Dwayne D. Hegedus

New Insights into Peritrophic Matrix Synthesis, Architecture, and Function

The peritrophic matrix (PM) is a chitin and glycoprotein layer that lines the invertebrate midgut. Although structurally different, it is functionally similar to the mucous secretions of the vertebrate digestive tract. The PM is a physical barrier, protecting the midgut epithelium from abrasive food particles, digestive enzymes, and pathogens infectious per os. It is also a biochemical barrier, sequestering and, in some cases, inactivating ingested toxins. Finally, the PM compartmentalizes digestive processes, allowing for efficient nutrient acquisition and reuse of hydrolytic enzymes. The PM consists of an organized lattice of chitin fibrils held together by chitin binding proteins. Glycans fill the interstitial spaces, creating a molecular sieve, the properties of which are dependent on the immediate ion content and pH. In this review, we have integrated recent structural and functional information to create a holistic model for the PM. We also show how this information may generate novel technologies for use in insect pest management.

Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris.

A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterative induction. P. pastoris was found to express several proteases; however, adjustment of medium pH to limit their activity did not correlate with increased scFv recovery. Induction medium pH values of 6.5-8.0 were most conducive to scFv production, despite significant differences in cell growth rates. Increasing inoculum density limited growth potential but gave rise to higher levels of scFv production. Three factors, medium composition, pre-induction osmotic stress, and temperature, had the greatest effects on protein production. Supplementation of the induction medium with arganine, casamino acids, or EDTA increased scFv production several fold, as did cultivation under osmotic stress conditions during pre-induction biomass accumulation. Incubation at 15 versus 30 degrees C extended the period whereby cells were capable of producing scFv from 1 to 7 days. Under optimal conditions, yeast cultures yielded 25 mg/L of functional scFv and could be subject to five reiterative inductions.

Repression of Seed Maturation Genes by a Trihelix Transcriptional Repressor in Arabidopsis Seedlings

The seed maturation program is repressed during germination and seedling development so that embryonic genes are not expressed in vegetative organs. Here, we describe a regulator that represses the expression of embryonic seed maturation genes in vegetative tissues. ASIL1 (for Arabidopsis 6b-interacting protein 1-like 1) was isolated by its interaction with the Arabidopsis thaliana 2S3 promoter. ASIL1 possesses domains conserved in the plant-specific trihelix family of DNA binding proteins and belongs to a subfamily of 6b-interacting protein 1-like factors. The seedlings of asil1 mutants exhibited a global shift in gene expression to a profile resembling late embryogenesis. LEAFY COTYLEDON1 and 2 were markedly derepressed during early germination, as was a large subset of seed maturation genes, such as those encoding seed storage proteins and oleosins, in seedlings of asil1 mutants. Consistent with this, asil1 seedlings accumulated 2S albumin and oil with a fatty acid composition similar to that of seed-derived lipid. Moreover, ASIL1 specifically recognized a GT element that overlaps the G-box and is in close proximity to the RY repeats of the 2S promoters. We suggest that ASIL1 targets GT-box–containing embryonic genes by competing with the binding of transcriptional activators to this promoter region.

De novo sequence assembly of Albugo candida reveals a small genome relative to other biotrophic oomycetes.

BackgroundAlbugo candida is a biotrophic oomycete that parasitizes various species of Brassicaceae, causing a disease (white blister rust) with remarkable convergence in behaviour to unrelated rusts of basidiomycete fungi.ResultsA recent genome analysis of the oomycete Hyaloperonospora arabidopsidis suggests that a reduction in the number of genes encoding secreted pathogenicity proteins, enzymes for assimilation of inorganic nitrogen and sulphur represent a genomic signature for the evolution of obligate biotrophy. Here, we report a draft reference genome of a major crop pathogen Albugo candida (another obligate biotrophic oomycete) with an estimated genome of 45.3 Mb. This is very similar to the genome size of a necrotrophic oomycete Pythium ultimum (43 Mb) but less than half that of H. arabidopsidis (99 Mb). Sequencing of A. candida transcripts from infected host tissue and zoosporangia combined with genome-wide annotation revealed 15,824 predicted genes. Most of the predicted genes lack significant similarity with sequences from other oomycetes. Most intriguingly, A. candida appears to have a much smaller repertoire of pathogenicity-related proteins than H. arabidopsidis including genes that encode RXLR effector proteins, CRINKLER-like genes, and elicitins. Necrosis and Ethylene inducing Peptides were not detected in the genome of A. candida. Putative orthologs of tat-C, a component of the twin arginine translocase system, were identified from multiple oomycete genera along with proteins containing putative tat-secretion signal peptides.ConclusionAlbugo candida has a comparatively small genome amongst oomycetes, retains motility of sporangial inoculum, and harbours a much smaller repertoire of candidate effectors than was recently reported for H. arabidopsidis. This minimal gene repertoire could indicate a lack of expansion, rather than a reduction, in the number of genes that signify the evolution of biotrophy in oomycetes.

Patterns of differential gene expression in Brassica napus cultivars infected with Sclerotinia sclerotiorum

SUMMARY The fungal pathogen Sclerotinia sclerotiorum infects a broad range of dicotyledonous plant species and causes stem rot in Brassica napus. To elucidate the mechanisms underlying the defence response, the patterns of gene expression in the partially resistant B. napus cultivar ZhongYou 821 (ZY821) and the susceptible cultivar Westar were studied using a B. napus oligonucleotide microarray. Although maximum differential gene expression was observed at 48 h post-inoculation (hpi) in both cultivars, increased transcript levels were detected in cv. ZY821 at the earlier stages of infection (6-12 hpi) for many genes, including those encoding defence-associated proteins, such as chitinases, glucanases, osmotins and lectins, as well as genes encoding transcription factors belonging to the zinc finger, WRKY, APETALA2 (AP2) and MYB classes. In both cultivars, genes encoding enzymes involved in jasmonic acid, ethylene and auxin synthesis were induced, as were those for gibberellin degradation. In addition, changes in the expression of genes encoding enzymes involved in carbohydrate and energy metabolism appeared to be directed towards shuttling carbon reserves to the tricarboxylic acid cycle and generating reactive oxygen species. Transcripts from genes encoding enzymes involved in glucosinolate and phenylpropanoid biosynthesis were highly elevated in both cultivars, suggesting that secondary metabolites are also components of the response to S. sclerotiorum in B. napus.

A comparison of the virulence, stability and cell-wall-surface characteristics of three spore types produced by the entomopathogenic fungus Beauveria bassiana

SummaryBeauveria bassiana can produce three spore types; aerial conidia, submerged conidia and blastospores. We have examined the spore surface characteristics (hydrophobicity and cell-wall surface lectins), thermal inactivation and the virulence towards the migratory grasshopper, Melanoplus sanguinipes, of each of the three spore types. The hydrophobicities of the aerial and submerged conidia were quite similar. Blastospores were less hydrophobic than either of the two types of conidia. Hydrophobic interactions are thought to play a significant role in attachment of the spore to the host organism. However, the less hydrophobic blastospores were slightly more virulent (LT50 of 6.50 days) when compared to the aerial and submerged conidia (7.12 and 7.24 days), respectively. The lectin-binding characteristics of the aerial and submerged conidia were very similar but differed from that of blastospores. Growth of blastospores on a variety of carbohydrates did not affect their lectin-binding characteristics. Spore viability measurements showed that aerial and submerged conidia retained their viability for a longer period than blastospores. The similarity in hydrophobicity, stability, virulence and lectin-binding of aerial and submerged conidia make the latter an ideal candidate for mycoinsecticide production since they can be recovered after growth on inexpensive substrates.

The impact of biotechnology on hyphomycetous fungal insect biocontrol agents

The potential for the control of insect pests by entomopathogenic fungi has been touted for decades, if not centuries. Only recently have advances in biotechnology provided the tools for indepth analysis of the mechanisms involved in pathogenesis and host death at the molecular level. This review outlines the current state of knowledge regarding the mode of infection and targets several key components that are amenable to improvement via biotechnology. Realization of the considerable economic potential of fungal bioinsecticides can occur only through a combined and coordinated effort involving fundamental science, formulation technology and field applications.

Beauveria bassiana submerged conidia production in a defined medium containing chitin, two hexosamines or glucose

SummaryBeauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH4Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 105/ ml after two days in GlcNAc medium as compared to 8.9 × 105/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 106/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.

Production of an extracellular lipase byBeauveria bassiana

SummaryThe entomopathogenic fungus,Beauveria bassiana, produces an extracellular lipase when grown on a yeast extract-peptone-dextrose broth (YPD) medium. The time course of lipase production in the presence of olive oil was studied and which was shown to induce lipase. The addition of fatty acids, such as, myristic, palmitic, stearic, oleic, linoleic and arachidic acids, inhibited both growth and lipase production. Lipase production was also assessed on YPD and glucose minimal salts (GMS) medium. The addition of olive oil increased the lipase induction much more on, YPD than on the GMS. The effect of the divalent metal ions; iron, copper and magnesium, on lipase activity was studied. Whereas the iron and copper inhibited lipase activity, magnesium slightly increased lipase activity. Compounds containing a hydrolyzable ester group, such as Tweens, were found to inhibit lipase activity.

Enhanced seed carotenoid levels and branching in transgenic Brassica napus expressing the Arabidopsis miR156b gene.

The Arabidopsis AtmiR156b gene was expressed in Brassica napus under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the seed-specific napin promoter. Seed carotenoid levels, branching habit, seed yield, and seed weight were examined in the transgenic B. napus. Our results demonstrated that constitutive expression of AtmiR156b in B. napus resulted in enhanced levels of seed lutein and beta-carotene and a 2-fold increase in the number of flowering shoots, whereas AtmiR156b driven by the napin promoter did not affect these traits. This suggested that enhancement of seed quality and shoot branching are both related to AtmiR156b expression patterns. Seed yield and seed weight varied significantly within the transgenic lines. However, one line was found to have enhanced seed carotenoid levels but unchanged seed weight or yield. These data suggest that AtmiR156b gene expression could be applied in plant breeding initiatives for enhancing carotenoid production in canola and other crop species.