Charles A. Maynard

Synthetic Antimicrobial Peptide Design

To guide the design of potential plant pathogen-resistance genes, synthetic variants of naturally occurring antimicrobial gene products were evaluated. Five 20-amino acid (ESF1, ESF4, ESF5, ESF6, ESF13), one 18-amino acid (ESF12), and one 17-amino acid (ESF17) amphipathic peptide sequences were designed, synthesized, and tested with in vitro bioassays. Positive charges on the hydrophilic side of the peptide were shown to be essential for antifungal activity, yet the number of positive charges could be varied with little or no change in activity. The size could be reduced to 18 amino acids, but at 17 amino acids a significant reduction in activity was observed. ESF1, 5, 6, and 12 peptides were inhibitory to the germination of conidia from Cryphonectria parasitica, Fusarium oxysporum f. sp. lycopersici, and Septoria musiva but did not inhibit the germination of pollen from Castanea mollissima and Salix lucida. ESF12 also had no effect on the germination of Malus sylvestris and Lycopersicon esculentum pollen, but inhibited the growth of the bacteria Agrobacterium tumefaciens, Erwinia amylovora, and Pseudomonas syringae. The minimal inhibitory concentrations of the active ESF peptides were similar to those of the naturally occurring control peptides, magainin II and cecropin B. The significant differential in sensitivity between the microbes and plant cells indicated that the active ESF peptides are potentially useful models for designing plant pathogen-resistance genes.

Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization

The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm.

Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh.) Borkh.) somatic embryos

These studies were designed to test if a binary vector containing the gfp, bar and oxalate oxidase genes could transform American chestnut somatic embryos; to see if a desiccation treatment during co-cultivation would affect the transformation frequency of different American chestnut somatic embryo clones; to explore the effects of more rapid desiccation; and to see if the antibiotics used to kill the Agrobacterium were interfering with the regeneration of the somatic embryos. Two days of gradual desiccation was found to significantly enhance transient GFP expression frequency. When this treatment was tested on six American chestnut clones, five were transformed and four of these remained embryogenic. Transformation was confirmed by Southern hybridization. Phenotypically normal transgenic shoots were regenerated and rooted. Vascular tissue specific expression of the oxalate oxidase gene was detected in one transgenic line. Carbenicillin, cefotaxime, and tricarcillin were found to not interfere with the regeneration of transformed embryos.

Development and germination of American chestnut somatic embryos

American chestnut (Castanea dentata (Marsh.) Borkh.) plants were regenerated from developing ovules through somatic embryogenesis. On an initiation medium containing 18.18 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine (BA), 25 out of 1,576 ovules were induced to form proembryogenic masses (PEMs). These PEMs were cultivated on a development medium for 4 weeks. Individual somatic embryos were then grown on a maturation medium for at least one month, until shoot meristems and radicles were developed. Both development and maturation media consisted of Gamborgs B-5 basal medium, 0.5 μM BA, and 0.5 μM α-naphthaleneacetic acid, but the former contained 20 g l−1 sucrose and the later contained 60 g l−1 sucrose. A range of 86 to 586 embryos per gram PEMs was observed beyond the cotyledonary stage. These embryos then germinated, resulting in plantlets with a 3.3% conversion rate. An additional 6.3% of the mature embryos produced shoots, which could also result in plantlets by rooting of microcuttings. Proembryogenic masses that were established in continuous culture and maintained on initiation medium for 17 months retained regenerability, though the embryo yield decreased over time. Twenty plantlets were acclimatized and grown in potting mix in a greenhouse. The largest 6 were transplanted, along with seedling controls, into a nursery bed in 1997. As of July, 1999, 4 out of the 6 were surviving.

Black Cherry (Prunus serotina Ehrh.)

The genus Prunus comprises approximately 400 species of trees and shrubs. Many species and cultivars are grown for their edible fruits, while others are planted for their ornamental value. Approximately 25 species are native to the United States, and of these 18 reach tree size. Black cherry (Prunus serotina Ehrh.) is the only member of this genus with commercial importance as a timber species in the United States (Fowells 1965).

A threshold level of oxalate oxidase transgene expression reduces Cryphonectria parasitica-induced necrosis in a transgenic American chestnut (Castanea dentata) leaf bioassay

American chestnut (Castanea dentata) was transformed with a wheat oxalate oxidase (oxo) gene in an effort to degrade the oxalic acid (OA) secreted by the fungus Cryphonectria parasitica, thus decreasing its virulence. Expression of OxO was examined under two promoters: a strong constitutive promoter, CaMV 35S, and a predominantly vascular promoter, VspB. Oxo gene transcription was quantified by RT-qPCR. Relative expression of OxO varied approximately 200 fold among events produced with the 35S-OxO. The lowest 35S-OxO event expressed approximately 3,000 fold higher than the highest VspB-OxO event. This was potentially due to the tissue-specific nature of the VspB-controlled expression, the strength of the CaMV 35S constitutive promoter, or position effects. Leaf assays measuring necrotic lesion length were conducted to better understand the relationship between OxO expression level and the blight fungus in planta. A threshold response was observed between the OxO expression level and the C. parasitica lesion length. Five events of the 35S-OxO line showed significantly reduced lesion length compared to the blight-susceptible American chestnut. More importantly, the lesion length in these five events was reduced to the same level as the blight-resistant Chinese chestnut, C. mollissima. This is the first report on enhanced pathogen resistance in transgenic American chestnut.

Micropropagation of American chestnut: increasing rooting rate and preventing shoot-tip necrosis

SummaryA three-step medium sequence was developed for rooting microcuttings of American chestnut [Castanea dentata (Marsh.) Borkh.]. First, individual shoots or clumps of shoots were cultivated on shoot-elongation medium for 4–8 wk until shoots were 2–3 cm long. The medium consisted of modified Woody Plant Medium, 500 mg/l polyvinylpyrrolidone (MW 40,000), and 0.89 µM benzyladenine. Microcuttings were then excised, vertically split at the base to approximately 2 mm through the pith, dipped in 5 or 10 mM indolebutyric acid for 1 min, and cultivated on half-strength Murashige and Skoog basal medium plus 0.2 g/l charcoal for 2 wk. During that time, roots were induced and became visible. Finally, the microcuttings were transferred back to shoot-elongation medium and cultivated for 3 wk, allowing growth of both roots and shoots. Using this protocol with 3 genotypes derived from one mature tree and two 1-yr-old seedlings, 57 to 73% rooting was obtained with less than 23% shoot-tip necrosis.

Cloning and characterization of promoters from American chestnut capable of directing reporter gene expression in transgenic Arabidopsis plants

Promoters play a central role in the regulation of gene expression, determining when, where and to what extent a gene is expressed. The aim of this study was to clone promoters from American chestnut capable of acting in wood-forming tissues (in this paper collectively referred to as stems). This hardwood species is besieged by the fungal pathogen, Cryphonectria parasitica, which causes lethal cankers on the stems of susceptible trees; therefore, stem-specific promoters would be useful in driving pathogen resistance transgenes. Using a genome walking technique and primers complementary to cDNA clones (CASdeBBP2, CASdeER10A, and CASdeHyp9A) isolated from a stem expression library, upstream flanking sequences were cloned and verified to be the promoters of the cDNAs from which they were derived. To study their activities, these promoters were subcloned into pCAMBIA vectors upstream of the GUS reporter gene. These recombinant plasmids were used to transform Arabidopsis thaliana, resulting in a total of 17 independent transgenic lines. The promoter of CASdeBBP2 showed consistent activity in the vascular tissues of all organs assayed upon both macroscopic and microscopic observation. The promoter of CASdeHyp9A acted constitutively, while that of CASdeER10A displayed inconsistent activity. The development of these promoters will allow researchers to regulate the expression of pathogen resistance transgenes and may also be useful in further research that focuses on the modification of wood quality in American chestnut and other species.

Molecular characterization of a gene encoding a cystatin expressed in the stems of American chestnut (Castanea dentata)

Abstract. A cDNA clone with similarity to genes encoding cystatin was recently isolated from a cDNA library created using mRNA extracted from stem tissues of Castanea dentata (Marsh.) Borkh. (CASde:Pic1). All of the requisite motifs for inhibitory activity were found upon examination of the deduced amino acid sequence. Reverse transcription–polymerase chain reaction was used to detect the cystatin transcript in healthy stem, leaf and seed tissues, as well as in diseased tissues. Gene fragments encoding this putative cystatin were cloned from American and Chinese (Castanea mollissima Blume) chestnuts and a comparison of these sequences revealed significant differences within the intron, including deletions and alterations in restriction-enzyme sites. The long-term goal of this study is to determine whether the cystatin allele in Chinese chestnut correlates to a resistance gene and, if so, if this allele could be used to enhance resistance in American chestnut.

Predicting within-family variability in juvenile height growth of Salix based upon similarity among parental AFLP fingerprints

Abstract.Willow is being developed as a crop for biomass plantations in the Northeast and North-central United States, but has only recently been the subject of controlled breeding to generate improved genotypes. Maximizing variability among progeny within full-sib families produced by controlled pollination may increase the probability of producing willow clones exhibiting desirable extreme phenotypes. Yet, predicting combinations of parents yielding highly variable progeny is not currently possible. Controlled pollinations were completed among 15 Salix eriocephala clones and the resulting progeny were vegetatively propagated and planted in a greenhouse progeny test. Heights of rooted cuttings were measured after 4 months of growth. Genetic similarity among parents was estimated based on 77 polymorphic AFLP bands. Strong negative correlation (r = –0.88) was detected between mean female-parent similarity indices and the standard deviation of height among half-sib progeny from those females. Parent combinations that had relatively low similarity indices tended to produce progeny that had greater variability in height. This negative relationship suggests that AFLP fingerprints of S. eriocephala parents may be useful for predicting parent combinations that will yield families with large variability.