Description of Cohnella zeiphila sp. nov., a bacterium isolated from maize callus cultures.

A Gram-stain positive, aerobic, motile, rod-shaped bacterium designated as strain CBP-2801T was isolated as a contaminant from a culture containing maize callus in Peoria, Illinois, United States. The strain is unique relative to other Cohnella species due to its slow growth and reduced number of sole carbon sources. Phylogenetic analysis using 16S rRNA indicated that strain CBP-2801T is a Cohnella bacterium and showed the highest similarity to Cohnella xylanilytica (96.8%). Genome-based phylogeny and genomic comparisons based on average nucleotide identity confirmed the strain to be a novel species of Cohnella. Growth occurs at 15-45 °C (optimum 40 °C), pH 5-7 (optimum pH 6) and with 0-1% NaCl. The predominant fatty acids are anteiso-15:0 and 18:1 ω6c. Genome mining for secondary metabolites identified a putative biosynthetic cluster that encodes for a novel lasso peptide. In addition, this study contributes five new genome assemblies of type strains of Cohnella species, a genus with less than 30% of the type strains sequenced. The DNA G + C content is 58.7 mol %. Based on the phenotypic, phylogenetic and biochemical data strain CBP-2801T represents a novel species, for which the name Cohnella zeiphila sp. nov. is proposed. The type strain is CBP-2801T (= DSM 111598 = ATCC TSD-230).

Maize peroxidase Px5 has a highly conserved sequence in inbreds resistant to mycotoxin producing fungi which enhances fungal and insect resistance.

Mycotoxin presence in maize causes health and economic issues for humans and animals. Although many studies have investigated expression differences of genes putatively governing resistance to producing fungi, few have confirmed a resistance role, or examined putative resistance gene structure in more than a couple of inbreds. The pericarp expression of maize Px5 has previously been associated with resistance to Aspergillus flavus growth and insects in a set of inbreds. Genes from 14 different inbreds that included ones with resistance and susceptibility to A. flavus, Fusarium proliferatum, F. verticillioides and F. graminearum and/or mycotoxin production were cloned using high fidelity enzymes, and sequenced. The sequence of Px5 from all resistant inbreds was identical, except for a single base change in two inbreds, only one of which affected the amino acid sequence. Conversely, the Px5 sequence from several susceptible inbreds had several base variations, some of which affected amino acid sequence that would potentially alter secondary structure, and thus enzyme function. The sequence of the maize peroxidase Px5 common to inbreds resistant to mycotoxigenic fungi was overexpressed in maize callus. Callus transformants overexpressing the gene caused significant reductions in growth for fall armyworms, corn earworms, and F. graminearum compared to transformant callus with a ß-glucuronidase gene. This study demonstrates rarer transcripts of potential resistance genes overlooked by expression screens can be identified by sequence comparisons. A role in pest resistance can be verified by callus expression of the candidate genes, which can thereby justify larger scale transformation and regeneration of transgenic plants expressing the resistance gene for further evaluation.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium.

The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.

Helicoverpa zea (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) Responses to Sorghum bicolor (Poales: Poaceae) Tissues From Lowered Lignin Lines.

The presence of lignin within biomass impedes the production of liquid fuels. Plants with altered lignin content and composition are more amenable to lignocellulosic conversion to ethanol and other biofuels but may be more susceptible to insect damage where lignin is an important resistance factor. However, reduced lignin lines of switchgrasses still retained insect resistance in prior studies. Therefore, we hypothesized that sorghum lines with lowered lignin content will also retain insect resistance. Sorghum excised leaves and stalk pith Sorghum bicolor (L.) Moench (Poales: Poaceae) from near isogenic brown midrib (bmr) 6 and 12 mutants lines, which have lowered lignin content and increased lignocellulosic ethanol conversion efficiency, were examined for insect resistance relative to wild-type (normal BTx623). Greenhouse and growth chamber grown plant tissues were fed to first-instar larvae of corn earworms, Helicoverpa zea (Boddie) and fall armyworms Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), two sorghum major pests. Younger bmr leaves had significantly greater feeding damage in some assays than wild-type leaves, but older bmr6 leaves generally had significantly less damage than wild-type leaves. Caterpillars feeding on the bmr6 leaves often weighed significantly less than those feeding on wild-type leaves, especially in the S. frugiperda assays. Larvae fed the pith from bmr stalks had significantly higher mortality compared with those larvae fed on wild-type pith, which suggested that bmr pith was more toxic. Thus, reducing lignin content or changing subunit composition of bioenergy grasses does not necessarily increase their susceptibility to insects and may result in increased resistance, which would contribute to sustainable production.

A non-autonomous insect piggyBac transposable element is mobile in tobacco.

The piggyBac transposable element, originally isolated from a virus in an insect cell line, is a valuable molecular tool for transgenesis and mutagenesis of invertebrates. For heterologous transgenesis in a variety of mammals, transfer of the piggyBac transposable element from an ectopic plasmid only requires expression of piggyBac transposase. To determine if piggyBac could function in dicotyledonous plants, a two-element system was developed in tobacco (Nicotiana tabacum) to test for transposable element excision and insertion. The first transgenic line constitutively expressed piggyBac transposase, while the second transgenic line contained at least two non-autonomous piggyBac transposable elements. Progeny from crosses of the two transgenic lines was analyzed for piggyBac excision and transposition. Several progeny displayed excision events, and all the sequenced excision sites exhibited evidence of the precise excision mechanism characteristic of piggyBac transposase. Two unique transposition insertion events were identified that each included diagnostic duplication of the target site. These data indicate that piggyBac transposase is active in a dicotyledonous plant, although at a low frequency.

Functional characterization of a veA-dependent polyketide synthase gene in Aspergillus flavus necessary for the synthesis of asparasone, a sclerotium-specific pigment.

The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few other metabolites have been identified. In the process of studying how the developmental regulator, VeA, affects A. flavus secondary metabolism we discovered that mutation of veA caused a dramatic down-regulation of transcription of a polyketide synthase gene belonging to cluster 27 and the loss of the ability of the fungi to produce sclerotia. Inactivation of the cluster 27 pks (pks27) resulted in formation of greyish-yellow sclerotia rather than the dark brown sclerotia normally produced by A. flavus while conidial pigmentation was unaffected. One metabolite produced by Pks27 was identified by thin layer chromatography and mass spectral analysis as the known anthraquinone, asparasone A. Sclerotia produced by pks27 mutants were significantly less resistant to insect predation than were the sclerotia produced by the wild-type and more susceptible to the deleterious effects of ultraviolet light and heat. Normal sclerotia were previously thought to be resistant to damage because of a process of melanization similar to that known for pigmentation of conidia. Our results show that the dark brown pigments in sclerotia derive from anthraquinones produced by Pks27 rather than from the typical tetrahydronapthalene melanin production pathway. To our knowledge this is the first report on the genes involved in the biosynthesis of pigments important for sclerotial survival.

Antimicrobial and antiinsectan phenolic metabolites of Dalea searlsiae.

Continued interest in the chemistry of Dalea spp. led to investigation of Dalea searlsiae, a plant native to areas of the western United States. Methanol extractions of D. searlsiae roots and subsequent chromatographic fractionation afforded the new prenylated and geranylated flavanones malheurans A-D (1-4) and known flavanones (5 and 6). Known rotenoids (7 and 8) and isoflavones (9 and 10) were isolated from aerial portions. Structure determination of pure compounds was accomplished primarily by extensive 1D- and 2D-NMR spectroscopy. The absolute configurations of compounds 1-5, 7, and 8 were assigned using electronic circular dichroism spectroscopy. Antimicrobial bioassays revealed significant activity concentrated in the plant roots. Compounds 1-6 exhibited MICs of 2-8 µg/mL against Streptococcus mutans, Bacillus cereus, and oxacillin-sensitive and -resistant Staphylococcus aureus. Aerial metabolites 7-10 were inactive against these organisms, but the presence of 7 and 8 prompted investigation of the antiinsectan activity of D. searlsiae metabolites toward the major crop pest Spodoptera frugiperda (fall armyworm). While compounds 1-10 all caused significant reductions in larval growth rates, associated mortality (33-66%) was highest with flavanone 4 and rotenoids 7 and 8. These findings suggest a differential allocation of antimicrobial and antiinsectan plant resources to root and aerial portions of the plant, respectively.

Expression of a wolf spider toxin in tobacco inhibits the growth of microbes and insects.

Lycotoxin I, from the wolf spider (Lycosa carolinensis), is an amphipathic pore-forming peptide that has antimicrobial and anti-insect activity. Constitutive expression of a lycotoxin I modified for oral toxicity to insects in tobacco (Nicotiana tabacum) conferred significantly enhanced resistance to larvae of the corn earworm (Helicoverpa zea) and cigarette beetle (Lasioderma serricorne). Gene expression levels of modified lycotoxin I were negatively correlated to the survival of corn earworm larvae. In addition, pathogenic symptoms caused by Pseudomonas syringae pathovar tabaci and Alternaria alternata on the modified lycotoxin I-expressing leaves were significantly less severe than on wild type leaves. These results indicate that modified lycotoxin I expression in tobacco can potentially protect leaf tissue from a broad spectrum of pests and pathogens.

Potential Biocontrol Agents of Corn Tar Spot Disease Isolated from Overwintered Phyllachora maydis Stromata.

Tar spot disease in corn, caused by Phyllachora maydis, can reduce grain yield by limiting the total photosynthetic area in leaves. Stromata of P. maydis are long-term survival structures that can germinate and release spores in a gelatinous matrix in the spring, which are thought to serve as inoculum in newly planted fields. In this study, overwintered stromata in corn leaves were collected in Central Illinois, surface sterilized, and caged on water agar medium. Fungi and bacteria were collected from the surface of stromata that did not germinate and showed microbial growth. Twenty-two Alternaria isolates and three Cladosporium isolates were collected. Eighteen bacteria, most frequently Pseudomonas and Pantoea species, were also isolated. Spores of Alternaria, Cladosporium, and Gliocladium catenulatum (formulated as a commercial biofungicide) reduced the number of stromata that germinated compared to control untreated stromata. These data suggest that fungi collected from overwintered tar spot stromata can serve as biological control organisms against tar spot disease.

Effects of elevated peroxidase levels and corn earworm feeding on gene expression in tomato.

Microarray analysis was used to measure the impact of herbivory by Helicoverpa zea, (corn earworm caterpillar) on wild-type and transgenic tomato, Solanum lycopersicum, plants that over-express peroxidase. Caterpillar herbivory had by far the greatest affect on gene expression, but the peroxidase transgene also altered the expression of a substantial number of tomato genes. Particularly high peroxidase activity resulted in the up-regulation of genes encoding proteinase inhibitors, pathogenesis-related (PR) proteins, as well as proteins associated with iron and calcium transport, and flowering. In a separate experiment conducted under similar conditions, real-time quantitative polymerase chain reaction (qPCR) analysis confirmed our microarray results for many genes. There was some indication that multiple regulatory interactions occurred due to the interaction of the different treatments. While herbivory had the greatest impact on tomato gene expression, our results suggest that levels of expression of a multifunctional gene, such as peroxidase and its products, can influence other gene expression systems distinct from conventional signaling pathways, further indicating the complexity of plant defensive responses to insects.

Comparative transcription profiling analyses of maize reveals candidate defensive genes for seedling resistance against corn earworm.

As maize seedlings germinate into the soil, they encounter an environment teeming with insects seeking rich sources of nutrition. Maize presumably has developed a number of molecular mechanisms to ensure survival at the beginning of its life cycle. Comparative transcription analysis using microarrays was utilized to document the expression of a number of genes with potential defensive functions in seedling tissue. In addition to elevated levels of the genes involved in the biosynthesis of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), an anti-insect resistance molecule, other highly expressed genes in the seedling encode the following putative defensive proteins: defensin, hydroxyproline and proline-rich protein, thaumatin-like protein, lipase, cystatin, protease inhibitor, and a variety of proteases. The potential resistance genes identified occurred mainly on chromosomes 1 and 5 in the B73 genome. Analysis of promoters of seven DIMBOA biosynthetic genes identified three transcription factor binding sites that are possibly involved in regulation of the DIMBOA biosynthetic pathway. The results indicate that maize employs a wide variety of potential resistance mechanisms in seedling tissue to resist a possible insect attack.

Differential activity of multiple saponins against omnivorous insects with varying feeding preferences.

A variety of glycosylated and unglycosylated saponins from seven different plant families (Aquifoliaceae, Asparagaceae, Caryophyllaceae, Dioscoreaceae, Leguminosae, Rosaceae, Sapindaceae) were tested against the corn earworm, Helicoverpa zea, and the fall armyworm, Spodoptera frugiperda. The corn earworm feeds readily on both monocots and dicots, while the fall armyworm is primarily a grass feeder. Most of the saponins were similarly effective or ineffective against both insect species, with the glycosides being the primary active form (compared to aglycones). However, one aglycone possessed antifeedant properties toward the fall armyworm. Thus, in contrast to many plant secondary metabolites effective against either of these two species where the aglycone is more effective, in the case of the saponins the opposite is generally true. This appears to be a contradictory strategy of plant defenses that requires further consideration. The activity of protodioscin against insects is reported for the first time and may be important in insect defense by the bioenergy crop switchgrass.

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system.

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.

Synergistic enhancement of beta-lactam antibiotics by modified tunicamycin analogs TunR1 and TunR2.

The ß-lactams are the most widely used group of antibiotics in human health and agriculture, but this is under threat due to the persistent rise of pathogenic resistance. Several compounds, including tunicamycin (TUN), can enhance the antibacterial activity of the ß-lactams to the extent of overcoming resistance, but the mammalian toxicity of TUN has precluded its use in this role. Selective hydrogenation of TUN produces modified compounds (TunR1 and TunR2), which retain the enhancement of ß-lactams while having much lower mammalian toxicity. Here we show that TunR1 and TunR2 enhance the antibacterial activity of multiple ß-lactam family members, including penems, cephems, and third-generation penicillins, to a similar extent as does the native TUN. Eleven of the ß-lactams tested were enhanced from 2 to >256-fold against Bacillus subtilis, with comparable results against a penicillin G-resistant strain. The most significant enhancements were obtained with third-generation aminothiazolidyl cephems, including cefotaxime, ceftazidime, and cefquinome. These results support the potential of low toxicity tunicamycin analogs (TunR1 and TunR2) as clinically valid, synergistic enhancers for a broad group of ß-lactam antibiotics.

Oral toxicity of beta-N-acetyl hexosaminidase to insects.

Insect chitin is a potential target for resistance plant proteins, but plant-derived chitin-degrading enzymes active against insects are virtually unknown. Commercial beta-N-acetylhexosaminidase (NAHA), a chitin-degrading enzyme from jack bean Canavalia ensiformis, caused significant mortality of fall armyworm Spodoptera frugiperda larvae at 75 microg/gm, but no significant mortality was noted with Aspergillus niger NAHA. Maize Zea mays callus transformed to express an Arabidopsis thaliana clone that putatively codes for NAHA caused significantly higher mortality of cigarette beetle Lasioderma serricorne larvae and significantly reduced growth rates (as reflected by survivor weights) of S. frugiperda as compared to callus that expressed control cDNAs. Tassels from model line Hi-II maize (Z. mays) plants transformed with the NAHA gene fed to S. frugiperda caused significantly higher mortality than tassels transformed to express glucuronidase; mortality was significantly correlated with NAHA expression levels detected histochemically. Leaf disks from inbred Oh43 maize plants transformed with the NAHA gene on average had significantly less feeding by caterpillars than null transformants. Leaf disks of Oh43 transformants caused significant mortality of both S. frugiperda and corn earworm Helicoverpa zea larvae, which was associated with higher expression levels of NAHA detected by isoelectric focusing, histochemically, or with antibody. Overall, these results suggest that plant NAHA has a role in insect resistance. Introduction of NAHA genes or enhancement of activity through breeding or genetic engineering has the potential to significantly reduce insect damage and thereby indirectly reduce mycotoxins that are harmful to animals and people.

Expression of a maize Myb transcription factor driven by a putative silk-specific promoter significantly enhances resistance to Helicoverpa zea in transgenic maize.

Hi II maize (Zea mays) plants were engineered to express maize p1 cDNA, a Myb transcription factor, controlled by a putative silk specific promoter, for secondary metabolite production and corn earworm resistance. Transgene expression did not enhance silk color, but about half of the transformed plant silks displayed browning when cut, which indicated the presence of p1-produced secondary metabolites. Levels of maysin, a secondary metabolite with insect toxicity, were highest in newly emerged browning silks. The insect resistance of transgenic silks was also highest at emergence, regardless of maysin levels, which suggests that other unidentified p1-induced molecules likely contributed to larval mortality. Mean survivor weights of corn earworm larvae fed mature browning transgenic silks were significantly lower than weights of those fed mature nonbrowning transgenic silks. Some transgenic pericarps browned with drying and contained similar molecules found in pericarps expressing a dominant p1 allele, suggesting that the promoter may not be silk-specific.

Penifulvin A: a sesquiterpenoid-derived metabolite containing a novel dioxa[5,5,5,6]fenestrane ring system from a fungicolous isolate of Penicillium griseofulvum.

[structure: see text] Penifulvin A (1), a new fungal metabolite with a previously undescribed ring system, has been isolated from cultures of an isolate of Penicillium griseofulvum (NRRL 35584) obtained from a white mycelial growth on a dead hardwood branch collected in a Hawaiian forest. The structure was assigned by analysis of NMR data and confirmed by single-crystal X-ray diffraction analysis. Penifulvin A (1) shows significant activity in dietary assays against the fall armyworm Spodoptera frugiperda.

Relative activity of a tobacco hybrid expressing high levels of a tobacco anionic peroxidase and maize ribosome-inactivating protein against Helicoverpa zea and Lasioderma serricorne.

Tobacco (Nicotiana tabacum) plants grown from seed obtained by crossing a tobacco line that expressed an activated maize ribosome-inactivating protein (RIP) with a line that overexpressed tobacco anionic peroxidase were tested for their effects on corn earworm Helicoverpa zea and cigarette beetle Lasioderma serricorne larvae as compared to the wild-type plant cross. Significant feeding reductions were noted for transgenic plants expressing both resistance proteins as compared to wild-type plants for both H. zea and L. serricorne. Significant increases in mortality were also noted for those insects fed on the transgenic cross as compared to wild-type plants in some cases. Levels of both peroxidase and maize RIP were significantly higher in transgenic as compared to wild-type plants (which did not produce maize RIP). The degree of feeding was significantly negatively correlated with the level of RIP or peroxidase individually.

A quantitative method for determining relative colonization rates of maize callus by Fusarium graminearum for resistance gene evaluations.

A quantitative PCR method was developed for detecting Fusarium graminearum growing in maize callus. Fungal DNA was detected 12h after inoculation (detection limit, 0.2pg) and was correlated with visual ratings. The method effectively quantified fungal growth in callus overexpressing a peroxidase gene conferring fungal resistance.