Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The information generated during this study could be valuable for application in breeding programs aimed at enhancingsoybean innate resistance. Soybean breeders will use the novel transgenic lines produced in this project to develop new soybean cultivars for soybean producers with increased yield stability, due to reduced infection, avoiding negative effects on environment and beneficial microorganisms caused by application of chemical fungicides and nematicides. Soybean producers will ultimately benefit from the release of soybean cultivars with increased disease resistance that can stabilize soybean production in the presence of high disease levels. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The research carried out helped to establish collaboration between individuals who worked on this project: Lozovaya V., Lygin A., Zernova O., Widholm J., (Dept. Crop Sciences, UIUC), G.L. Hartman (National Soybean Research Center, USDA-ARS, UIUC), and C. Hill (National Soybean Research Center, UIUC) and provided a good training basis for graduate and undergraduatestudents. How have the results been disseminated to communities of interest? Results and progress of studies were included in published paper and in manuscripts which are currently in preparation, quarterly reported to the United Soybean Board, and presented at seminars and at the CPBR Symposium (Washington DC, March 5-6, 2013) with the goal to leverage currentfunding from combined CPBR/DOE and industry program. What do you plan to do during the next reporting period to accomplish the goals? Determine genetic segregation of transgenes in at least two transgenic lines transformed with the transgenes (AhRS3 - Resveratrol synthase 3 gene from peanut; ROMT - Resveratrol o-methyltransferase from Vitis vinifera; G4DT - Pterocarpan 4-dimethylallyltransferase from soybean), which control phytoalexin synthesis (native – glyceollin and non-native - resveratrol and pterostilbene). Test detached leaves from transformed soybean lines expressing targeted transgenes with Macrophomina phaseolina, causing charcoal rot; Rhizoctonia solani, causing Rhizoctonia damping off and root rot; andSclerotinia sclerotiorum, causing Sclerotinia stem rot; to validate the efficacy of phytoalexin manipulation on controlling pathogen colonization. Study metabolic and functional interaction between the production of native and non-native phytoalexins in transgenic soybean.
Impacts
What was accomplished under these goals?
Over 30 transgenic T0 plants were produced during the reporting period from selected hygromycin resistant soybean clones, generated following the previously reported DNA bombardments with different plasmids or mixture of cassettes, containing transgenes encoding formation of phytoalexins. PCR tests showed the presence of the G4DT gene (regulates glyceollin synthesis) driven by lectin promoter in 13 transgenic plants of two independent lines. We showed that eight plants of one transgenic line contain AhRS3 and ROMT genes both under CsVMV promoter and four plants of one line contain these genes under actin or CsVMV promoter respectively. Eight of newly generated putative transgenic plants were not PCR analyzed yet. We carried out the gene segregation test by planting all seeds from five T0 plants of lines transformed with the G4DT (pterocarpan 4- dimethylallytransferase) gene under soybean lectin promoter and from six T0 plants of line transformed with the AhRS3 (peanut resveratrol synthase) and the ROMT (resveratrol o-methyltransferase from Vitis vinifera) genes, both under CsVMV promoter. We have collected T2 seeds fromthree lines expressing the AhRS3 gene under actin promoter, one line expressing two target transgenes - the AhRS3 gene under actin promoter and the ROMT (resveratrol o-methyltransferase from Vitis vinifera) under CsVMV promoter, one line expressing the AhRS3 gene under actin promoter and the G4DT (pterocarpan 4- dimethylallytransferase) under CsVMV promoter and also one line with the G4DT gene under lectin promoter. The PCR gene segregation analysis of T2 plants of one line, transformed with actin/AhRS3and CsVMV/ROMT, as well as of another line transformed with actin/AhRS3 and CsVMV/G4DT, showed that these lines were not homozygous in this generation. PCR positive plants will be analyzed in future using next generation. PCR analysis of newly established hairy root lines, transformed with the G4DT gene (which controls glyceollin synthesis) under CsVMV promoter was carried out and confirmed the presence of this gene in selected lines. Treatment of these hairy roots with HgCl2 resulted in increased accumulation of glyceollin in some transformed lines compared to lines without the G4DT transgene, indicating that transformed hairy roots have increased capacity to produce native phytoalexin glyceollin in response to abiotic stress and thus, G4DT transgene could be the appropriate target for plant transformation aimed at enhancing plant innate immunity. We started biochemical analysis of hairy roots transformed with the AhRS3 and the ROMT genes to see if phytoalexins that are normally not present in soybean could be found in these transformed roots. We showed that 10 of 17 lines tested accumulated resveratrol and 4 lines accumulated the resveratrol derivative pterostilbene. Isoflavone concentration in pterostilbene accumulated lines was less compared to other lines tested. Treatment with HgCl2 showed that accumulation of non-native phytoalexins does not affect considerably the induction of native phytoalexin glyceollin in transformed roots compared to untransformed roots in response to this abiotic stress. We have repeated experiments on resveratrol and pterostilbene effects on growth of important soybean fungal pathogens and found that they strongly suppress the growth of Sclerotinia sclerotiorum, Macrophomina phaseolina and Rhizoctonia solani when present in growth medium. Pterostilbene reduced fungal growth at concentrations several-fold lower than that of resveratrol, indicating this resveratrol derivative had high potential to contribute to increased innate resistance. A poster summarizing these results was presented at the North Central Region APS meeting in Manhattan, KS. We carried out the extensive analyses of the chemical products which were present in resveratrol amended liquid medium over time during fungal incubation to identify enzymatic activities used by fungi to degrade/metabolize resveratrol to make it less toxic for their development. Several products of resveratrol metabolization by fungi Sclerotinia sclerotiorum (Sclerotinia stem rot), Rhizoctonia solani (Rs, damping off and root rot) and Macrophomina phaseolina (Mp, charcoal rot) were identified using liquid chromatography-mass spectrometry (LC-MS). p-Hydroxybenzaldehyde, p-hydroxybenzoic acid, 3,5-dihydroxybenzaldehyde, 3,5-dihydroxybenzoic acid and two dimers of resveratrol ([M-H]- m/z 453) were identified in medium amended with resveratrol of all three fungi. We also found 3,4-dihydroxybenzoic acid and 3,4,3’,5’-tetrahydroxy stilbene ([M+H]+ m/z 245), which is the product of resveratrol hydroxylation, and one oxidized dimer of resveratrol ([M-H]- m/z 471) in the Ss and Mp medium with resveratrol, whereas Rs medium lacks of these products of resveratrol degradation. Three experiments were completed to study the susceptibility of soybean hairy roots transformed with genes for expression of resveratrol and pterostilbene synthesis in comparison with control hairy roots not expressing these foreign phytoalexins to Rhizoctonia solani. We measured percent of necrosis on 2 cm segments of the hairy roots tested 48 hours after inoculation with R. solani. These hairy root tests clearly indicated that expression of both non-native phytoalexins decreased root colonization of the fungus. The expression of pterostilbene synthesis resulted in dramatically improved soybean root resistance in all tests since the level of necrosis caused by R. solani was only 3-10% of the control roots. We also started testing the response of transformed hairy roots accumulated stilbenoids to Macrophomina phaseolina using the same assay and found that roots accumulating pterostilbene had only 35% of the necrosis of control roots. Experiments were recently initiated that tested the effects of different levels of glyceollin, resveratrol and pterostilbene on soybean cyst nematode development on inoculated soybean hairy roots. Preliminary results suggested that the number of nematode cysts was several-fold less on transformed soybean hairy roots expressing resveratrol and pterostilbene, and on hairy roots grown on resveratrol and pterostilbene amended medium, than on un-transformed hairy root controls without transgenes grown on regular medium. Hairy roots with a transgene that suppressed glyceollin synthesis had higher numbers of nematode cysts compared to hairy root controls without any transgenes. We also evaluated the effect of resveratrol and pterostilbene on cyst nematode development and found that number of cysts that formed on soybean hairy roots (cv. Spencer) grown on the phytoalexin amended medium 30 days after inoculation was about 8-fold less compared to that grown on medium without phytoalexins. Results of these tests described above demonstrated that molecular engineering of soybean to enable plants to synthesize non-native phytoalexins has high potential to increase broad spectrum and durable innate immunity. This approach to improving innate soybean defense against diseases and pests through genetic engineering is novel and to the best of our knowledge, it is not being used by any other organizations, including major soybean biotech seed producers.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Lygin AV, Zernova OV, Hill CB, Kholina NA, Widholm JM, Hartman GL, and Lozovaya VV. Glyceollin is an important component of soybean plant defense against Phytophthora sojae and Macrophomina phaseolina. Phytopathology 103: 984-994
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