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.

In vivo proteome analysis of Xanthomonas campestris pv. campestris in the interaction with the host plant Brassica oleracea.

The genus Xanthomonas is composed of several species that cause severe crop losses around the world. In Latin America, one of the most relevant species is Xanthomonas campestris pv. campestris, which is responsible for black rot in cruciferous plants. This pathogen causes yield losses in several cultures, including cabbage, cauliflower and broccoli. Although the complete structural genome of X. campestris pv. campestris has been elucidated, little is known about the protein expression of this pathogen in close interaction with the host plant. Recently, a method for in vivo analysis of Xanthomonas axonopodis pv. citri was developed. In the present study, this technique was employed for the characterization of the protein expression of X. campestris pv. campestris in close interaction with the host plant Brassica oleracea. The bacterium was infiltrated into leaves of the susceptible cultivar and later recovered for proteome analysis. Recovered cells were used for protein extraction and separated by two-dimensional electrophoresis. Proteins were analysed by peptide mass fingerprinting or de novo sequencing and identified by searches in public databases. The approach used in this study may be extremely useful in further analyses in order to develop novel strategies to control this important plant pathogen.

Analysis of Phaseolus vulgaris response to its association with Trichoderma harzianum (ALL-42) in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani.

The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively.

Production and biochemical characterization of insecticidal enzymes from Aspergillus fumigatus toward Callosobruchus maculatus.

In the present work, Aspergillus fumigatus is described as a higher producer of hydrolytic enzymes secreted in response to the presence of the Callosobruchus maculatus bruchid pest. This fungus was able to grow over cowpea weevil shells as a unique carbon source, secreting alkaline proteolytic and chitinolytic enzymes. Enzyme secretion in A. fumigatus was induced by both C. maculatus exoskeleton as well as commercial chitin, and alkaline proteolytic and chitinolytic activities were detected after 48 hours of growth. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the production of specific proteins. Among them, two extracellular alkaline proteinases from culture enriched with C. maculatus exoskeleton were purified after chromatographic procedures using ion exchange and affinity columns. These proteins, named AP15 and AP30, had apparent molecular masses of 15,500 and 30,000 Da, respectively, as estimated by SDS-PAGE electrophoresis and mass spectrometry. AP30 was classified as a serine proteinase because it was inhibited by 5 mM: phenylmethylsulfonyl fluoride (100%) and 50 microM leupeptin (67.94%).