Bioengineering potato plants to produce benzylglucosinolate for improved broad-spectrum pest and disease resistance.

In traditional, small-scale agriculture in the Andes, potatoes are frequently co-cultivated with the Andean edible tuber Tropaeolum tuberosum, commonly known as mashua, which is believed to exert a pest and disease protective role due to its content of the phenylalanine-derived benzylglucosinolate (BGLS). We bioengineered the production of BGLS in potato by consecutive generation of stable transgenic events with two polycistronic constructs encoding for expression of six BGLS biosynthetic genes from Arabidopsis thaliana. First, we integrated a polycistronic construct coding for the last three genes of the pathway (SUR1, UGT74B1 and SOT16) into potato driven by the cauliflower mosaic virus 35S promoter. After identifying the single-insertion transgenic event with the highest transgene expression, we stacked a second polycistronic construct coding for the first three genes in the pathway (CYP79A2, CYP83B1 and GGP1) driven by the leaf-specific promoter of the rubisco small subunit from chrysanthemum. We obtained transgenic events producing as high as 5.18 pmol BGLS/mg fresh weight compared to the non-transgenic potato plant producing undetectable levels of BGLS. Preliminary bioassays suggest a possible activity against Phytophthora infestans, causing the late blight disease and Premnotrypes suturicallus, referred to as the Andean potato weevil. However, we observed altered leaf morphology, abnormally thick and curlier leaves, reduced growth and tuber production in five out of ten selected transgenic events, which indicates that the expression of BGLS biosynthetic genes has an undesirable impact on the potato. Optimization of the expression of the BGLS biosynthetic pathway in potato is required to avoid alterations of plant development.

Partial purification of a GTP-insensitive (1-->3)-beta-glucan synthase from Phytophthora sojae.

A (1 --> 3)-beta-glucan synthase activity was identified in cell membrane preparations from the oomycete Phytophthora sojae, a soybean pathogen. The activity could be solubilized using the zwitterionic detergent CHAPS at relatively low concentrations (3 mg/ml). High salt concentrations were not effective in removing the activity from the membranes. Detergent solubilization of the enzyme resulted in a six-fold increase of calculated Vmax values (2.5 vs. 0.4 nkat/mg protein) but only minor alteration of the Km (10.6 vs. 10.7 mM). Analysis of the reaction product of the solubilized enzyme by enzymatic degradation and by 2D NMR spectroscopy confirmed its identity as a linear high molecular weight (1 --> 3)-beta-glucan. Glucan synthase activity in both membrane and solubilized preparations was not activated by GTP or divalent cations as reported for other fungal or plant glucan synthases, The activity was inhibited, as expected, in a competitive manner by UDP with a Ki of 2.9 mM. Partial purification of the enzyme was achieved by anion exchange chromatography followed by product entrapment. This procedure resulted in the selective enrichment of a protein band with apparent Mr 108,000 in SDS-PAGE which was not visible in any of the steps preceding product entrapment. The glucan pellets from product entrapment contained up to 3% of the initial enzyme activity present in the fraction used for the procedure.

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.

Release of highly elicitor-active glucans by germinating zoospores of Phytophthora megasperma f. sp. glycinea.

An in-vitro culture system allowing the simultaneous germination of cysts was used to study the early host-independent release of phytoalexin elicitors by Phytophthora megasperma f. sp. glycinea, a soybean pathogen. Significant elicitor activity could be detected in the culture medium as early as 2 h after germination of P.m. f. sp. glycinea, race 1, cysts. The phytoalexin elicitor was heat-stable and heterogeneous in size. The apparent molecular mass ranged from 3 to 80 kDa. Anion exchange and lectin-affinity chromatography followed by sugar analysis confirmed that the elicitor activity resided primarily in glucans. The time course of elicitor release could then be accurately monitored by means of a competitive radioligand-displacement assay using the ß-glucan elicitor-binding sites of soybean (Glycine max (L.) Merr.) membranes. Linkage-composition analysis of the glucan elicitors showed that they were primarily (1 → 3)ß-linked with (1 → 6)-ß-branches, a composition similar to that of glucans obtained by heat release from mature mycelium but different from that of elicitors obtained by acid hydrolysis or from spontaneous autohydrolytic release by senescent cultures. The naturally released elicitors displayed a biological activity in soybean cotyledon bioassays higher than purified acid-hydrolysed glucan elicitor or than the hepta-(1 → 3, 1 → 6)-ß-glucoside, the smallest known carbohydrate elicitor for soybean. The present results demonstrate that elicitor release from the pathogen and perception by the potential host can take place in this system as early as during germ-tube formation and independent of the presence of host-produced endoglucanases.

High-affinity binding of a synthetic heptaglucoside and fungal glucan phytoalexin elicitors to soybean membranes.

Soybean membranes possess high-affinity binding sites for fungal beta-glucans that elicit phytoalexin synthesis. The ability of 1,3-1,6-beta-glucans, released by acid hydrolysis from mycelial walls of Phytophthora megasperma f.sp. glycinea, to compete for the putative phytoalexin elicitor receptors increases with their average degree of polymerization (DP). The results suggest a function where the probability for glucan fragments of containing a structural determinant that is optimal for binding approaches 1 as the DP tends to infinity. Ligand displacement data obtained against a 125I-labeled glucan elicitor (average DP = 18) provided a theoretical minimum IC50 (50% inhibitory concentration) for 1,3-1,6-beta-glucans of 3 nM. The IC50 value obtained for a synthetic hepta-beta-glucoside having a known elicitor-active structure was 8 nM, remarkably close to the predicted value. Displacement of the 125I-glucan of large DP was uniform and complete showing that the heptaglucoside had access, with similar affinity, to all sites available to the radioligand. Further analysis using a 125I-labeled aminophenethylamine derivative of the heptaglucoside suggested that the putative glucan-elicitor receptors bind a basic structural determinant present in all elicitor-active glucans from the soybean pathogen P. megasperma.

Solubilization of soybean membrane binding sites for fungal beta-glucans that elicit phytoalexin accumulation.

Soybean membranes contain high-affinity binding sites for fungal beta-glucans. These sites may play a role in the recognition by soybean tissues of fungal phytoalexin elicitors. We have solubilized beta-glucan-binding activity from microsomal membranes using two C12-alkyl zwitterionic detergents, Zwittergent 3-12 (ZW 3-12) and the lysolecithin analog 1-dodecanoyl propanediol-3-phosphorylcholine [corrected] (ES12H). The solubilized binding sites displayed identical affinity for beta-glucans as that found in membranes (KD = 11-34 nM). Detergent-protein micelles with glucan binding activity eluted with approximate Mr values of 300,000 in ZW 3-12 and 380,000 in ES12H in gel permeation chromatography. Maximal binding activity eluted from a chromatofocusing column in the pH range between 6.2 and 6.6 with both ES12H and ZW 3-12, suggesting an apparent pI close to neutral.

High-affinity binding of fungal beta-glucan fragments to soybean (Glycine max L.) microsomal fractions and protoplasts.

We have recently reported the existence of binding sites in soybean membranes for a beta-glucan fraction derived from the fungal pathogen Phytophthora megasperma f. sp. glycinea, which may play a role in the elicitor-mediated phytoalexin response of this plant [Schmidt, W. E. & Ebel, J. (1987) Proc. Natl Acad. Sci. USA 84, 4117-4121]. The specificity of beta-glucan binding to soybean membranes has now been investigated using a variety of competing polyglucans and oligoglucans of fungal origin. P. megasperma beta-glucan binding showed high apparent affinity for branched glucans with degrees of polymerization greater than 12. Binding affinity showed good correlation with elicitor activity as measured in a soybean cotyledon bioassay. Modification of the glucans at the reducing end with phenylalkylamine reagents had no effect on binding affinity. This characteristic was used to synthesize an oligoglucosyl tyramine derivative suitable for radioiodination. The 125I-glucan (15-30 Ci/mmol) provided higher sensitivity and lower detection limits for the binding assays while behaving in a manner identical to the [3H]glucan used previously. More accurate determinations of the Kd value for glucan binding indicated a higher affinity than previously shown (37 nM versus 200 nM). The 125I-glucan was used to provide the first reported evidence of specific binding of a fungal beta-glucan fraction in vivo to soybean protoplasts. The binding affinity to protoplasts proved identical to that found in microsomal fractions.

Acifluorfen-induced isoflavonoids and enzymes of their biosynthesis in mature soybean leaves : whole leaf and mesophyll responses.

Mature soybean (Glycine max L. cv Harosoy 63) leaves normally contain kaempferol-3-glycosides but they accumulate no other flavonoids. Whole leaves sprayed with the diphenyl ether herbicide Acifluorfen and maintained in the light developed small necrotic lesions and accumulated isoflavone aglycones, isoflavone glucosides, and pterocarpans. Isoflavonoid accumulation was preceded by induced activity for chalcone synthase (CHS) and by increased activity for phenylalanine ammonia-lyase (PAL) and UDP-glucose:isoflavone 7-O-glucosyl transferase (IGT). PAL and CHS activity was highest between 24 and 30 hours after treatment, isoflavone aglycones and pterocarpans at 48 hours, IGT at 72 hours, and isoflavone glucosides at 96 hours.Mesophyll cells isolated from control leaves contained no activity for PAL, CHS, or IGT and no flavonoids of any class. Cells isolated from treated leaves at the stage of maximal enzyme activity or isoflavonoid content contained PAL (12% of the whole leaf activity), CHS (24%), IGT (20%), and 25% of the whole leaf isoflavone glucosides, but only traces, presumably as contaminants, of the other flavonoids. We suggest that the isoflavone glucosides were synthesized and accumulated in intact mesophyll cells as soluble detoxification products, while the isoflavone aglycones and pterocarpans accumulated in the epidermis or extracellularly within the mesophyll. To our knowledge this is the first report of tissue-specific induction of isoflavonoid glucosides and key enzymes of their biosynthesis in any plant.

Kaempferol glycosides and enzymes of flavonol biosynthesis in leaves of a soybean strain with low photosynthetic rates.

Soybean (Glycine max L.) strains which accumulate kaempferol 3-(2(G)-glucosylgentiobioside) in their leaves fix CO(2) at rates significantly lower than those lacking this compound (Buttery, Buzzell 1976 Crop Sci 16: 547-550), and kaempferol aglycone is a well known inhibitor of photosynthesis in vitro. However, since neither kaempferol nor any of its glycosides could be detected in mesophyll cells isolated from mature soybean leaves we suspect that kaempferol 3-(2(G)-glucosylgentiobioside) has no direct inhibitory effect on photosynthesis. The most rapid stage of flavonoid accumulation, and the highest level of activity for several enzymes of phenolic biosynthesis, occurs in leaflets 2.5 to 3 centimeters long. Mesophyll cells isolated from these leaflets contain about 70% of the whole leaf activity for shikimate dehydrogenase, 24% of the 4-coumarate:CoA ligase activity, 35% of the activity for chalcone-flavanone isomerase, but no demonstrable activity for phenylalanine ammonialyase. Our results suggest a highly tissue-specific pattern of secondary phenolic metabolism in soybean leaves.