Characterization of a Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) - derived chitinase and its potential for pest control

The larval endoparasitoid Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) has a toolbox of biological weapons to secure for host colonization and the successful parasitization of its host Heliothis virescens (F.) (Lepidoptera: Noctuidae). The cDNA of a putative chitinase has been previously isolated and initially characterized from teratocytes of this parasitoid among the plethora of molecules available in the venom and calyx fluids injected by females, oral and/or anal secretions released by the parasitoid larvae and/or produced by the expression of genes of the symbiotic associated polydnavirus. This putative chitinase has been initially associated with the host cuticle digestion to allow for parasitoid egression and with the asepsis of the host environment, acting as an antimicrobial. As chitinases are commonly expressed in plants against plant pathogens, the chitinase derived from the teratocytes of T. nigriceps is a potential tool for the development of insect pest control methods based on the disruption of the perithrophic membrane of herbivores. Therefore, we aimed to characterize the activity of the putative chitinase from teratocytes of T. nigriceps (Tnchi) produced using the Escherichia coli expression system and its potential to control H. virescens larvae when expressed into transgenic tobacco plants. The purified E. coli-produced Tnchi protein showed no chitinolitic activity, but was active in binding with colloidal and crystalline chitins in water and with colloidal chitin in buffered solution (pH = 6.74). Transgenic tobacco plants showed no enhanced chitinolitic activity relative to control plants, but survival of three-day old larvae of H. virescens was severely affected when directly fed on transgenic tobacco leaves expressing the recombinant Tnchi protein. Some properties of the Tnchi protein and the potential use of Tnchi-transgenic plants to control plant pests are discussed. (c) 2012 Elsevier Inc. All rights reserved.

RNA Interference of Endochitinases in the Sugarcane Endophyte Trichoderma virens 223 Reduces Its Fitness as a Biocontrol Agent of Pineapple Disease

The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-beta-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-beta-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-beta-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.

Sugarcane Growth Promotion by the Endophytic Bacterium Pantoea agglomerans 33.1

The promotion of sugarcane growth by the endophytic Pantoea agglomerans strain 33.1 was studied under gnotobiotic and greenhouse conditions. The green fluorescent protein (GFP)-tagged strain P. agglomerans 33.1: pNKGFP was monitored in vitro in sugarcane plants by microscopy, reisolation, and quantitative PCR (qPCR). Using qPCR and reisolation 4 and 15 days after inoculation, we observed that GFP-tagged strains reached similar density levels both in the rhizosphere and inside the roots and aerial plant tissues. Microscopic analysis was performed at 5, 10, and 18 days after inoculation. Under greenhouse conditions, P. agglomerans 33.1-inoculated sugarcane plants presented more dry mass 30 days after inoculation. Cross-colonization was confirmed by reisolation of the GFP-tagged strain. These data demonstrate that 33.1:pNKGFP is a superior colonizer of sugarcane due to its ability to colonize a number of different plant parts. The growth promotion observed in colonized plants may be related to the ability of P. agglomerans 33.1 to synthesize indoleacetic acid and solubilize phosphate. Additionally, this strain may trigger chitinase and cellulase production by plant roots, suggesting the induction of a plant defense system. However, levels of indigenous bacterial colonization did not vary between inoculated and noninoculated sugarcane plants under greenhouse conditions, suggesting that the presence of P. agglomerans 33.1 has no effect on these communities. In this study, different techniques were used to monitor 33.1:pNKGFP during sugarcane cross-colonization, and our results suggested that this plant growth promoter could be used with other crops. The interaction between sugarcane and P. agglomerans 33.1 has important benefits that promote the plant's growth and fitness.

Proteomic analysis of banana fruit reveals proteins that are differentially accumulated during ripening

Bananas (Musa spp.) are highly perishable fruit of notable economic and nutritional relevance. Because the identification of proteins involved in metabolic pathways could help to extend green-life and improve the quality of the fruit, this study aimed to compare the proteins of banana pulp at the pre-climacteric and climacteric stages. The use of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) revealed 50 differentially expressed proteins, and comparing those proteins to the Mass Spectrometry Protein Sequence Database (MSDB) identified 26 known proteins. Chitinases were the most abundant types of proteins in unripe bananas, and two isoforms in the ripe fruit have been implicated in the stress/defense response. In this regard, three heat shock proteins and isoflavone reductase were also abundant at the climacteric stage. Concerning fruit quality, pectate lyase, malate dehydrogenase, and starch phosphorylase accumulated during ripening. In addition to the ethylene formation enzyme amino cyclo carboxylic acid oxidase, the accumulation of S-adenosyl-L-homocysteine hydrolase was needed because of the increased ethylene synthesis and DNA methylation that occurred in ripening bananas. Differential analysis provided information on the ripening-associated changes that occurred in proteins involved in banana flavor, texture, defense, synthesis of ethylene, regulation of expression, and protein folding, and this analysis validated previous data on the transcripts during ripening. In this regard, the differential proteomics of fruit pulp enlarged our understanding of the process of banana ripening. (C) 2012 Elsevier B.V. All rights reserved.

Purification, characterization and structural determination of chitinases produced by Moniliophthora perniciosa

The enzyme chitinase from Moniliophthora perniciosa the causative agent of the witches' broom disease in Theobroma cacao, was partially purified with ammonium sulfate and filtration by Sephacryl S-200 using sodium phosphate as an extraction buffer. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes were obtained: ChitMp I, ChitMp II, ChitMp III and ChitMp IV. ChitMp I had an optimum temperature at 44-73ºC and an optimum pH at 7.0-8.4. ChitMp II had an optimum temperature at 45-73ºC and an optimum pH at 7.0-8.4. ChitMp III had an optimum temperature at 54-67ºC and an optimum pH at 7.3-8.8. ChitMp IV had an optimum temperature at 60ºC and an optimum pH at 7.0. For the computational biology, the primary sequence was determined in silico from the database of the Genome/Proteome Project of M. perniciosa, yielding a sequence with 564 bp and 188 amino acids that was used for the three-dimensional design in a comparative modeling methodology. The generated models were submitted to validation using Procheck 3.0 and ANOLEA. The model proposed for the chitinase was subjected to a dynamic analysis over a 1 ns interval, resulting in a model with 91.7% of the residues occupying favorable places on the Ramachandran plot and an RMS of 2.68.