Carbohydrate-active enzymes revealed in Coptotermes formosanus (Isoptera: Rhinotermitidae) transcriptome.

Coptotermes formosanus is one of the most destructive wood-feeding termites. To understand the molecular mechanisms that regulate the development of the termite, a normalized C. formosanus cDNA library was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. The sequencing of this library generated 131 636 expressed sequence tags (ESTs) and 25 939 assembled unigenes. The carbohydrate-active enzymes (CAZymes) revealed in this library were analysed in the present report. A total of 509 putative CAZymes were identified. Diverse cellulolytic enzymes were uncovered from both the host termite and from symbionts harboured by the termite, which were possibly the result of the high efficiency of cellulose utilization. CAZymes associated with trehalose biosynthetic and metabolic pathways were also identified, which are potential regulators of the physiological activities of trehalose, an important insect blood sugar. Representative CAZyme coding genes in glycoside hydrolase family 1 (GH1) were quantitatively analysed. The results showed that the five GH1 ß-glucosidase genes were expressed differentially among different castes and one of them was female alate-specific. Overall, the normalized EST library provides a comprehensive genetic resource of C. formosanus and will serve a diverse range of research areas. The CAZymes represent one of the repositories of enzymes useful for physiological studies and applications in sugar-based biofuel production.

Solid-phase microextraction for the detection of termite cuticular hydrocarbons.

Solid-phase microextraction (SPME)-gas chromatography-mass spectrometry was used to identify the cuticular hydrocarbons of the subterranean termite Coptotermes formosanus Shiraki. Headspace SPME and direct contact SPME methods were evaluated and compared to the hexane extraction method. Variables, such as temperature, time, number of termites, condition of the termites, and the type of SPME fiber were evaluated. Methods were refined to increase the reproducibility as well as the sensitivity. Both SPME methods were successfully used for the identification of all the major termite cuticular hydrocarbons. Using the headspace SPME method, other compounds of interest could also be identified, such as fatty acids. Using the direct contact SPME method, termites could be repeatedly studied over time to monitor chemical changes.

A Corn Trypsin Inhibitor with Antifungal Activity Inhibits Aspergillus flavus alpha-Amylase.

ABSTRACT In this study, we found that the inhibition of fungal growth in potato dextrose broth (PDB) medium by the 14-kDa corn trypsin inhibitor (TI) protein, previously found to be associated with host resistance to aflatoxin production and active against various fungi, was relieved when exogenous alpha-amylase was added along with TI. No inhibitory effect of TI on fungal growth was observed when Aspergillus flavus was grown on a medium containing either 5% glucose or 1% gelatin as a carbon source. Further investigation found that TI not only inhibited fungal production of extracellular alpha-amylase when A. flavus was grown in PDB medium containing TI at 100 mug ml(-1) but also reduced the enzymatic activity of A. flavus alpha-amylase by 27%. At a higher concentration, however, TI stimulated the production of alpha-amylase. The effect of TI on the production of amyloglucosidase, another enzyme involved in starch metabolism by the fungus, was quite different. It stimulated the production of this enzyme during the first 10 h at all concentrations studied. These studies suggest that the resistance of certain corn genotypes to A. flavus infection may be partially due to the ability of TI to reduce the production of extracellular fungal alpha-amylase and its activity, thereby limiting the availability of simple sugars for fungal growth. However, further investigation of the relationship between TI levels and fungal alpha-amylase expression in vivo is needed.

Resistance to Aspergillus flavus in Corn Kernels Is Associated with a 14-kDa Protein.

ABSTRACT Corn genotypes resistant or susceptible to Aspergillus flavus were extracted for protein analysis using a pH 2.8 buffer. The profile of protein extracts revealed that a 14-kDa protein is present in relatively high concentration in kernels of seven resistant corn genotypes, but is absent or present only in low concentration in kernels of six susceptible ones. The N-terminal sequence of this 14-kDa protein showed 100% homology to a corn trypsin inhibitor. The 14-kDa protein purified from resistant varieties also demonstrated in vitro inhibition of both trypsin activity and the growth of A. flavus. This is the first demonstration of antifungal activity of a corn 14-kDa trypsin inhibitor protein. The expression of this protein among tested genotypes may be related to their difference in resistance to A. flavus infection and subsequent aflatoxin contamination.

Germination induces accumulation of specific proteins and antifungal activities in corn kernels.

ABSTRACT This study examined protein induction and accumulation during imbibition and germination of corn kernels, as well as antifungal activities of extracts from germinating kernels against Aspergillus flavus and Fusarium moniliforme. Genotypes studied included GT-MAS:gk and Mp420, which are resistant to A. flavus infection and aflatoxin accumulation, and Pioneer 3154 and Deltapine G-4666, which are susceptible to A. flavus infection and aflatoxin accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved five protein bands that were present at higher concentrations in germinated kernels than in nongerminated kernels. Western blot analyses revealed that one of these proteins reacted with the 22-kDa zeamatin antiserum, and a zeamatin-like protein accumulated to a higher concentration in germinated kernels. Two protein bands from dry kernels that reacted with ribosome-inactivating protein (RIP) antiserum were identified as the 32-kDa proRIP-like form and an 18-kDa peptide of the two peptides that form active RIP. However, in germinated kernels, two protein bands that reacted with RIP antiserum were identified as two RIP-like peptides with a molecular mass of approximately 18 and 9 kDa. Purified RIP and zeamatin from corn inhibited growth of A. flavus. Bioassays of germinated kernel extracts from all four genotypes exhibited antifungal activity against A. flavus and F. moniliforme, with extracts from the susceptible genotypes showing greater inhibition zones. This study provides evidence of protein induction in corn kernels during imbibition or the early stages of germination, and the induced proteins may be related to our previous findings of germination-associated resistance in the corn kernel, especially in the susceptible kernels.

Isolation and characterization of a peanut maturity-associated protein.

Using reversed-phase high-performance liquid chromatography (RP-HPLC), the peanut protein profile was shown to be related to the maturity, drying time, and drying procedure of the peanut. Differences were seen between (a) immature and mature seeds for untreated and windrow-dried peanuts, (b) untreated and windrow-dried peanuts for immature and mature seeds, and (c) windrow- and stackpole-dried peanuts. The most pronounced HPLC peak that increased in size as the peanut matured and decreased in size with longer drying times was isolated and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry to have a molecular weight of 62 500. Since maturity is related to the sensory quality of peanuts, this protein may be a marker for peanuts that will produce a higher quality flavor when roasted.

Protein profiles and antifungal activities of kernel extracts from corn genotypes resistant and susceptible to Aspergillus flavus.

Mechanisms of resistance to infection by the fungus Aspergillus flavus and accumulation of aflatoxin were studied in kernels of resistant (GT-MAS:gk, Mp420) and susceptible ( Pioneer 3154, Deltapine G-4666) corn genotypes. Proteins from kernel extracts of corn genotypes were analyzed by several methods of polyacrylamide gel electrophoresis. Consistent differences in protein profiles were detected among genotypes. Several proteins were unique to or present in greater concentration in resistant genotypes, whereas others were present only in susceptible genotypes. Extracts of resistant kernels showed markedly greater antifungal activity against A. flavus than did susceptible kernel extracts. Results from the present study suggest a role for kernel proteins in resistance to A. flavus infection and aflatoxin contamination in corn genotypes.

Insecticide susceptibility in Coptotermes formosanus and Reticulitermes virginicus (Isoptera: Rhinotermitidae).

Lethal time to mortality responses were established for eight insecticides against workers and soldiers of the Formosan subterranean termite, Coptotermes formosanus Shiraki, and workers of Reticulitermes virginicus (Banks). There were significant differences in the tolerance ratios between workers of C. formosanus colonies to all toxicants tested except fipronil. One colony was 16 times more tolerant than another to deltamethin. C. formosanus soldiers had significant differences in tolerance ratios among colonies exposed to all toxicants except chlorpyrifos. Methoxychlor, permethrin, deltamethrin, and fipronil did not kill soldiers from two, one, one, and three colonies, respectively, within 8 h. Seventy-five percent of R. virginicus colonies were significantly less susceptible than the most susceptible colony to chlordane, methoxychlor, chlorpyrifos, cypermethrin, and fipronil, with 50% of the colonies less susceptible to permethrin and bendiocarb. In 50% of C. formosanus colonies the worker lethal time curves displayed substantial flattening in response to permethrin, and deltamethrin. Lethal time curses for C. formosanus soldiers exposed to chlordane, chlorpyrifos, permethrin, cypermethrin, deltamethrin, and bendiocarb showed substantial flattening. R. virginicus workers demonstrated substantial curve flattening when exposed to chlordane, methoxychlor, chlorpyrifos, deltamethrin, and fipronil. These findings indicate substantial intercolony and intra-colony differences in susceptibility to insecticides.

Colocalization of Polyphenol Oxidase and Photosystem II Proteins.

Polyphenol oxidase (PPO) appears to be ubiquitous in higher plants but, as yet, no function has been ascribed to it. Herein, we report on the localization of PPO based upon biochemical fractionation of chloroplast membranes in Vicia faba (broad bean) into various complexes and immunocytochemical electron microscopic investigations. Sucrose density gradient fractionations of thylakoid membranes after detergent solubilization reveals that PPO protein (by reactivity with anti-PPO antibody) and activity (based upon ability to oxidize di-dihydroxyphenylalanine) are found only in fractions enriched in photosystem II (PSII). Furthermore, of the PSII particles isolated using three different protocols utilizing several plant species, all had PPO. Immunogold localization of PPO on thin sections reveals exclusive thylakoid labeling with a distribution pattern consistent with other PSII proteins (80% grana, 20% stroma). These data strongly indicate that PPO is at least peripherally associated with the PSII complex.

Partial deletion of transposon Tn4560 integrated into the genome of Streptomyces tendae.

Polymerase chain reaction (PCR), Southern hybridization and DNA sequencing experiments were done to determine whether all of Tn4560, a Streptomyces transposon, integrated into the genomes of three nikkomycin non-producing mutants. A deletion of 279 bases occurred at one end of Tn4560 while present in the genome of one of the mutants.

Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase.

Three cDNA clones were isolated which code for the ubiquitous chloroplast enzyme, polyphenol oxidase (PPO), from Vicia faba. Analysis of the cloned DNA reveals that PPO is synthesized with an N-terminal extension of 92 amino acid residues, presumed to be a transit peptide. The mature protein is predicted to have a molecular mass of 58 kDa which is in close agreement to the molecular mass estimated for the in vivo protein upon SDS-PAGE. Differences in the DNA sequence of two full-length and one partial cDNA clones indicate that PPO is encoded by a gene family. Analysis of the deduced amino acid sequence shows that the chloroplast PPO shares homology with the 59 kDa PPOs in glandular trichomes of solanaceous species. A high degree of sequence conservation was found with the copper-binding domains of the 59 kDa tomato PPO as well as hemocyanins and tyrosinases from a wide diversity of taxa.

Inhibition of some mycotoxigenic fungi by iturin A, a peptidolipid produced by Bacillus subtilis.

Bacillus subtilis produces peptidolipid compounds of the iturin group that have been shown to have antifungal properties, but not all fungal species are sensitive to these compounds. In this study, the activity of iturin A, produced by B. subtilis strain B-3, was tested. Paper disks impregnated with various concentrations of iturin A were placed on agar plates seeded with conidia of toxigenic species of Fusarium, Gerlacia, Penicillium or Aspergillus. Most isolates were inhibited at iturin A concentrations as low as 4 micrograms/disk. Penicillium italicum, P. viridicatum, A. ochraceus and A. versicolor were most strongly inhibited by the iturin whereas P. citrinum and A. parasiticus were least sensitive to iturin A.

Comparison of the enzymatic composition of cell-free extracts of non-aflatoxigenic Aspergillus parasiticus with respect to late stages of aflatoxin biosynthesis.

Cell-free extracts of fungal mycelia of two non-aflatoxigenic isolates of Aspergillus parasiticus (SRRC 163 and SRRC 2043) were examined for enzyme activities involved in the latter stages of aflatoxin biosynthesis. Post-microsomal fractions (105 Kxg supernatant) of both SRRC 163 (ATTC 56774) and SRRC 2043 were able to convert sterigmatocystin (ST) to O-methylsterigmatocystin (OMST); whereas the microsomal (105 Kxg pellet) preparation of only SRRC 163 was able to convert OMST to aflatoxin B1 (AFB1). A mixture of the microsomal fraction of SRRC 163 and post-microsomal fraction of SRRC 2043 converted ST to AFB1. Electrophoretic protein separations of the microsomal fractions of the two A. parasiticus isolates on non-denaturing or denaturing polyacrylamide gel electrophoresis (PAGE) provided equivalent protein profiles.

Ferredoxin-NADP(+) reductase, a nuclearly-coded enzyme unaffected by tentoxin treatment.

Previous studies in our laboratory have shown that tentoxin prevents the incorporation of polyphenol oxidase (PPO), a nuclearly-coded protein, into the chloroplasts of sensitive species. In this study, we show, by comparison of electrophoretically separated isozymes, that ferredoxin-NADP(+) reductase (FNR) is nuclearly coded in Nicotiana. Electrophoresis of FNR isozymes from tentoxin treated seedlings of a sensitive and a resistant species demonstrated that, unlike PPO, ferredoxin-NADP(+) reductase was unaffected by tentoxin treatment. These data indicate that tentoxin selectively inhibits transport of cytoplasmically synthesized proteins into the chloroplast, and does not produce a generalized disruption of cellular integration.

Iturin A: a potential new fungicide for stored grains.

The removal of many synthetic fungicides from the market has created a demand for new, environmentally safe fungicides. Iturin A, a cyclic lipopeptide produced by Bacillus subtilis, has strong antifungal properties and low mammalian toxicity. To determine the efficacy of this compound as a potential fungicide on stored feed grains, lots of corn, peanuts and cottonseed were treated with varying concentrations of iturin A. The mycoflora of treated seed was assayed along with that of untreated seed and seed treated with fungicides used commercially for planting seed. Fungal species varied considerably in their sensitivity to iturin A. Significant reductions in total mycoflora occurred in most seed lots tested at iturin A concentrations of 50 to 100 ppm.

Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures.

Two activities involved in terminal pathway conversion of sterigmatocystin to aflatoxin B1 were isolated from an aflatoxin-nonproducing mutant of Aspergillus parasiticus (avn-1), and the time course of appearance of the activities in culture was determined. Subcellular fractionation of fungal mycelia resolved the two activities into a postmicrosomal activity which catalyzed conversion of sterigmatocystin to O-methylsterigmatocystin and a microsomal activity which converted O-methylsterigmatocystin to aflatoxin B1. The two activities were absent in 24-h-old cells, increased to optimum levels during the stationary phase, and then declined.

Inhibition of plant-pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli.

The cDNA of a 14-kDa trypsin inhibitor (TI) from corn was subcloned into an Escherichia coli overexpression vector. The overexpressed TI was purified based on its insolubility in urea and then refolded into the active form in vitro. This recombinant TI inhibited both conidium germination and hyphal growth of all nine plant pathogenic fungi studied, including Aspergillus flavus, Aspergillus parasiticus, and Fusarium moniliforme. The calculated 50% inhibitory concentration of TI for conidium germination ranged from 70 to more than 300 microgram/ml, and that for fungal growth ranged from 33 to 124 microgram/ml depending on the fungal species. It also inhibited A. flavus and F. moniliforme simultaneously when they were tested together. The results suggest that the corn 14-kDa TI may function in host resistance against a variety of fungal pathogens of crops.