A Comprehensive Review of the Coffee Leaf Miner Leucoptera coffeella (Lepidoptera: Lyonetiidae)-A Major Pest for the Coffee Crop in Brazil and Others Neotropical Countries.

The coffee leaf miner (CLM) Leucoptera coffeella moth is a major threat to coffee production. Insect damage is related to the feeding behavior of the larvae on the leaf. During the immature life stages, the insect feeds in the mesophyll triggering necrosis and causing loss of photosynthetic capacity, defoliation and significant yield loss to coffee crops. Chemical control is used to support the coffee production chain, though market requirements move toward conscious consumption claiming for more sustainable methods. In this overview, we discuss aspects about the CLM concerning biology, history, geographical distribution, economic impacts, and the most relevant control strategies in progress. Insights to develop an integrated approach for a safer and eco-friendly control of the CLM are discussed here, including bio-extracts, nanotechnology, pheromones, and tolerant cultivars.

Alpha-amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits alpha-amylases from the coffee berry borer pest.

BACKGROUND: 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 alpha-amylase inhibitor gene (alpha-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants. RESULTS: We transformed C. arabica with the alpha-amylase inhibitor-1 gene (alpha-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the alpha-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against alpha-AI1 inhibitor showed a maximum alpha-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the alpha-AI1 protein against H. hampei alpha-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity. CONCLUSIONS: 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.

Methodological evaluation of 2-DE to study root proteomics during nematode infection in cotton and coffee plants.

The identification of plant proteins expressed in response to phytopathogens is a remaining challenge to proteome methodology. Proteomic methods, such as electrophoresis and mass spectrometry have been extensively used for protein differential expression studies in several plants including Arabidopsis thaliana, rice, and wheat. However, in coffee (Coffea canephora) and cotton (Gossypium hirsutum), bidimensional electrophoresis (2-DE) analysis has been rarely employed. Moreover, global protein expression in both agricultural plants in response to biotic stress conditions had not been reported until now. In this study, Meloidogyne paranaensis and M. incognita, two devastating phytonematodes for numerous crop cultures, were used to infect resistant genotypes of coffee and cotton plants. The protein expression of infected- and non-infected roots were evaluated by 2-DE following in silico experiments. Additionally, gels were stained with silver nitrate and/or Coomassie brilliant blue in order to obtain an optimized method for proteomic analysis of plant-nematode interaction. The 2-DE analysis revealed an enhanced number of protein spots, as well as differentially expressed proteins, when Coomassie brilliant blue was used. The results obtained here could be extended to other plant species, providing valuable information to root-nematode interactions.

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut.

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a approximately 120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots.

Comparative proteomical and metalloproteomical analyses of human plasma from patients with laryngeal cancer.

Laryngeal cancer is a significant disease worldwide, which presents an increasing incidence. Two contrasting ideas of the immune system role during cancer development are accepted: (1) it fights tumor cells, and (2) it aids tumor progression. Thus, there is no clear understanding about the immune response in laryngeal cancer. Furthermore, since tobacco is the main cause of laryngeal cancer and it contains various carcinogenic components, including metallic elements, these may play a role on cancer development. Plasmas of patients with laryngeal cancer and of healthy smokers were evaluated by 2D gel electrophoresis and mass spectrometry. Proteins were detected on every gel around pH 4.0-10.0 from molecular mass of 10-60 kDa. Few differences were found among cancer and control patients. However, three spots gathered between pI 7.3 and 7.6 with different molecular masses appeared exclusively in cancer profiles. From ten spots identified, six correspond to immune system components, including the three differential ones. The latter were observed only in cancer patients. The presence of several trace elements in the identified proteins was determined by inductively coupled plasma mass spectrometry, where chromium was increased in all proteins analyzed from patients with cancer. This study reinforces the importance of the immune response as target in the understanding and treatment of laryngeal cancer and the possibility that chromium is important in the carcinogenic progress.

Plant-pathogen interactions: what is proteomics telling us?

Over the years, several studies have been performed to analyse plant-pathogen interactions. Recently, functional genomic strategies, including proteomics and transcriptomics, have contributed to the effort of defining gene and protein function and expression profiles. Using these 'omic' approaches, pathogenicity- and defence-related genes and proteins expressed during phytopathogen infections have been identified and enormous datasets have been accumulated. However, the understanding of molecular plant-pathogen interactions is still an intriguing area of investigation. Proteomics has dramatically evolved in the pursuit of large-scale functional assignment of candidate proteins and, by using this approach, several proteins expressed during phytopathogenic interactions have been identified. In this review, we highlight the proteins expressed during plant-virus, plant-bacterium, plant-fungus and plant-nematode interactions reported in proteomic studies, and discuss these findings considering the advantages and limitations of current proteomic tools.

Rooteomics: the challenge of discovering plant defense-related proteins in roots.

In recent years, a strong emphasis has been given in deciphering the function of genes unraveled by the completion of several genome sequencing projects. In plants, functional genomics has been massively used in order to search for gene products of agronomic relevance. As far as root-pathogen interactions are concerned, several genes are recognized to provide tolerance/resistance against potential invaders. However, very few proteins have been identified by using current proteomic approaches. One of the major drawbacks for the successful analysis of root proteomes is the inherent characteristics of this tissue, which include low volume content and high concentration of interfering substances such as pigments and phenolic compounds. The proteome analysis of plant-pathogen interactions provides important information about the global proteins expressed in roots in response to biotic stresses. Moreover, several pathogenic proteins superimpose the plant proteome and can be identified and used as targets for the control of viruses, bacteria, fungi and nematode pathogens. The present review focuses on advances in different proteomic strategies dedicated to the challenging analysis of plant defense proteins expressed during bacteria-, fungi- and nematode-root interactions. Recent developments, limitations of the current techniques, and technological perspectives for root proteomics aiming at the identification of resistance-related proteins are discussed.

Identification of a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from marine Zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa, Zoanthidea).

beta-N-Acetylhexosaminidases (EC 3.2.1.52) belong to an enzyme family that hydrolyzes terminal beta-d-N-glucosamine and beta-d-N-galactosamine residues from oligosaccharides. In this report, we purified a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from the marine zoanthid Palythoa caribaeorum by applying ammonium sulfate fractionation, affinity chromatography on a chitin column, followed by two rounds of size exclusion chromatography. SDS-PAGE analysis indicated a single band protein of apparent homogeneity with a molecular mass of 25kDa. The purified enzyme preferentially hydrolyzed p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylglucosamide (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylgalactosamide (pNP-GalNAc). Detailed kinetic analysis using pNP-GlcNAc resulted in a specific activity of 57.9 U/mg, a K(m) value of 0.53 mM and a V(max) value of 88.1 micromol/h/mg and k(cat) value of 0.61s(-1). Furthermore, purified Pcb-NAHA1 enzyme activity was decreased by Hg Cl(2) or maltose and stimulated in the presence of Na(2)SeO(4,) BaCl(2), MgCl(2,) chondroitin 6-sulfate, and phenylmethylsulfonylfluoride. The optimum activity of Pcb-NAHA1 was observed at pH 5.0 and elevated temperatures (45-60 degrees C). Direct sequencing of proteolytic fragments generated from Pcb-NAHA1 revealed remarkable similarities to plant chitinases, which belong to family 18, although no chitinase activity was detected with Pcb-NAHA1. We conclude that beta-N-acetylhexosaminidases, representing a type of exochitinolytic activity, and endo-chitinases share common functional domains and/or may have evolved from a common ancestor.

Molecular and structural characterization of a trypsin highly expressed in larval stage of Zabrotes subfasciatus.

The Mexican bean weevil, Zabrotes subfasciatus, feeds on several seeds such as Vigna unguiculata, Phaseolus vulgaris, and Pisum sativum, causing severe crop losses. This ability to obtain essential compounds from different diets could possibly be explained due to a wide variability of digestive proteinases present in the weevil's midgut. These may improve digestion of many different dietary proteins. Coleopteran serine-like proteinases have not been thoroughly characterized at the molecular level. In this report, a full-length cDNA encoding a trypsin-like protein, named ZsTRYP, was isolated from Z. subfasciatus larvae using RT-PCR, 5' and 3' RACE techniques. The quantitative real-time PCR analysis strongly correlated the Zstryp transcript accumulation to the major feeding developmental larval stage. Zstryp cDNA was subcloned into pET101 vector and expressed in a Escherichia coli BL21(DE3) strain. Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was used to purify a 29.0-kDa recombinant enzyme. The purified ZsTRYP was then assayed with several synthetic peptide substrates and also challenged with different inhibitors. The biochemical data allowed us to classify ZsTRYP as a trypsin. Moreover, homology modeling analysis indicated a typical trypsin structural core and a conserved catalytic triad (His(41), Asp(86), and Ser(182)).

Screening of entomopathogenic Metarhizium anisopliae isolates and proteomic analysis of secretion synthesized in response to cowpea weevil (Callosobruchus maculatus) exoskeleton.

Cowpea crops are severely attacked by Callosobruchus maculatus, a Coleopteran that at the larval stage penetrates into stored seeds and feeds on cotyledons. Cowpea weevil control could be based in utilization of bacteria and fungi to reduce pest development. Entomopathogenic fungi, such as Metarhizium anisopliae, are able to control insect-pests and are widely applied in biological control. This report evaluated ten M. anisopliae isolates according to their virulence, correlating chitinolytic, proteolytic and alpha-amylolytic activities, as well proteomic analysis by two dimensional gels of fungal secretions in response to an induced medium containing C. maculatus shells, indicating novel biotechnological tools capable of improving cowpea crop resistance.