Siderophores from the Entomopathogenic Fungus Beauveria bassiana.

We report NMR- and MS-based structural characterizations of siderophores and related compounds from Beauveria bassiana (Balsamo-Crivelli) Vuillemin, including ten new chemical entities (2-4, 6-9, 11-12, and 15) and five known compounds, (1, 5, 10, 13, and 14). The siderophore mixture from ARSEF strain #2680 included two compounds in which N5-mevalonyl-N5-hydroxyornithine replaces both (2) or one (3) of the N5-anhydromevalonyl-N5-hydroxyornithine units of dimerumic acid (1). Mevalonolactone (14) was present as a degradation product of 2 and 3. ARSEF #2860 also produced compounds that have mannopyranose (5, 6) or 4-O-methyl-mannopyranose units (4, 7), two compounds (8, 9) that can be rationalized as 4-O-methyl-mannopyranosyl analogues of the esterifying acid moieties of metachelins A and B, respectively, and two probable decomposition products of 1, a nitro compound (11) and a formate (12). Beauverichelin A (15), a coprogen-type siderophore that represents the di-4-O-methyl-mannopyranosyl analogue of metachelin A, was detected in crude extracts of ARSEF #2860, but only in trace amounts. ARSEF strains #252 and #1955 yielded beauverichelin A in quantities that were sufficient for NMR analysis. Only the di- (1-7) and trihydroxamate (15) siderophores showed iron-binding activity in the CAS assay and, when ferrated, showed strong ESIMS signals consistent with 1:1 ligand/iron complexes.

Starvation and Imidacloprid Exposure Influence Immune Response by Anoplophora glabripennis (Coleoptera: Cerambycidae) to a Fungal Pathogen.

In several insect systems, fungal entomopathogens synergize with neonicotinoid insecticides which results in accelerated host death. Using the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky), an invasive woodborer inadvertently introduced into North America and Europe, we investigated potential mechanisms in the synergy between the entomopathogenic fungus Metarhizium brunneum Petch and the insecticide imidacloprid. A potential mechanism underlying this synergy could be imidacloprid's ability to prevent feeding shortly after administration. We investigated whether starvation would have an impact similar to imidacloprid exposure on the mortality of fungal-inoculated beetles. Using real-time PCR to quantify fungal load in inoculated beetles, we determined how starvation and pesticide exposure impacted beetles' ability to tolerate or resist a fungal infection. The effect of starvation and pesticide exposure on the encapsulation and melanization immune responses of the beetles was also quantified. Starvation had a similar impact on the survival of M. brunneum-inoculated beetles compared to imidacloprid exposure. The synergy, however, was not completely due to starvation, as imidacloprid reduced the beetles' melanotic encapsulation response and capsule area, while starvation did not significantly reduce these immune responses. Our results suggest that there are multiple interacting mechanisms involved in the synergy between M. brunneum and imidacloprid.

Swainsonine Biosynthesis Genes in Diverse Symbiotic and Pathogenic Fungi.

Swainsonine-a cytotoxic fungal alkaloid and a potential cancer therapy drug-is produced by the insect pathogen and plant symbiont Metarhizium robertsii, the clover pathogen Slafractonia leguminicola, locoweed symbionts belonging to Alternaria sect. Undifilum, and a recently discovered morning glory symbiont belonging to order Chaetothyriales. Genome sequence analyses revealed that these fungi share orthologous gene clusters, designated "SWN," which included a multifunctional swnK gene comprising predicted adenylylation and acyltransferase domains with their associated thiolation domains, a ß-ketoacyl synthase domain, and two reductase domains. The role of swnK was demonstrated by inactivating it in M. robertsii through homologous gene replacement to give a ∆swnK mutant that produced no detectable swainsonine, then complementing the mutant with the wild-type gene to restore swainsonine biosynthesis. Other SWN cluster genes were predicted to encode two putative hydroxylases and two reductases, as expected to complete biosynthesis of swainsonine from the predicted SwnK product. SWN gene clusters were identified in six out of seven sequenced genomes of Metarhzium species, and in all 15 sequenced genomes of Arthrodermataceae, a family of fungi that cause athlete's foot and ringworm diseases in humans and other mammals. Representative isolates of all of these species were cultured, and all Metarhizium spp. with SWN clusters, as well as all but one of the Arthrodermataceae, produced swainsonine. These results suggest a new biosynthetic hypothesis for this alkaloid, extending the known taxonomic breadth of swainsonine producers to at least four orders of Ascomycota, and suggest that swainsonine has roles in mutualistic symbioses and diseases of plants and animals.

Disruptions of the genes involved in lysine biosynthesis, iron acquisition, and secondary metabolisms affect virulence and fitness in Metarhizium robertsii.

Based on genomic analysis, polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways account for biosynthesis of the majority of the secondary metabolites produced by the entomopathogenic fungus Metarhizium robertsii. To evaluate the contribution of these pathways to M. robertsii fitness and/or virulence, mutants deleted for mrpptA, the Sfp-type 4' phosphopantetheinyl transferase gene required for their activation were generated. ΔmrpptA strains were deficient in PKS and NRPS activity resulting in colonies that lacked the typical green pigment and failed to produce the nonribosomal peptides (destruxins, serinocylins, and the siderophores ferricrocin and metachelins) as well as the hybrid polyketide-peptides (NG-39x) that are all produced by the wild type (WT) M. robertsii. The ΔmrpptA colonies were also auxotrophic for lysine. Two other mutant strains were generated: ΔmraarA, in which the α-aminoadipate reductase gene critical for lysine biosynthesis was disrupted, and ΔmrsidA, in which the L-ornithine N5-oxygenase gene that is critical for hydroxamate siderophore biosynthesis was disrupted. The phenotypes of these mutants were compared to those of ΔmrpptA to separate effects of the loss of lysine or siderophore production from the overall effect of losing all polyketide and non-ribosomal peptide production. Loss of lysine biosynthesis marginally increased resistance to H2O2 while it had little effect on the sensitivity to the cell wall disruptor sodium dodecyl sulfate (SDS) and no effect on sensitivity to iron deprivation. In contrast, combined loss of metachelin and ferricrocin through the inactivation of mrsidA resulted in mutants that were as hypersensitive or slightly more sensitive to H2O2, iron deprivation, and SDS, and were either identical or marginally higher in ΔmrpptA strains. In contrast to ΔmrpptA, loss of mrsidA did not completely abolish siderophore activity, which suggests the production of one or more non-hydroxamate iron-chelating compounds. Deletion of mrpptA, mrsidA, and mraarA reduced conidium production and conidia of a GFP-tagged ΔmrpptA strain displayed a longer germination delay than WT on insect cuticles, a deficiency that was rescued by lysine supplementation. Compared with WT, ΔmrpptA strains displayed ∼19-fold reduction in virulence against Drosophila suzukii. In contrast, lysine auxotrophy and loss of siderophores accounted for ∼2 and ∼6-fold decreases in virulence, respectively. Deletion of mrpptA had no significant effect on growth inhibition of Bacillus cereus. Our results suggest that PKS and NRPS metabolism plays a significant role in M. robertsii virulence, depresses conidium production, and contributes marginally to resistance to oxidative stress and iron homeostasis, but has no significant antibacterial effect.

Insights into Adaptations to a Near-Obligate Nematode Endoparasitic Lifestyle from the Finished Genome of Drechmeria coniospora.

Nematophagous fungi employ three distinct predatory strategies: nematode trapping, parasitism of females and eggs, and endoparasitism. While endoparasites play key roles in controlling nematode populations in nature, their application for integrated pest management is hindered by the limited understanding of their biology. We present a comparative analysis of a high quality finished genome assembly of Drechmeria coniospora, a model endoparasitic nematophagous fungus, integrated with a transcriptomic study. Adaptation of D. coniospora to its almost completely obligate endoparasitic lifestyle led to the simplification of many orthologous gene families involved in the saprophytic trophic mode, while maintaining orthologs of most known fungal pathogen-host interaction proteins, stress response circuits and putative effectors of the small secreted protein type. The need to adhere to and penetrate the host cuticle led to a selective radiation of surface proteins and hydrolytic enzymes. Although the endoparasite has a simplified secondary metabolome, it produces a novel peptaibiotic family that shows antibacterial, antifungal and nematicidal activities. Our analyses emphasize the basic malleability of the D. coniospora genome: loss of genes advantageous for the saprophytic lifestyle; modulation of elements that its cohort species utilize for entomopathogenesis; and expansion of protein families necessary for the nematode endoparasitic lifestyle.

Intracellular siderophore but not extracellular siderophore is required for full virulence in Metarhizium robertsii.

Efficient iron acquisition mechanisms are fundamental for microbial survival in the environment and for pathogen virulence within their hosts. M. robertsii produces two known iron-binding natural products: metachelins, which are used to scavenge extracellular iron, and ferricrocin, which is strictly intracellular. To study the contribution of siderophore-mediated iron uptake and storage to M. robertsii fitness, we generated null mutants for each siderophore synthase gene (mrsidD and mrsidC, respectively), as well as for the iron uptake transcriptional repressor mrsreA. All of these mutants showed impaired germination speed, differential sensitivity to hydrogen peroxide, and differential ability to overcome iron chelation on growth-limiting iron concentrations. RT-qPCR data supported regulation of mrsreA, mrsidC, and mrsidD by supplied iron in vitro and during growth within the insect host, Spodoptera exigua. We also observed strong upregulation of the insect iron-binding proteins, transferrins, during infection. Insect bioassays revealed that ferricrocin is required for full virulence against S. exigua; neither the loss of metachelin production nor the deletion of the transcription factor mrsreA significantly affected M. robertsii virulence.

Discovering the secondary metabolite potential encoded within entomopathogenic fungi.

This highlight discusses the secondary metabolite potential of the insect pathogens Metarhizium and Beauveria, including a bioinformatics analysis of secondary metabolite genes for which no products are yet identified.

Metachelins, mannosylated and N-oxidized coprogen-type siderophores from Metarhizium robertsii.

Under iron-depleted culture conditions, the entomopathogenic fungus Metarhizium robertsii (Bischoff, Humber, and Rehner) (= M. anisopliae) produces a complex of extracellular siderophores including novel O-glycosylated and N-oxidized coprogen-type compounds as well as the known fungal siderophores N(α)-dimethylcoprogen (NADC) and dimerumic acid (DA). Metachelin A (1), the most abundant component in the M. robertsii siderophore mixture, was characterized as a 1094 Da analogue of NADC that is O-glycosylated by ß-mannose at both terminal hydroxyl groups and N-oxidized at the dimethylated α-nitrogen. The mixture also contained a 1078 Da analogue, metachelin B (2), which lacks the N-oxide modification. Also characterized were the aglycone of 1, i.e., the N-oxide of NADC (3), and the monomannoside of DA (6). N-Oxide and O-glycosyl substituents are unprecedented among microbial siderophores. At high ESIMS source energy and at room temperature in DMSO, 1 underwent Cope elimination, resulting in loss of the N(α)-dimethyl group and dehydration of the α-ß bond. High-resolution ESIMS data confirmed that all tri- and dihydroxamate siderophores (1-6) complex with trivalent Fe, Al, and Ga. In a chrome azurol S assay, all of the M. robertsii siderophores showed iron-binding activity roughly equivalent to that of desferrioxamine B.

Resistant ticks inhibit Metarhizium infection prior to haemocoel invasion by reducing fungal viability on the cuticle surface.

We studied disease progression of, and host responses to, four species in the Metarhizium anisopliae complex expressing green fluorescent protein (GFP). We compared development and determined their relative levels of virulence against two susceptible arthropods, the cattle tick Rhipicephalus annulatus and the lepidopteran Galleria mellonella, and two resistant ticks, Hyalomma excavatum and Rhipicephalus sanguineus. Metarhizium brunneum Ma7 caused the greatest mortality of R. annulatus, Metarhizium robertsii ARSEF 2575 and Metarhizium pingshaense PPRC51 exhibited intermediate levels of virulence, and Metarhizium majus PPRC27 caused low mortality of cattle ticks. Conidia of all four species germinated on all hosts examined, but on resistant hosts, sustained hyphal growth was inhibited and GFP emission steadily and significantly decreased over time, suggesting a loss of fungal viability. Cuticle penetration was observed only for the three most virulent species infecting susceptible hosts. Cuticles of resistant and susceptible engorged female ticks showed significant increases in red autofluorescence at sites immediately under fungal hyphae. This is the first report (i) of tick mortality occurring after cuticle penetration but prior to haemocoel colonization and (ii) that resistant ticks do not support development of Metarhizium germlings on the outer surface of the cuticle. Whether reduced Metarhizium viability on resistant tick cuticles is due to antibiosis or limited nutrient availability is unknown.

Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence.

Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. Herein, we report identification of three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum, and functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a class I and class II hydrophobin, respectively. To examine function, we deleted all three separately, from the M. brunneum strain KTU-60 genome, using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in wild-type pigmentation, but all retained wild-type hydrophobicity, except for one individual hyd3 mutant. Complementation with the wild-type HYD3 gene restored hydrophobicity. Each gene, present as a single copy in the genome, showed differential expression patterns dependent on the developmental stage of the fungus. When Spodoptera exigua (beet armyworm) larvae were treated with either conidia or blastospores of each hyd mutant, reductions in virulence and delayed mortality were observed as compared to WT. Together, these results suggest that hydrophobins are differentially expressed and may have distinct, but compensating roles, in conidiation, pigmentation, hydrophobicity, and virulence.

Genetic basis of destruxin production in the entomopathogen Metarhizium robertsii.

Destruxins are among the most exhaustively researched secondary metabolites of entomopathogenic fungi, yet definitive evidence for their roles in pathogenicity and virulence has yet to be shown. To establish the genetic bases for the biosynthesis of this family of depsipeptides, we identified a 23,792-bp gene in Metarhizium robertsii ARSEF 2575 containing six complete nonribosomal peptide synthetase modules, with an N-methyltransferase domain in each of the last two modules. This domain arrangement is consistent with the positioning of the adjacent amino acids N-methyl-L: -valine and N-methyl-L: -alanine within the depsipeptide structure of destruxin. DXS expression levels in vitro and in vivo exhibited comparable patterns, beginning at low levels during the early growth phases and increasing with time. Targeted gene knockout using Agrobacterium-mediated transformation produced mutants that failed to synthesize destruxins, in comparison with wild type and ectopic control strains, indicating the involvement of this gene in destruxin biosynthesis. The destruxin synthetase (DXS) disruption mutant was as virulent as the control strain when conidial inoculum was topically applied to larvae of Spodoptera exigua, Galleria mellonella, and Tenebrio molitor indicating that destruxins are dispensable for virulence in these insect hosts. The DXS mutants exhibited no other detectable changes in morphology and development.

Metacridamides A and B, macrocycles from conidia of the entomopathogenic fungus Metarhizium acridum.

Metarhizium acridum, an entomopathogenic fungus, has been commercialized and used successfully for biocontrol of grasshopper pests in Africa and Australia. Its conidia produce two novel 17-membered macrocycles, metacridamides A and B, which consist of a Phe unit condensed with a nonaketide. Planar structures were elucidated by a combination of mass spectrometric and NMR techniques. Following hydrolysis of 1, chiral amino acid analysis assigned the L-configuration to the Phe unit. A crystal structure established the absolute configuration of the eight remaining stereogenic centers in 1. Metacridamide A showed cytotoxicity to three cancer lines with IC50's of 6.2, 11.0, and 10.8 µM against Caco-2 (epithelial colorectal adenocarcinoma), MCF-7 (breast cancer), and HepG2/C3A (hepatoma) cell lines, respectively. In addition, metacridamide B had an IC50 of 18.2 µM against HepG2/C3A, although it was inactive at 100 µM against Caco-2 and MCF-7. Neither analogue showed antimicrobial, phytotoxic, or insecticidal activity.

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.

Identification of a hybrid PKS-NRPS required for the biosynthesis of NG-391 in Metarhizium robertsii.

The fungal entomopathogen Metarhizium robertsii (formerly known as M. anisopliae var. anisopliae) is a prolific producer of secondary metabolites of which very little is known at the genetic level. To establish the genetic bases for the biosynthesis of the mutagenic compound NG- 391, we identified a 19,818 kb genomic region harboring the predicted hybrid polyketide synthase-nonribosomal peptide synthetase NGS1, plus five additional ORFs. NGS1 knockouts generated by Agrobacterium-mediated transformation failed to produce detectable levels of NG-391, indicating the involvement of this locus in its biosynthesis. NGS1 deletion mutants had no significant changes in virulence levels against larvae of Spodoptera exigua and in resistance to hydrogen peroxide-generated oxidative stress compared to the wild-type strain. All 6 ORFs were expressed in medium supporting production of NG-391, and NGS1 was expressed during the interaction with the S. exigua host. The use of an NGS1 promoter-GFP reporter fusion showed that during in vitro growth in still broth cultures, NGS1 expression is restricted to the early exponential phase and is affected by M. robertsii cell density.

Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in the invertebrate pathogen Metarhizium anisopliae reveals a peptide spore factor.

Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins, cyclic heptapeptides, in the extracts of conidia of control strains, whereas the compounds were undetectable in DeltaManps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in DeltaManps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. DeltaManps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences in pathogenicity of the DeltaManps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison to control strains. This is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor.

Serinocyclins A and B, cyclic heptapeptides from Metarhizium anisopliae.

Two new cyclic heptapeptides, serinocyclins A (1) and B (2), were isolated from conidia of the entomopathogenic fungus Metarhizium anisopliae. Structures were elucidated by a combination of mass spectrometric, NMR, and X-ray diffraction techniques. Serinocyclin A (1) contains three serine units, a hydroxyproline (Hyp), a beta-alanine (beta-Ala), and two uncommon nonproteinogenic amino acids, 1-aminocyclopropane-1-carboxylic acid (Acc) and gamma-hydroxylysine (HyLys). The peptide sequence established for 1 by NMR is cyclo-(Acc-Hyp-Ser1-HyLys-beta-Ala-Ser2-Ser3). Serinocyclin B (2) has Lys in place of the HyLys unit found in 1. Chiral amino acid analysis indicated the presence in both compounds of one (2 S,4 R)-Hyp, two L-Ser, and one D-Ser residue. A Lys found in the hydrolyzate of 2 was established as D-configured. A crystal structure of 1 established the position of the D-Ser (Ser2) and the absolute configuration of the HyLys unit (2 R,4 S). The absence of methyl groups is unusual among fungal peptides and, along with the charged lysyl side chain and multiple hydroxyl groups, contributes to the polar nature of the compounds. Serinocyclin A produced a sublethal locomotory defect in mosquito larvae at an EC 50 of 59 ppm.

A Quantitative Assay Using Mycelial Fragments to Assess Virulence of Mycosphaerella fijiensis.

ABSTRACT We describe a method to evaluate the virulence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease (BLSD) of banana and plantain. The method is based on the delivery of weighed slurries of fragmented mycelia by camel's hair brush to 5-by-5-cm areas on the abaxial surface of banana leaf blades. Reliable BLSD development was attained in an environmental growth chamber with stringent lighting and humidity controls. By localizing inoculum onto small areas of large leaves, we achieved a dramatic increase in the number of strains that can be tested on each leaf and plant, which is critical for comparing the virulence of numerous strains concurrently. Image analysis software was used to measure the percentage of each inoculated leaf section showing BLSD symptoms over time. We demonstrated that the level of disease of four isolates was correlated with the weight of the mycelium applied and relatively insensitive to the degree of fragmentation of hyphae. This is the first report demonstrating that weighed mycelial inoculum, combined with image analysis software to measure disease severity, can be used to quantitatively assess the virulence of M. fijiensis under rigorously controlled environmental conditions.

Production of mutagenic metabolites by Metarhizium anisopliae.

NG-391 (1) and NG-393 (2), previously reported from undescribed Fusarium species as nerve-cell growth stimulants, were identified from fermentation extracts of the entomopathogenic fungus Metarhizium anisopliae. These compounds are 7-desmethyl analogues of fusarin C and (8Z)-fusarin C, mutagenic toxins from Fusarium species that contaminate corn. A mutant strain of M. anisopliae (KOB1-3) overproduces 1 and 2 by ca. 10-fold relative to the wild-type strain, ARSEF 2575, from which it was derived. Overproduction of these compounds in KOB1-3 imparts a yellow pigmentation to the culture medium of the fungus. These compounds were inactive at 100 mug/disk in antimicrobial disk diffusion assays. Compound 1 was inactive at 100 ppm in a mosquitocidal assay. However, like their fusarin analogues, 1 and 2 exhibited potent S9-dependent mutagenic activity in the Salmonella mutagenicity test. Discovery of these highly mutagenic mycotoxins in M. anisopliae suggests that screening for production of NG-391 and NG-393 in strains that are used as biocontrol agents would be a prudent course of action. The impact of these findings on the use of M. anisopliae as a biocontrol agent is currently unknown and requires further investigation.

Biolistic transformation of grapevine using minimal gene cassette technology.

The use of minimal gene cassettes (MCs), which are linear DNA fragments (promoter+open reading frame+terminator) lacking the vector backbone sequence, was compared to the traditional use of whole circular plasmids (CPs) for transformation of grapevine. Embryogenic cell suspensions of 'Chardonnay' (Vitis vinifera L.) were transformed via particle co-bombardment using two nonlinked genes in either MCs or CPs. One construct contained the npt-II selectable marker and the second construct contained the MSI99 antimicrobial peptide gene. A total of five lines each from MC and CP treatments that showed positive signals by PCR for both the npt-II and MSI99 genes were selected. Southern blot analyses revealed up to five integration events in the DNA treatments. Transcription levels determined by semi-quantitative RT-PCR varied among transgenic lines. No significant differences were found in transgene transcription between lines from MC and CP transformation. The correlation between npt-II and MSI99 transcription levels was positive (P<0.05), however, no correlation between the transcription level and the number of integration events was observed. Transgenic lines presented a similar phenotype in leaf morphology and plant vigor compared to non-transgenic lines. Moreover, transgenic lines from both MC and CP DNA treatments produced fruit as did the non-transgenic lines in the third year of growth in the greenhouse. Our data confirm the effectiveness of the minimal cassette technology for genetic transformation of grapevine cultivars.

Enhanced enzymatic hydrolysis of langostino shell chitin with mixtures of enzymes from bacterial and fungal sources.

A combination of enzyme preparations from Trichoderma atroviride and Serratia marcescens was able to completely degrade high concentrations (100 g/L) of chitin from langostino crab shells to N-acetylglucosamine (78%), glucosamine (2%), and chitobiose (10%). The result was achieved at 32 degrees C in 12 days with no pre-treatment (size reduction or swelling) of the substrate and without removal of the inhibitory end-products from the mixture. Enzymatic degradation of three forms of chitin by Serratia/Trichoderma and Streptomyces/Trichoderma blends was carried out according to a simplex-lattice mixture design. Fitted polynomial models indicated that there was synergy between prokaryotic and fungal enzymes for both hydrolysis of crab chitin and reduction of turbidity of colloidal chitin (primarily endo-type activity). Prokaryotic/fungal enzymes were not synergistic in degrading chitosan. Enzymes from prokaryotic sources had much lower activity against chitosan than enzymes from T. atroviride.