Regulation of plant immunity through modulation of phytoalexin synthesis.

Soybean hairy roots transformed with the resveratrol synthase and resveratrol oxymethyl transferase genes driven by constitutive Arabidopsis actin and CsVMV promoters were characterized. Transformed hairy roots accumulated glycoside conjugates of the stilbenic compound resveratrol and the related compound pterostilbene, which are normally not synthesized by soybean plants. Expression of the non-native stilbenic phytoalexin synthesis in soybean hairy roots increased their resistance to the soybean pathogen Rhizoctonia solani. The expression of the AhRS3 gene resulted in 20% to 50% decreased root necrosis compared to that of untransformed hairy roots. The expression of two genes, the AhRS3 and ROMT, required for pterostilbene synthesis in soybean, resulted in significantly lower root necrosis (ranging from 0% to 7%) in transgenic roots than in untransformed hairy roots that had about 84% necrosis. Overexpression of the soybean prenyltransferase (dimethylallyltransferase) G4DT gene in soybean hairy roots increased accumulation of the native phytoalexin glyceollin resulting in decreased root necrosis.

Inhibitory effects of stilbenes on the growth of three soybean pathogens in culture.

The effects of resveratrol and pterostilbene on in vitro growth of three soybean pathogens were tested to determine whether these stilbenic compounds could potentially be targets to increase innate resistance in transgenic soybean plants. Growth of Macrophomina phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum was measured on solid and in liquid media amended with resveratrol and pterostilbene (concentration in the media of resveratrol at 100 µg/ml and pterostilbene at 25 µg/ml). All three fungi were very sensitive to pterostilbene in potato dextrose agar (PDA), which reduced colony area of each of the three pathogens to less than half of the control 3 days after incubation. The three fungal pathogens were less sensitive to resveratrol compared with pterostilbene; however, area under the curve (AUC) calculated from colony areas measured over 3 days was significantly (P < 0.05) less than the control for S. sclerotiorum and R. solani on PDA with resveratrol or pterostilbene. AUC for M. phaseolina on PDA with pterostilbene was significantly (P < 0.05) lower than the control whereas, on PDA with resveratrol, AUC for M. phaseolina was lower than the control but the difference was nonsignificant (P > 0.05). AUC for all three fungi was significantly lower (P < 0.05) on PDA with pterostilbene than with resveratrol. In potato dextrose broth (PDB) shake cultures, AUC for all three fungi was significantly (P < 0.01) lower in pterostilbene than in the control. AUC for R. solani and S. sclerotiorum was significantly lower (P < 0.01) in resveratrol than the control, whereas AUC for M. phaseolina in resveratrol was lower, but not significantly (P > 0.05) different from the control. AUC in pterostilbene was highly significantly (P < 0.01) lower than in resveratrol for M. phaseolina and significantly (P < 0.05) lower for R. solani but the difference for S. sclerotiorum was nonsignificant (P > 0.05). There was a trend for lower mass accumulation of all three fungi in either pterostilbene or resveratrol compared with the control during the course of the experiment; however, S. sclerotiorum appeared to recover from the effects of pterostilbene between days 2 and 4. Results of biochemical analyses of the PDB over time indicated that the three fungi degraded resveratrol, with nearly 75% reduction in concentration in M. phaseolina, 80% in S. sclerotiorum, and 60% in R. solani PDB cultures by day 4 of fungal growth. M. phaseolina and S. sclerotiorum were able to resume growth after early inhibition by resveratrol after its concentration was reduced in the cultures through degradation, whereas R. solani was less efficient in resveratrol degradation and was not able to overcome its inhibitory effects on growth. The capacity to degrade pterostilbene was lowest in M. phaseolina compared with S. sclerotiorum and R. solani and the recovery of M. phaseolina cultures after initial growth inhibition by pterostilbene was minimal. The potential products of resveratrol and pterostilbene degradation by fungi were identified to be dimers and various oxidation products.

Glyceollin is an important component of soybean plant defense against Phytophthora sojae and Macrophomina phaseolina.

The response of soybean transgenic plants, with suppressed synthesis of isoflavones, and nontransgenic plants to two common soybean pathogens, Macrophomina phaseolina and Phytophthora sojae, was studied. Transgenic soybean plants of one line used in this study were previously generated via bombardment of embryogenic cultures with the phenylalanine ammonia lyase, chalcone synthase, and isoflavone synthase (IFS2) genes in sense orientation driven by the cotyledon-preferable lectin promoter (to turn genes on in cotyledons), while plants of another line were newly produced using the IFS2 gene in sense orientation driven by the Cassava vein mosaic virus constitutive promoter (to turn genes on in all plant parts). Nearly complete inhibition of isoflavone synthesis was found in the cotyledons of young seedlings of transgenic plants transformed with the IFS2 transgene driven by the cotyledon-preferable lectin promoter compared with the untransformed control during the 10-day observation period, with the precursors of isoflavone synthesis being accumulated in the cotyledons of transgenic plants. These results indicated that the lectin promoter could be active not only during seed development but also during seed germination. Downregulation of isoflavone synthesis only in the seed or in the whole soybean plant caused a strong inhibition of the pathogen-inducible glyceollin in cotyledons after inoculation with P. sojae, which resulted in increased susceptibility of the cotyledons of both transgenic lines to this pathogen compared with inoculated cotyledons of untransformed plants. When stems were inoculated with M. phaseolina, suppression of glyceollin synthesis was found only in stems of transgenic plants expressing the transgene driven by a constitutive promoter, which developed more severe infection. These results provide further evidence that rapid glyceollin accumulation during infection contributes to the innate soybean defense response.

Response of soybean pathogens to glyceollin.

Plants recognize invading pathogens and respond biochemically to prevent invasion or inhibit colonization in plant cells. Enhancing this response in crop plants could improve sustainable methods to manage plant diseases. To enhance disease resistance in soybean, the soybean phytoalexin glyceollin was assessed in soybean hairy roots of two soybean genotypes, Spencer and PI 567374, transformed with either soybean isoflavone synthase (IFS2) or chalcone synthase (CHS6) genes that were inoculated with the soybean pathogens Diaporthe phaseolorum var. meridionales, Macrophomina phaseolina, Sclerotinia sclerotiorum, and Phytophthora sojae. The hairy-root-transformed lines had several-fold decreased levels of isoflavone daidzein, the precursor of glyceollin, and considerably lower concentrations of glyceollin induced by pathogens measured 5 days after fungal inoculation compared with the nontransformed controls without phenolic transgenes. M. phaseolina, P. sojae, and S. sclerotiorum grew much more on IFS2- and CHS6-transformed roots than on control roots, although there was no significant difference in growth of D. phaseolorum var. meridionales on the transformed hairy-root lines. In addition, glyceollin concentration was lower in D. phaseolorum var. meridionales-inoculated transformed and control roots than roots inoculated with the other pathogens. Glyceollin inhibited the growth of D. phaseolorum var. meridionales, M. phaseolina, P. sojae, S. sclerotiorum, and three additional soybean pathogens: Cercospora sojina, Phialophora gregata, and Rhizoctonia solani. The most common product of glyceollin conversion or degradation by the pathogens, with the exception of P. sojae, which had no glyceollin degradation products found in the culture medium, was 7-hydroxyglyceollin.

Modification of isoflavones in soybean seeds via expression of multiple phenolic biosynthetic genes.

To modify the level and composition of isoflavones, the important bioactive constituents of soybean seeds, soybean was transformed via co-bombardment of embryogenic cultures with three DNA cassettes containing the CHS6-chalcone synthase and IFS2-isoflavone synthase genes, and a fragment of PAL5-phenylalanine ammonia-lyase gene, all in sense orientation under the lectin promoter mixed with the selectable marker gene, HPT (hygromycin phosphotransferase) under the 35S promoter. Four of six fertile lines produced integrated all four genes. Isoflavone levels were lower in T1 mature seeds of 5 of the 6 lines compared to the control. Transgene segregation was found in one selected line, with formation of additional sublines with different transgene composition found also in the homozygous plants. Decreased isoflavone concentrations (by about 70%) were found in T4 homozygous seeds of the two lines studied in detail here. The embryo axes accumulated most of the glycitein and contained a higher isoflavone concentration than the cotyledons. Expression of transgenes driven by the lectin promoter reduced the isoflavone concentration only in the cotyledons and not in embryo axes, indicating that this promoter is preferably active in cotyledons.

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase.

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen.

Isoflavonoid accumulation in soybean hairy roots upon treatment with Fusarium solani.

Hairy roots were initiated from two soybean [Glycine max (L.) Merr.] genotypes with different susceptibility (susceptible 'Spencer' and partially resistant 'PI567.374') to the disease sudden death syndrome (SDS) caused by the soil-borne fungal pathogen Fusarium solani f. sp. glycines (FSG) to study the role of isoflavonoids in the plant response to FSG infection. Hairy root cultures obtained by transformation with Agrobacterium rhizogenes allows normal root growth that can be visually monitored. The principal isoflavones (genistin, daidzin, glycitin and their malonyl conjugates and aglycones) and also isoflavonoid phytoalexins (coumestrol and glyceollin) were measured by HPLC in extracts of the FSG-inoculated and non-inoculated hairy roots. FSG mycelia grew more slowly on inoculated PI567.374 hairy roots than on Spencer hairy roots. The glyceollin content was higher in FSG-inoculated PI567.374 hairy roots than in Spencer hairy roots even though the glyceollin precursor, the isoflavone daidzein, was higher in Spencer. The de novo synthesis of isoflavones and glyceollin was confirmed by [(14)C]Phe incorporation into glyceollin, which was higher both in the FSG-inoculated roots and surrounding medium of the cv. PI567.374 than that of Spencer. Glyceollin was the most inhibitory to FSG growth among eight isoflavonoids tested. The levels of coumestrol, a putative phytoalexin, did not change upon FSG inoculation. The defense response was also elicited by FSG culture filtrates in hairy roots grown in liquid culture. The data obtained indicate that the ability of soybean roots to rapidly produce sufficient amounts of glyceollin in response to FSG infection might be important in providing partial resistance to this fungus.