erosion
Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.
We transformed C. arabica with the α-amylase inhibitor-1 gene (α-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the α-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against α-AI1 inhibitor showed a maximum α-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the α-AI1 protein against H. hampei α-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity.
This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee.
Coffee is one of the most important plantation crops, grown in about 80 countries across the world. The genus Coffea comprises approximately 100 species of which only two species, that is, Coffea arabica (commonly known as arabica coffee) and Coffea canephora (known as robusta coffee), are commercially cultivated. Genetic improvement of coffee through traditional breeding is slow due to the perennial nature of the plant. Genetic transformation has tremendous potential in developing improved coffee varieties with desired agronomic traits, which are otherwise difficult to achieve through traditional breeding. During the last twenty years, significant progress has been made in coffee biotechnology, particularly in the area of transgenic technology. This paper provides a detailed account of the advances made in the genetic transformation of coffee and their potential applications.
Endo-β-1,4-xylanases (EC 18.104.22.168; endoxylanases), key enzymes in the degradation of xylan, are considered to play an important role in phytopathogenesis, as they occupy a prominent position in the arsenal of hydrolytic enzymes secreted by phytopathogens to breach the cell wall and invade the plant tissue. Plant endoxylanase inhibitors are increasingly being pinpointed as part of a counterattack mechanism. To understand the surprising XIP-type endoxylanase inhibitor insensitivity of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum, an extensive mutational study of these enzymes was performed. Using combinatorial and site-directed mutagenesis, the XIP insensitivity of XylA as well as XylB was proven to be solely due to amino acid sequence adaptations in the “thumb” structural region. While XylB residues Cys141, Asp148, and Cys149 were shown to prevent XIP interaction, the XIP insensitivity of XylA could be ascribed to the occurrence of only one aberrant residue, i.e., Val151. This study, in addition to providing a thorough explanation for the XIP insensitivity of both F. graminearum endoxylanases at the molecular level, generated XylA and XylB mutants with altered inhibition specificities and pH optima. As this is the first experimental elucidation of the molecular determinants dictating the specificity of the interaction between endoxylanases of phytopathogenic origin and a plant inhibitor, this work sheds more light on the ongoing evolutionary arms race between plants and phytopathogenic fungi involving recognition of endoxylanases.
Surveying invasive species can be highly resource intensive, yet near-real-time evaluations of invasion progress are important resources for management planning. In the case of the soybean rust invasion of the United States, a linked monitoring, prediction, and communication network saved U.S. soybean growers approximately $200 M/yr. Modeling of future movement of the pathogen (Phakopsora pachyrhizi) was based on data about current disease locations from an extensive network of sentinel plots. We developed a dynamic network model for U.S. soybean rust epidemics, with counties as nodes and link weights a function of host hectarage and wind speed and direction. We used the network model to compare four strategies for selecting an optimal subset of sentinel plots, listed here in order of increasing performance: random selection, zonal selection (based on more heavily weighting regions nearer the south, where the pathogen overwinters), frequency-based selection (based on how frequently the county had been infected in the past), and frequency-based selection weighted by the node strength of the sentinel plot in the network model. When dynamic network properties such as node strength are characterized for invasive species, this information can be used to reduce the resources necessary to survey and predict invasion progress.
Water is essential for all living organisms. Aquaporin proteins are the major facilitator of water transport activity through cell membranes of plants including soybean. These proteins are diverse in plants and belong to a large major intrinsic (MIP) protein family. In higher plants, MIPs are classified into five subfamilies including plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), NOD26-like intrinsic proteins (NIP), small basic intrinsic proteins (SIP), and the recently discovered X intrinsic proteins (XIP). This paper reports genome wide assembly of soybean MIPs, their functional prediction and expression analysis. Using a bioinformatic homology search, 66 GmMIPs were identified in the soybean genome. Phylogenetic analysis of amino acid sequences of GmMIPs divided the large and highly similar multi-gene family into 5 subfamilies: GmPIPs, GmTIPs, GmNIPs, GmSIPs and GmXIPs. GmPIPs consisted of 22 genes and GmTIPs 23, which showed high sequence similarity within subfamilies. GmNIPs contained 13 and GmSIPs 6 members which were diverse. In addition, we also identified a two member GmXIP, a distinct 5th subfamily. GmMIPs were further classified into twelve subgroups based on substrate selectivity filter analysis. Expression analyses were performed for a selected set of GmMIPs using semi-quantitative reverse transcription (semi-RT-qPCR) and qPCR. Our results suggested that many GmMIPs have high sequence similarity but diverse roles as evidenced by analysis of sequences and their expression. It can be speculated that GmMIPs contains true aquaporins, glyceroporins, aquaglyceroporins and mixed transport facilitators.
Coffea arabica L. (arabica coffee), the only tetraploid species in the genus Coffea, represents the majority of the world's coffee production and has a significant contribution to Nicaragua's economy. The present paper was conducted to determine the genetic diversity of arabica coffee in Nicaragua for its conservation and breeding values. Twenty-six populations that represent eight varieties in Nicaragua were investigated using simple sequence repeat (SSR) markers. A total of 24 alleles were obtained from the 12 loci investigated across 260 individual plants. The total Nei's gene diversity (HT) and the within-population gene diversity (HS) were 0.35 and 0.29, respectively, which is comparable with that previously reported from other countries and regions. Among the varieties, the highest diversity was recorded in the variety Catimor. Analysis of variance (AMOVA) revealed that about 87% of the total genetic variation was found within populations and the remaining 13% differentiate the populations (FST = 0.13; P < 0.001). The variation among the varieties was also significant. The genetic variation in Nicaraguan coffee is significant enough to be used in the breeding programs, and most of this variation can be conserved through ex situ conservation of a low number of populations from each variety.
A cDNA encoding the catalytic site of a phosphatidylinositol-specific phospholipase C (PI-PLC) was isolated from Coffea arabica suspension cells. The cDNA (designated CaPLC) encodes a polypeptide of 308 amino acids, containing the catalytic X and Y domains, and has 99% identity to the soybean gene. Recombinant CaPLC protein was expressed in Escherichia coli, purified, and used to produce a polyclonal antibody. The peptide has a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses. Immunoblots revealed the presence of PLC-like proteins in the tissues of different plant species.
cloning; Coffea arabica; phospholipase C; recombinant protein; purification
Two experiments were conducted to assess nutrient partitioning in coffee (Coffea arabica cv. Typica land race Guatemala) infected with Meloidogyne konaensis. Nutrient levels were quantified from soil, roots, and leaves. In the first experiment, 500-cm3 aliquants of a Kealakekua Andisol were infested with four initial population densities of M. konaensis ranging from 0 to 1,500 freshly hatched second-stage juveniles. Coffee plants (~3 months old) were transplanted into the soil and grown for 25 weeks. Plants responded to nematode infection with decreases (P < 0.05) in concentrations of Ca, Mg, P, and B and increases (P < 0.05) in concentrations of Mn, Cu, Zn, and Ca/B in the roots. Mn and Cu uptake by roots was decreased (P < 0.05) by nematode infection even though concentrations of Mn and Cu increased (P < 0.05) in the roots. Concentrations of Ca and Mg also decreased (P < 0.05) in the leaves, whereas the concentration of Zn increased (P < 0.05). In the second experiment, the soil was amended with Zn at 0 or 5 mg/kg soil and infested with M. konaensis at 0, 100, 1,000 or 10,000 eggs/1,200 cm3 soil. Three-month-old coffee seedlings of similar height were weighed and transplanted into pots and then placed in a greenhouse and grown under 50% shade for 23 weeks. Concentrations of P, K, Ca, Mg, Mn, B, and Zn increased in roots of nematode-free plants growing in Zn-amended soil. The beneficial effects due to the Zn amendment were not apparent in nematode-infected plants. Mn, B, and Zn uptake by coffee roots and P and B concentrations in coffee leaves responded similarly. Management of M. konaensis is necessary to achieve optimal nutrient management in coffee.
aluminum; boron; calcium; Coffea arabica; coffee; copper; kona coffee root-knot nematode; macronutrient; magnesium; manganese; ?; micronutrient; nematode; phosphorus; plant nutrition; potassium; zinc
β-tubulins are structural components of microtubules and the targets of benzimidazole fungicides used to control many diseases of agricultural importance. Intron polymorphisms in the intron-rich genes of these proteins have been used in phylogeographic investigations of phytopathogenic fungi. In this work, we sequenced 2764 nucleotides of the β-tubulin gene (Pp tubB) in samples of Phakopsora pachyrhizi collected from seven soybean fields in Brazil. Pp tubB contained an open reading frame of 1341 nucleotides, including nine exons and eight introns. Exon length varied from 14 to 880 nucleotides, whereas intron length varied from 76 to 102 nucleotides. The presence of only four polymorphic sites limited the usefulness of Pp tubB for phylogeographic studies in P. pachyrhizi. The gene structures of Pp tubB and orthologous β-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in β-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His 6 , Glu 198 and Phe 200 , were absent from the predicted sequence of the P. pachyrhizi β-tubulin protein.
Asian soybean rust; beta-tubulin; genetic diversity; Phakopsora pachyrhizi; soybeans
The chloroplast genome sequence of Coffea arabica L., the first sequenced member of the fourth largest family of angiosperms, Rubiaceae, is reported. The genome is 155 189 bp in length, including a pair of inverted repeats of 25 943 bp. Of the 130 genes present, 112 are distinct and 18 are duplicated in the inverted repeat. The coding region comprises 79 protein genes, 29 transfer RNA genes, four ribosomal RNA genes and 18 genes containing introns (three with three exons). Repeat analysis revealed five direct and three inverted repeats of 30 bp or longer with a sequence identity of 90% or more. Comparisons of the coffee chloroplast genome with sequenced genomes of the closely related family Solanaceae indicated that coffee has a portion of rps19 duplicated in the inverted repeat and an intact copy of infA. Furthermore, whole-genome comparisons identified large indels (> 500 bp) in several intergenic spacer regions and introns in the Solanaceae, including trnE (UUC)–trnT (GGU) spacer, ycf4–cemA spacer, trnI (GAU) intron and rrn5–trnR (ACG) spacer. Phylogenetic analyses based on the DNA sequences of 61 protein-coding genes for 35 taxa, performed using both maximum parsimony and maximum likelihood methods, strongly supported the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids, asterids, eurosids II, and euasterids I and II. Coffea (Rubiaceae, Gentianales) is only the second order sampled from the euasterid I clade. The availability of the complete chloroplast genome of coffee provides regulatory and intergenic spacer sequences for utilization in chloroplast genetic engineering to improve this important crop.
chloroplast genetic engineering; chloroplast genome; coffee; phylogeny; Rubiaceae
We present an electromagnetic model of plant leaves which describes their permittivity at terahertz frequencies. The complex permittivity is investigated as a function of the water content of the leaf. Our measurements on coffee leaves (Coffea arabica L.) demonstrate that the dielectric material parameters can be employed to determine the leaf water status and, therefore, to monitor drought stress in plant leaves. The electromagnetic model consists of an effective medium theory, which is implemented by a third order extension of the Landau, Lifshitz, Looyenga model. The influence of scattering becomes important at higher frequencies and is modeled by a Rayleigh roughness factor.
Terahertz time-domain spectroscopy; Dielectric mixture; Permittivity model; Leaf water content; Drought stress; Coffea arabica
The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.
Coffea canephora; differential expression; drought acclimation; proteomic; real-time PCR; candidate gene
In Brazil, coffee (Coffea arabica) husks are reused in several ways due to their abundance, including as stall bedding. However, field veterinarians have reported that horses become intoxicated after ingesting the coffee husks that are used as bedding. The objective of this study was to evaluate whether coffee husk consumption causes intoxication in horses.
Six horses fed coast cross hay ad libitum were given access to coffee husks and excitability, restlessness, involuntary muscle tremors, chewing movements and constant tremors of the lips and tongue, excessive sweating and increased respiration and heart rates were the most evident clinical signs. Caffeine levels were measured in the plasma and urine of these horses on two occasions: immediately before the coffee husks were made available to the animals (T0) and at the time of the clinical presentation of intoxication, 56 h after the animals started to consume the husks (T56). The concentrations of caffeine in the plasma (p < 0.001) and urine (p < 0.001) of these animals were significantly greater at T56 than at T0.
It was concluded that consumption of coffee husks was toxic to horses due to the high levels of caffeine present in their composition. Therefore, coffee husks pose a risk when used as bedding or as feed for horses.