Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties.

The orchid Gastrodia elata depends on the fungus Armillaria mellea to complete its life cycle. In the interaction, fungal hyphae penetrate older, nutritive corms but not newly formed corms. From these corms, a protein fraction with in vitro activity against plant-pathogenic fungi has previously been purified. Here, the sequence of gastrodianin, the main constituent of the antifungal fraction, is reported. Four isoforms that encoded two different mature proteins were identified at the cDNA level. Another isoform was detected in sequenced peptides. Because the antifungal activity of gastrodianins produced in and purified from Escherichia coli and Nicotiana tabacum was comparable to that of gastrodianin purified from the orchid, gastrodianins are the active component of the antifungal fractions. Gastrodianin accumulation is probably an important part of the mechanism by which the orchid controls Armillaria penetration. Gastrodianin was found to be homologous to monomeric mannose-binding proteins of other orchids, of which at least one (Epipactis helleborine mannose-binding protein) also displayed in vitro antifungal activity. This establishes the gastrodianin-like proteins (GLIPs) as a novel class of antifungal proteins.

Analysis of bruchid resistance in the wild common bean accession G02771: no evidence for insecticidal activity of arcelin 5.

Arcelins are abundant seed storage proteins thought to be implicated in the resistance of wild Phaseolus vulgaris (L.) genotypes against Zabrotes subfasciatus (Boheman), an important storage insect pest of common bean. Here, the insecticidal activity of the arcelin-5 variant that is present in the highly resistant P. vulgaris accession G02771 was investigated. No correlation could be established between the presence of arcelin 5 and the insecticidal effects observed in G02771 seeds. Insect feeding assays with artificial seeds into which purified arcelin-5 protein was incorporated and with transgenic P. acutifolius (A. Gray) seeds in which the arcelin-5 genes were expressed, showed that the presence of arcelin-5 proteins, even at elevated levels, was not sufficient to achieve adequate resistance against Z. subfasciatus. The same might apply to other arcelin variants. Nevertheless, as resistance is clearly closely linked to the presence of the arcelin-1 or arcelin-5 locus, arcelins remain useful markers in breeding programmes aimed at introgressing high levels of resistance to Z. subfasciatus in P. vulgaris cultivars.

The arcelin-5 gene of Phaseolus vulgaris directs high seed-specific expression in transgenic Phaseolus acutifolius and Arabidopsis plants.

The regulatory sequences of many genes encoding seed storage proteins have been used to drive seed-specific expression of a variety of proteins in transgenic plants. Because the levels at which these transgene-derived proteins accumulate are generally quite low, we investigated the utility of the arcelin-5 regulatory sequences in obtaining high seed-specific expression in transgenic plants. Arcelin-5 is an abundant seed protein found in some wild common bean (Phaseolus vulgaris L.) genotypes. Seeds of Arabidopsis and Tepary bean (Phaseolus acutifolius A. Gray) plants transformed with arcelin-5 gene constructs synthesized arcelin-5 to levels of 15% and 25% of the total protein content, respectively. To our knowledge, such high expression levels directed by a transgene have not been reported before. The transgenic plants also showed low plant-to-plant variation in arcelin expression. Complex transgene integration patterns, which often result in gene silencing effects, were not associated with reduced arcelin-5 expression. High transgene expression was the result of high mRNA steady-state levels and was restricted to seeds. This indicates that all requirements for high seed-specific expression are cis elements present in the cloned genomic arcelin-5 sequence and trans-acting factors that are available in Arabidopsis and Phaseolus spp., and thus probably in most dicotyledonous plants.

Plant regeneration from embryo-derived callus in Phaseolus vulgaris L. (common bean) and P. acutifolius A. Gray (tepary bean).

Regeneration-competent callus of Phaseolus vulgaris and P. acutifolius was obtained from mature embryo explants on a medium containing thidiazuron and indole-3-acetic acid. For the P. vulgaris genotype Xan-159, regeneration was achieved from cotyledon explants, but not from embryonic axis explants. Both explants could be used for the P. acutifolius genotype NI 574 but embryonic axes gave the best results. In-vitro-rooted plantlets of P. acutifolius could readily be established in the greenhouse. For P. vulgaris hardening problems with in-vitro-rooted plantlets could be overcome by means of in vitro grafting. The potential of the described procedure for obtaining transgenic P. vulgaris plants is discussed.

Shoot regeneration in long-term callus cultures derived from mature flowering plants of Cyclamen persicum Mill.

In long-term callus cultures of Cyclamen persicum Mill. two types of tissue could be distinguished. One type featured a brown suberised outer layer and was poorly organogenic. The other type was yellowish in appearance and gave rise to many shoot buds. Both types co-existed on the same callus, the former prevailing. Selection for organogenic tissue resulted in cultures yielding approximately three times more petioles than random subcultures. Callus-derived shoots could be rooted and established in the greenhouse. The method allowed for the production of thousands of plants but the regenerants often showed deviant phenotypes and genotypes.

Electroporation-mediated DNA delivery to seedling tissues ofPhaseolus vulgaris L. (common bean).

DNA was delivered to intact embryonic axes of the legumePhaseolus vulgaris L. through electroporation. Expression of the ß-glucuronidase reporter gene was observed in hypocotyl and epicotyl tissue in a spot-like manner. Transgene expression was high when a single pulse of 260 ms at a field strength of 225 V·cm(-1) was applied but could be achieved within a wide range of electrical conditions. Linearization of plasmid DNA greatly enhanced transient expression levels. The procedure was successful for embryonic axes of all testedP. vulgaris cultivars, for similar explants of several large-seeded leguminous species, as well as for some other tissues ofP. vulgaris.