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1.  Cloning and expression of an endo-1,4-β-xylanase from the coffee berry borer, Hypothenemus hampei 
BMC Research Notes  2012;5:23.
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.
PMCID: PMC3283504  PMID: 22233686
2.  Analysis of Phakopsora pachyrhizi transcript abundance in critical pathways at four time-points during infection of a susceptible soybean cultivar using deep sequencing 
BMC Genomics  2013;14:614.
Phakopsora pachyrhizi, the causal agent responsible for soybean rust, is among the top hundred most virulent plant pathogens and can cause soybean yield losses of up to 80% when appropriate conditions are met. We used mRNA-Seq by Illumina to analyze pathogen transcript abundance at 15 seconds (s), 7 hours (h), 48 h, and 10 days (d) after inoculation (ai) of susceptible soybean leaves with P. pachyrhizi to gain new insights into transcript abundance in soybean and the pathogen at specific time-points during the infection including the uredinial stage.
Over three million five hundred thousand sequences were obtained for each time-point. Energy, nucleotide metabolism, and protein synthesis are major priorities for the fungus during infection and development as indicated by our transcript abundance studies. At all time-points, energy production is a necessity for P. pachyrhizi, as indicated by expression of many transcripts encoding enzymes involved in oxidative phosphorylation and carbohydrate metabolism (glycolysis, glyoxylate and dicarboxylate, pentose phosphate, pyruvate). However, at 15 sai, transcripts encoding enzymes involved in ATP production were highly abundant in order to provide enough energy for the spore to germinate, as observed by the expression of many transcripts encoding proteins involved in electron transport. At this early time-point, transcripts encoding proteins involved in RNA synthesis were also highly abundant, more so than transcripts encoding genes involved in DNA and protein synthesis. At 7 hai, shortly after germination during tube elongation and penetration, transcripts encoding enzymes involved in deoxyribonucleotide and DNA synthesis were highly abundant. At 48 hai, transcripts encoding enzymes involved in amino acid metabolism were highly abundant to provide for increased protein synthesis during haustoria maturation. During sporulation at 10 dai, the fungus still required carbohydrate metabolism, but there also was increased expression of transcripts encoding enzymes involved in fatty acid metabolism.
This information provides insight into molecular events and their timing throughout the life cycle of the P. pachyrhizi, and it may be useful in the development of new methods of broadening resistance of soybean to soybean rust.
PMCID: PMC3847679  PMID: 24025037
Deep sequencing; Transcript abundance; Phakopsora pachyrhizi; Plant-pathogen interaction; Soybean; Soybean rust
3.  Characterization of a newly identified rice chitinase-like protein (OsCLP) homologous to xylanase inhibitor 
BMC Biotechnology  2013;13:4.
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.
PMCID: PMC3571981  PMID: 23331415
Antifungal activity; Chitinase; Oryza sativa; Xylanase inhibitor
4.  Gene expression and proteomic analysis of the formation of Phakopsora pachyrhizi appressoria 
BMC Genomics  2012;13:269.
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.
PMCID: PMC3431228  PMID: 22727213
5.  Expression analysis reveals a role for hydrophobic or epicuticular wax signals in pre-penetration structure formation of Phakopsora pachyrhizi 
Plant Signaling & Behavior  2013;8(11):e26959.
Asian soybean rust (ASR) caused by the fungus Phakopsora pachyrhizi is one of the most devastating foliar diseases affecting soybean production worldwide. Even though several resistance sources have been identified in soybean, they do not show resistance to all races of P. pachyrhizi. Identification of genes that confer nonhost resistance (NHR) against P. pachyrhizi in another legume species will provide an avenue to engineer soybean to have durable and broad spectrum resistance against P. pachyrhizi strains. Recently, we identified a Medicago truncatula gene, IRG1 (INHIBITOR OF RUST GERM-TUBE DIFFERENTIATION1), that when mutated inhibits the growth of P. pachyrhizi. IRG1 encodes a Cys(2)His(2) zinc finger transcription factor that controls wax-biosynthesis-related genes. The irg1 mutant shows a complete loss of abaxial epicuticular wax crystals and surface hydrophobicity, resulting in the inhibition of pre-penetration structure formation. In order to confirm the role of surface hydrophobicity in the formation of pre-penetration structures, we examined the expression profiles of P. pachyrhizi putative pre-penetration structure-development-related genes on a solid surface or a M. truncatula abaxial leaf surface. Interestingly, the expression of kinase family genes was upregulated on the hydrophobic surface and M. truncatula wild-type leaf surface, but not on the M. truncatula irg1 mutant leaf surface, suggesting that these genes play a role in P. pachyrhizi pre-penetration structure development. In addition, our results suggest that hydrophobicity on the M. truncatula leaf surface may function as a key signal to induce the P. pachyrhizi genes involved in pre-penetration structure development.
PMCID: PMC4091598  PMID: 24184888
IRG1; Epicutuciular WAX; nonhost resistance; Asian Soybean Rust; Phakopsora pachyrhizi; Medicago truncatula
6.  Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection 
BMC Plant Biology  2014;14:236.
Many previous studies have shown that soybean WRKY transcription factors are involved in the plant response to biotic and abiotic stresses. Phakopsora pachyrhizi is the causal agent of Asian Soybean Rust, one of the most important soybean diseases. There are evidences that WRKYs are involved in the resistance of some soybean genotypes against that fungus. The number of WRKY genes already annotated in soybean genome was underrepresented. In the present study, a genome-wide annotation of the soybean WRKY family was carried out and members involved in the response to P. pachyrhizi were identified.
As a result of a soybean genomic databases search, 182 WRKY-encoding genes were annotated and 33 putative pseudogenes identified. Genes involved in the response to P. pachyrhizi infection were identified using superSAGE, RNA-Seq of microdissected lesions and microarray experiments. Seventy-five genes were differentially expressed during fungal infection. The expression of eight WRKY genes was validated by RT-qPCR. The expression of these genes in a resistant genotype was earlier and/or stronger compared with a susceptible genotype in response to P. pachyrhizi infection. Soybean somatic embryos were transformed in order to overexpress or silence WRKY genes. Embryos overexpressing a WRKY gene were obtained, but they were unable to convert into plants. When infected with P. pachyrhizi, the leaves of the silenced transgenic line showed a higher number of lesions than the wild-type plants.
The present study reports a genome-wide annotation of soybean WRKY family. The participation of some members in response to P. pachyrhizi infection was demonstrated. The results contribute to the elucidation of gene function and suggest the manipulation of WRKYs as a strategy to increase fungal resistance in soybean plants.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0236-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4172953  PMID: 25201117
Glycine max; Genetic transformation; Fungus resistance; Transcription factors; Asian Soybean Rust; Functional analysis
7.  Novel Phakopsora pachyrhizi Extracellular Proteins Are Ideal Targets for Immunological Diagnostic Assays 
Applied and Environmental Microbiology  2012;78(11):3890-3895.
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.
PMCID: PMC3346385  PMID: 22447596
8.  Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing 
Plant Signaling & Behavior  2013;8(12):e27543.
Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars.
PMCID: PMC4091232  PMID: 24401541
soybean rust; transcription factor; disease resistance; gene silencing; Rpp1
9.  Identification of the soybean HyPRP family and specific gene response to Asian soybean rust disease 
Genetics and Molecular Biology  2013;36(2):214-224.
Soybean [Glycine max (L.) Merril], one of the most important crop species in the world, is very susceptible to abiotic and biotic stress. Soybean plants have developed a variety of molecular mechanisms that help them survive stressful conditions. Hybrid proline-rich proteins (HyPRPs) constitute a family of cell-wall proteins with a variable N-terminal domain and conserved C-terminal domain that is phylogenetically related to non-specific lipid transfer proteins. Members of the HyPRP family are involved in basic cellular processes and their expression and activity are modulated by environmental factors. In this study, microarray analysis and real time RT-qPCR were used to identify putative HyPRP genes in the soybean genome and to assess their expression in different plant tissues. Some of the genes were also analyzed by time-course real time RT-qPCR in response to infection by Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease. Our findings indicate that the time of induction of a defense pathway is crucial in triggering the soybean resistance response to P. pachyrhizi. This is the first study to identify the soybean HyPRP group B family and to analyze disease-responsive GmHyPRP during infection by P. pachyrhizi.
PMCID: PMC3715288  PMID: 23885204
fungal disease; HyPRP genes; Glycine max; real time RT-qPCR
10.  Identification and characterization of the Non-race specific Disease Resistance 1 (NDR1) orthologous protein in coffee 
BMC Plant Biology  2011;11:144.
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.
PMCID: PMC3212813  PMID: 22023696
11.  Mutational Analysis of Endoxylanases XylA and XylB from the Phytopathogen Fusarium graminearum Reveals Comprehensive Insights into Their Inhibitor Insensitivity▿  
Applied and Environmental Microbiology  2007;73(14):4602-4608.
Endo-β-1,4-xylanases (EC; 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.
PMCID: PMC1932832  PMID: 17513587
12.  Differential regulation of caffeine metabolism in Coffeaarabica (Arabica) and Coffea canephora (Robusta) 
Planta  2014;241:179-191.
Caffeine is a metabolite of great economic importance, especially in coffee, where it influences the sensorial and physiological impacts of the beverage. Caffeine metabolism in the Coffea species begins with the degradation of purine nucleotides through three specific N-methyltransferases: XMT, MXMT and DXMT. A comparative analysis was performed to clarify the molecular reasons behind differences in caffeine accumulation in two Coffea species, namely Coffea arabica and Coffea canephora var. robusta. Three different genes encoding N-methyltransferase were amplified in the doubled haploid Coffea canephora: CcXMT1, CcMXMT1 and CcDXMT. Six genes were amplified in the haploid Coffea arabica: CaXMT1, CaXMT2, CaMXMT1, CaMXMT2, CaDXMT1, and CaDXMT2. A complete phylogenic analysis was performed to identify specific key amino acids defining enzymatic function for each protein identified. Furthermore, a quantitative gene-expression analysis was conducted on leaves and on maturing coffee beans, simultaneously analyzing caffeine content. In the different varieties analyzed, caffeine accumulation is higher in leaves than in the coffee bean maturation period, higher in Robusta than in Arabica. In Robusta, CcXMT1 and CcDXMT gene expressions are predominant and transcriptional activity is higher in leaves than in maturing beans, and is highly correlated to caffeine accumulation. In Arabica, the CaXMT1 expression level is high in leaves and CaDXMT2 as well to a lesser extent, while global transcriptional activity is weak during bean maturation, suggesting that the transcriptional control of caffeine-related genes differs within different organs and between Arabica and Robusta. These findings indicate that caffeine accumulation in Coffea species has been modulated by a combination of differential transcriptional regulation and genome evolution.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-014-2170-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4282694  PMID: 25249475
Arabica; Beans; Caffeine; Expression; Leaves; N-methyltransferase; Robusta
13.  Nonhost resistance to rust pathogens – a continuation of continua 
The rust fungi (order: Pucciniales) are a group of widely distributed fungal plant pathogens, which can infect representatives of all vascular plant groups. Rust diseases significantly impact several crop species and considerable research focuses on understanding the basis of host specificity and nonhost resistance. Like many pathogens, rust fungi vary considerably in the number of hosts they can infect, such as wheat leaf rust (Puccinia triticina), which can only infect species in the genera Triticum and Aegilops, whereas Asian soybean rust (Phakopsora pachyrhizi) is known to infect over 95 species from over 42 genera. A greater understanding of the genetic basis determining host range has the potential to identify sources of durable resistance for agronomically important crops. Delimiting the boundary between host and nonhost has been complicated by the quantitative nature of phenotypes in the transition between these two states. Plant–pathogen interactions in this intermediate state are characterized either by (1) the majority of accessions of a species being resistant to the rust or (2) the rust only being able to partially complete key components of its life cycle. This leads to a continuum of disease phenotypes in the interaction with different plant species, observed as a range from compatibility (host) to complete immunity within a species (nonhost). In this review we will highlight how the quantitative nature of disease resistance in these intermediate interactions is caused by a continuum of defense barriers, which a pathogen needs to overcome for successfully establishing itself in the host. To illustrate continua as this underlying principle, we will discuss the advances that have been made in studying nonhost resistance towards rust pathogens, particularly cereal rust pathogens.
PMCID: PMC4263244  PMID: 25566270
host range; disease resistance; nonadapted pathogen; NHR; formae speciales; Pucciniales
14.  Genome-wide analysis of major intrinsic proteins in the tree plant Populus trichocarpa: Characterization of XIP subfamily of aquaporins from evolutionary perspective 
BMC Plant Biology  2009;9:134.
Members of major intrinsic proteins (MIPs) include water-conducting aquaporins and glycerol-transporting aquaglyceroporins. MIPs play important role in plant-water relations. The model plants Arabidopsis thaliana, rice and maize contain more than 30 MIPs and based on phylogenetic analysis they can be divided into at least four subfamilies. Populus trichocarpa is a model tree species and provides an opportunity to investigate several tree-specific traits. In this study, we have investigated Populus MIPs (PtMIPs) and compared them with their counterparts in Arabidopsis, rice and maize.
Fifty five full-length MIPs have been identified in Populus genome. Phylogenetic analysis reveals that Populus has a fifth uncharacterized subfamily (XIPs). Three-dimensional models of all 55 PtMIPs were constructed using homology modeling technique. Aromatic/arginine (ar/R) selectivity filters, characteristics of loops responsible for solute selectivity (loop C) and gating (loop D) and group conservation of small and weakly polar interfacial residues have been analyzed. Majority of the non-XIP PtMIPs are similar to those in Arabidopsis, rice and maize. Additional XIPs were identified from database search and 35 XIP sequences from dicots, fungi, moss and protozoa were analyzed. Ar/R selectivity filters of dicots XIPs are more hydrophobic compared to fungi and moss XIPs and hence they are likely to transport hydrophobic solutes. Loop C is longer in one of the subgroups of dicot XIPs and most probably has a significant role in solute selectivity. Loop D in dicot XIPs has higher number of basic residues. Intron loss is observed on two occasions: once between two subfamilies of eudicots and monocot and in the second instance, when dicot and moss XIPs diverged from fungi. Expression analysis of Populus MIPs indicates that Populus XIPs don't show any tissue-specific transcript abundance.
Due to whole genome duplication, Populus has the largest number of MIPs identified in any single species. Non-XIP MIPs are similar in all four plant species considered in this study. Small and weakly polar residues at the helix-helix interface are group conserved presumably to maintain the hourglass fold of MIP channels. Substitutions in ar/R selectivity filter, insertion/deletion in loop C, increasing basic nature of loop D and loss of introns are some of the events occurred during the evolution of dicot XIPs.
PMCID: PMC2789079  PMID: 19930558
15.  Identification of Two Metallothioneins as Novel Inhalative Coffee Allergens Cof a 2 and Cof a 3 
PLoS ONE  2015;10(5):e0126455.
Dust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust.
Our aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis.
A Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers.
In addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test.
In addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy.
PMCID: PMC4427360  PMID: 25962169
16.  Organization and molecular evolution of a disease-resistance gene cluster in coffee trees 
BMC Genomics  2011;12:240.
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.
PMCID: PMC3113787  PMID: 21575174
17.  On the current status of Phakopsora pachyrhizi genome sequencing 
Recent advances in the field of sequencing technologies and bioinformatics allow a more rapid access to genomes of non-model organisms at sinking costs. Accordingly, draft genomes of several economically important cereal rust fungi have been released in the last 3 years. Aside from the very recent flax rust and poplar rust draft assemblies there are no genomic data available for other dicot-infecting rust fungi. In this article we outline rust fungus sequencing efforts and comment on the current status of Phakopsora pachyrhizi (Asian soybean rust) genome sequencing.
PMCID: PMC4147182  PMID: 25221558
fungal genomics; rust fungi; Asian soybean rust; next-generation sequencing; herterozygosity; genome size; k-mer analysis
18.  Using an ensemble of statistical metrics to quantify large sets of plant transcription factor binding sites 
Plant Methods  2013;9:12.
From initial seed germination through reproduction, plants continuously reprogram their transcriptional repertoire to facilitate growth and development. This dynamic is mediated by a diverse but inextricably-linked catalog of regulatory proteins called transcription factors (TFs). Statistically quantifying TF binding site (TFBS) abundance in promoters of differentially expressed genes can be used to identify binding site patterns in promoters that are closely related to stress-response. Output from today’s transcriptomic assays necessitates statistically-oriented software to handle large promoter-sequence sets in a computationally tractable fashion.
We present Marina, an open-source software for identifying over-represented TFBSs from amongst large sets of promoter sequences, using an ensemble of 7 statistical metrics and binding-site profiles. Through software comparison, we show that Marina can identify considerably more over-represented plant TFBSs compared to a popular software alternative.
Marina was used to identify over-represented TFBSs in a two time-point RNA-Seq study exploring the transcriptomic interplay between soybean (Glycine max) and soybean rust (Phakopsora pachyrhizi). Marina identified numerous abundant TFBSs recognized by transcription factors that are associated with defense-response such as WRKY, HY5 and MYB2. Comparing results from Marina to that of a popular software alternative suggests that regardless of the number of promoter-sequences, Marina is able to identify significantly more over-represented TFBSs.
PMCID: PMC3639912  PMID: 23578135
19.  Probing Protein Sequences as Sources for Encrypted Antimicrobial Peptides 
PLoS ONE  2012;7(9):e45848.
Starting from the premise that a wealth of potentially biologically active peptides may lurk within proteins, we describe here a methodology to identify putative antimicrobial peptides encrypted in protein sequences. Candidate peptides were identified using a new screening procedure based on physicochemical criteria to reveal matching peptides within protein databases. Fifteen such peptides, along with a range of natural antimicrobial peptides, were examined using DSC and CD to characterize their interaction with phospholipid membranes. Principal component analysis of DSC data shows that the investigated peptides group according to their effects on the main phase transition of phospholipid vesicles, and that these effects correlate both to antimicrobial activity and to the changes in peptide secondary structure. Consequently, we have been able to identify novel antimicrobial peptides from larger proteins not hitherto associated with such activity, mimicking endogenous and/or exogenous microorganism enzymatic processing of parent proteins to smaller bioactive molecules. A biotechnological application for this methodology is explored. Soybean (Glycine max) plants, transformed to include a putative antimicrobial protein fragment encoded in its own genome were tested for tolerance against Phakopsora pachyrhizi, the causative agent of the Asian soybean rust. This procedure may represent an inventive alternative to the transgenic technology, since the genetic material to be used belongs to the host organism and not to exogenous sources.
PMCID: PMC3461044  PMID: 23029273
20.  An EST-based analysis identifies new genes and reveals distinctive gene expression features of Coffea arabica and Coffea canephora 
BMC Plant Biology  2011;11:30.
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 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.
PMCID: PMC3045888  PMID: 21303543
21.  α-Amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits α-amylases from the coffee berry borer pest 
BMC Biotechnology  2010;10:44.
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.
PMCID: PMC2914071  PMID: 20565807
22.  SNP assay to detect the ‘Hyuuga’ red-brown lesion resistance gene for Asian soybean rust 
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.
PMCID: PMC2938421  PMID: 20532750
23.  Proteomic analysis of apoplastic fluid of Coffea arabica leaves highlights novel biomarkers for resistance against Hemileia vastatrix 
A proteomic analysis of the apoplastic fluid (APF) of coffee leaves was conducted to investigate the cellular processes associated with incompatible (resistant) and compatible (susceptible) Coffea arabica-Hemileia vastatrix interactions, during the 24–96 hai period. The APF proteins were extracted by leaf vacuum infiltration and protein profiles were obtained by 2-DE. The comparative analysis of the gels revealed 210 polypeptide spots whose volume changed in abundance between samples (control, resistant and susceptible) during the 24–96 hai period. The proteins identified were involved mainly in protein degradation, cell wall metabolism and stress/defense responses, most of them being hydrolases (around 70%), particularly sugar hydrolases and peptidases/proteases. The changes in the APF proteome along the infection process revealed two distinct phases of defense responses, an initial/basal one (24–48 hai) and a late/specific one (72–96 hai). Compared to susceptibility, resistance was associated with a higher number of proteins, which was more evident in the late/specific phase. Proteins involved in the resistance response were mainly, glycohydrolases of the cell wall, serine proteases and pathogen related-like proteins (PR-proteins), suggesting that some of these proteins could be putative candidates for resistant markers of coffee to H. vastatrix. Antibodies were produced against chitinase, pectin methylesterase, serine carboxypeptidase, reticuline oxidase and subtilase and by an immunodetection assay it was observed an increase of these proteins in the resistant sample. With this methodology we have identified proteins that are candidate markers of resistance and that will be useful in coffee breeding programs to assist in the selection of cultivars with resistance to H. vastatrix.
PMCID: PMC4484983  PMID: 26175744
coffee leaf rust (CLR); cytology; MALDI-TOF/TOF MS; 2-DE; antibody production; ELISA assay
24.  Genome-Wide Sequence Characterization and Expression Analysis of Major Intrinsic Proteins in Soybean (Glycine max L.) 
PLoS ONE  2013;8(2):e56312.
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.
PMCID: PMC3577755  PMID: 23437113
25.  Ubiquitous urease affects soybean susceptibility to fungi 
Plant Molecular Biology  2012;79(1-2):75-87.
The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-012-9894-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3332383  PMID: 22382992
Glycine max; Functional analysis; Fungal resistance; Genetic transformation; Overexpression; Co-suppression; Life Sciences; Plant Pathology; Biochemistry, general; Plant Sciences

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