Progress 02/01/11 to 01/31/17
Outputs
Target Audience:The scientific community, bioenergy energy sector, sorghum industry (producers and private ag. companies) were target audiences reached. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Four undergraduate students annually and one graduate student have been trained in scientific techniques and experimental design through experiments designed and funded by this project. A post-doc continued her scientific training through her work on Objectives 4 and 5. She was successfully mentored and trained, which led to placement as a Category 1 ARS Research Molecular Biologist. The scientific meetings attended by the PD and Co-PIs provided opportunities for professional development and scientific networking. How have the results been disseminated to communities of interest?The results from this project have been presented at national and regional scientific meetings as either poster or research talks. As studies have completed, the results were published in peer-reviewed journals (see products). Funnell-Harris D, O'Neill PM, Sattler SE, Gries TL. Response of sorghum stalk pathogens to brown midrib plants and soluble phenolic extracts from near isogenic lines 2017 American Phytopathological Society Annual meeting, San Antonio, Texas, August 5-9, 2017. Tetreault HM, Scully ED, Clemente TE, Sattler SE. Modifying lignin composition and content of sorghum biomass for improved bioenergy conversion. Genome to Phenome 2017 Symposium, Lincoln, Nebraksa 68508, April 6-7, 2017. Scully ED, Palmer NA, Sarath G, Funnell-Harris D, Clemente TE, Sattler SE. Overexpression of SbMyb60 in Sorghum bicolor alters secondary cell wall composition and impacts transcriptional expression of both primary and secondary metabolic pathways. Plant and Animal Genome Conference 2017; San Diego, CA; January 14-18 2017. Tetreault HM, Scully ED, Clemente TE, Sattler SE. Modifying lignin composition and content of sorghum biomass for improved bioenergy conversion. International Plant & Animal Genome (PAG) Conference XXV, San Diego, CA. January 14-18, 2017. Sattler SE. Changing sorghum lignin for bioenergy uses. Center for Sorghum Improvement, Kansas State University, Manhattan, KS July 11, 2016. Sattler SE, Funnell-Harris D, Clemente TE, Scully ED. Modifying Lignin Content and Composition to Improve Sorghum for Bioenergy. 5th Pan-American Congress on Plants and Bioenergy 2016, Santa Fe, NM, August 4-7, 2016 Dowd P, Clemente TE, Sattler SE. Effects of overexpressing individual lignin biosynthetic enzymes on feeding and growth of corn earworms (Helicoverpa zea) and fall armyworms (Spodoptera frugiperda). International Congress of Entomology 2016, Orlando, FL, September 25-30, 2016 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This research addressed whether modifying lignin content and composition in sorghum increases its vulnerability to fungal pathogens or insect pests. A series of 20 brown midrib (bmr) sorghum mutants were characterized by PD Sattler to test the effects of reduced lignin content and altered lignin composition on insect pests and fungal pathogens. Eleven transgenic constructs were developed to overexpress genes involved in lignin synthesis by co-PI Clemente to test the effects of increased lignin or related compounds on sorghum insect pests and fungal pathogens. Three sorghum insect pests: a sucking-insect greenbugs (an aphid), and two chewing-insects fall armyworms and corn earworms were tested by co-PIs Huang and Dowd on these lignin-modified sorghum lines. Likewise, three sorghum fungal pathogens that cause the diseases stalk rot, charcoal rot and anthracnose, were introduced by co-PIs Funnell-Harris and Prom to these lignin-modified sorghum lines. Overall, the lignin-modified lines were not more vulnerable to these insect pests or fungal pathogens than normal plants. In some instances, specific tissues of a few lignin-modified lines showed increased resistance to specific pests or pathogens when compared to normal sorghum plants. In cases where lignin-modified plants were more resistant, plant compounds were analyzed to identify specific compounds linked to the increased resistance to the pest. In bmr sorghum stalk, a phenolic compound related to lignin synthesis was identified by co-PI Dowd, which may be a defense compound against corn earworms and fall army worms. Likewise, gene expression was analyzed to identify genes linked to plant defenses against these pathogens and insects. PD Sattler identified a regulatory protein which induced lignin biosynthesis and turned on the expression of approximately 5,000 genes. Overall, the modification of lignin biosynthesis did not significantly impair the ability of sorghum to defend itself from the fungal pathogens or insect pests tested. In fact, there were a few instances of increased resistance observed in sorghum plants where lignin synthesis was changed by a bmr mutation or transgenic overexpression of a gene involved in lignin synthesis. Based on these data, lignin modification appears to be a relatively low risk for increasing the vulnerability of bioenergy feedstock plants to insect pests or pathogens. Specific Objectives 1. To develop transgenic lines overexpressing phenylpropanoid biosynthetic enzymes. To increase carbon flux through the phenylpropanoid pathway, seven constitutive expression cassettes under control of the 35S promoter were constructed to overexpress the sorghum genes encoding 7 enzymes in the sorghum monolignol pathway. Previously, we had undertaken a transgenic approach and overexpressed 3 key enzymes and a regulatory protein in phenylpropanoid metabolism. We developed a set of transgenic lines that overexpress all 11 steps of the monolignol biosynthesis pathway of sorghum, which were evaluated in Specific Objective 2-5. This comprehensive set of lines is being used to develop novel strategies to alter phenylpropanoid metabolism in sorghum and test its effects on insect pests and fungal pathogens. Specific Objective 2. To determine the effects of modifications to lignin biosynthesis through bmr loci or transgenic overexpression on phloem-feeding and chewing insects of sorghum. For the chewing insects fall armyworm and corn earworm, A series of assays were performed with sucking (greenbugs) and chewing insects (fall armyworm and corn earworm). Overall, none of the lignin modified lines assayed showed dramatically increased susceptibility to the insects relative to WT. However, insect death was dramatically increased for both fall armyworms and corn earworms when fed bmr6 and to a lesser extent bmr12 stalk pith compared to WT pith tissue, which suggest bmr6 and bmr12 pith contain compounds toxic to these insects. Overall, bmr6 and bmr12 did not show increased susceptibility to any insect tested, which indicates that mutations in these 2 genes are good tools to reduce lignin for sustainable bioenergy feedstock production. Specific Objective 3. To determine the effects these lignin modifications have on fungi causing foliar or stalk diseases in sorghum. The bmr lines and transgenic overexpression lines were inoculated with the foliar disease anthracnose and stalk diseases stock rot and charcoal rot. The CCoAOMT overexpression had reduced leaf area infected compared to WT for anthracnose. None of the lignin modified lines appear to have increased susceptibility to the foliar and stalk pathogens tested. bmr6 and bmr12 actually appear more resistant to charcoal rot, hence impairing lignin synthesis may result in an increase in a defense compound that affects charcoal rot fungi. Specific Objective 4. To determine the alterations in metabolite profiles of relevant lines with significantly increased resistance to either insects or fungi. To address the impact of phenolic metabolites on insect and fungal interaction, in vitro assays were performed with phenolic compounds elevated in bmr6 and bmr12. The addition phenolic compounds inhibited the growth stalk rot fungi and fall armyworms (Funnell-Harris et 2014; Dowd & Sattler 2015). Extracts from bmr6 pith tissue inhibited the insect growth, but promoted fungal growth. A novel phenol compound was identified from bmr6 pith that may be toxic to insect. Soluble phenolic compounds likely play a role in inhibiting these insects, but bmr6 extracts were not inhibitory to the fungi. Wall-bound phenolic are being investigated for their inhibitory effects on these fungal pathogens. Modifying phenylpropanoid metabolism may provide new defense strategies against fungal pathogens and insect pests. Specific Objective 5. To determine the changes in global gene expression profiles of relevant lines with significantly increased resistance to either insects or fungi. RNAseq was performed on CCoAOMT and SbMyb60 overexpression lines, which showed instances of increased resistance to either insects or fungal pathogens. Monolignol pathway gene expression was determined by RT-qPCR for SbMyb60. The RNAseq reads were mapped to the sorghum genome and analyzed. SbMyb60 overexpression induced monolignol pathway gene expression (Scully et al 2016). SbMyb60 overexpression also increased the expression of ~5000 genes and repressed expression of ~4000 genes. CCoAOMT overexpression altered the expression of less than 500 genes. SbyMyb60 overexpression induced numerous defense related genes, but due to this large number of other potential pathways affected, this gene is not a good candidate to augment sorghum defenses against pathogens and insects. In contrast, CCoAOMT has a relatively small effect on global transcription, which makes this gene a potential candidate to enhance plant defenses against insect pests and fungal pathogens.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Sattler, S. A., Walker, A. M., Vermerris, W., Sattler, S. E. and Kang, C. 2017. Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases. Plant Physiology 173: 1031-1044.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Donze-Reiner, T., Palmer, N. A., Scully, E. D., Prochaska, T. J., Koch, K. G., Heng-Moss, T., Bradshaw, J. D., Twigg, P., Amundsen, K., Sattler, S. E. and Sarath, G. 2017. Transcriptional analysis of defense mechanisms in upland tetraploid switchgrass to greenbugs. BMC Plant Biology 17: 46.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2017
Citation:
Funnell-Harris, D.L., ONeill, P.M., Sattler, S.E., Gries, T., Berhow, M.A., Pedersen, J.F. Response of sorghum stalk pathogens to brown midrib plants and soluble phenolic extracts from near isogenic lines (2017) European Journal of Plant Pathology, pp. 1-13. Article in Press.
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Progress 02/01/15 to 01/31/16
Outputs
Target Audience:The scientific community, bioenergy energy sector, sorghum industry (producers and private ag. companies) were target audiences reached. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Four undergraduate students and one graduate student have been trained in scientific techniques and experimental design through experiments designed and funded by this project. A post-doc continued her scientific training through her work on Objectives 4 and 5. She was successfully mentored and trained, which led to placement as a Category 1 ARS Research Molecular Biologist. The scientific meetings attended by the PD and Co-PIs provided opportunities for professional development and scientific networking. How have the results been disseminated to communities of interest?The results from this project have been presented at national and regional scientific meetings as either poster or research talks. As studies have completed, the results were published in peer-reviewed journals (see products). Dowd PF, Berhow MA, Sattler SE. 2015. Reduction of lignin levels in mutant sorghum lines developed for saccharification leads to increased production of insecticidal compounds in stalk pith. 250th National Meeting, American Chemical Society. Boston, MA. Dowd, P.F., Sattler, S.E. 2016. Sorghum studies by USDA Peoria Ag Lab in 2015. Central Illinois Irrigated Growers Association, Irrigation Clinic. Sattler SE, Scully ED, Clemente TE, Funnell-Harris DL. 2015. Increasing lignin content to improve sorghum for bioenergy. Plant Biology 2015. Minneapolis, MN. Sattler SE. 2016. The Sorghum brown midrib (bmr) Mutants: A Forward Genetics Approach to Lignin. Plant & Animal Genome Conference. San Diego, CA. What do you plan to do during the next reporting period to accomplish the goals?Charcoal rot and Fusarium stalk rot assays on transgenic lines will commence this year in the spring. Additional insect and anthracnose bioassays will be performed on the lignin biosynthesis overexpression lines to confirm previous significant results. The lines that display the greatest differences in response to the insect or pathogen will be characterized as described in Objectives 4 and 5 to discover biological pathways involved in plant resistances.
Impacts
What was accomplished under these goals?
For Objective 2, transgenic sorghum lines that overexpressed genes in monolignol biosynthesis were evaluated for their attractiveness to aphids (greenbugs; Schizaphis graminum). The aphid preferred transgenic plants overexpressing F5H, COMT, CCoAOMT, C4H and 4CL compared to the non-transgenic plants. No aphid preference was observed for the CAD and SbMyb60 lines. These results suggest that manipulating this critical biochemical pathway could increase attractiveness of plants to aphids. However, the aphids fed these transgenic plants did not have increased reproductive rates compared to those fed non-transgenic plants in most cases. The reproductive rates were significantly greater for aphids on 4CL and C4H leaf tissue, but significantly reduced on COMT and SbMyb60 leaf tissue, compared to non-transgenic leaf tissue. Together, these results indicate that altering this biochemical pathway may potentially increase the threat of aphids. Overall, SbMyb60 overexpression appears to negatively affect aphids. Corn earworms (Helicoverpa zea) and fall armyworms (Spodoptera frugiperda) were also fed tissues from these transgenic plants, which resulted in both increases and decreases in feeding rates and weights relative to non-transgenic plants. Responses of the two insects to the same overexpressed gene varied. Fall armyworms fed leaves from plants overexpressing CCoAOMT weighed significantly less than those fed normal leaves, while the opposite effect was observed for corn earworms. These results indicate that insect responses to alterations of this pathway may be species specific. In field studies, bmr6 plants had less caterpillar-caused leaf damage than normal plants. In addition, European corn borer incidence and stalk damage were lower in the bmr6 plants compared to bmr12 and wild type. For Objective 3, the transgenic plants mentioned above were screened for resistance to Colletotrichum sublineolum, the causal agent of sorghum anthracnose in the greenhouse, and all exhibited a susceptible reaction to the pathogen. However, the infected leaf area was significantly reduced in the plants overexpressing the CCoAOMT gene, which suggests this gene may impart some resistance to this pathogen. For Objective 4, chemical analysis of the plant tissue overexpressing CCoAOMT showed significant increases in phenolic compounds compared to the non-transgenic plants. The changes could in part be responsible for increased resistance to the specific insect pests or fungal pathogens we observed. LC-MS based metabolite profiling was performed on SbMyb60 overexpressing plants, but preliminary analysis did not indicate any global or systematic metabolic changes within the tissues analyzed. For Objective 5, RNAseq was performed on plants overexpressing MYB and CCoAOMT, which has showed lignin modification affects multiple pathways that could be involved in insect and pathogen resistance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Dowd PF, Funnell-Harris DF, Sattler SE. 2016. Field damage of sorghum (Sorghum bicolor) with reduced lignin levels by naturally occurring insect pests and pathogens. J. Pest Sci. DOI 10.1007/S10340-015-0728-1
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Scully, E. D., T. Gries, D. L. Funnell-Harris, Z. Xin, F. A. Kovacs, W. Vermerris, and S. E. Sattler. Characterization of novel brown midrib 6 mutations affecting lignin biosynthesis in sorghum. Journal of Integrative Plant Biology 58: 136149. 2016.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Scully, E. D., T. Gries, G. Sarath, N. A. Palmer, L. Baird, M. J. Serapiglia, B. S. Dien, A. A. Boateng, D. L. Funnell-Harris, P. Twigg, T. E. Clemente and S. E. Sattler. Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in Sorghum bicolor. Plant Journal 85: 378-395. 2016.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Scully E.D., Donze-Reiner T., Wang H., Eickhoff T., Baxendale F., Twigg P., Kovacs F., Hang-Moss T., Sattler S.E., Sarath G. A related clade of peroxidases are expressed in C4 bioenergy grasses in response to feeding by greenbugs (Schizaphis graminum). Biomed Central (BMC) Plant Biology (submitted 9/14/2015)
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Progress 02/01/14 to 01/31/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Four undergraduate students and one graduate student have been trained in scientific techniques and experimental design through experiments designed and funded by this project. One of these undergraduate students is a member of an underrepresented group (black female). A post-doc continued her scientific training through her work on Objectives 4 and 5, and she received additional bioinformatics training through workshops from the Software Carpentry Organization. The scientific meetings (section below) attended by the PD and Co-PIs provided opportunities for professional development and scientific networking. How have the results been disseminated to communities of interest? The results from this project have been presented at national and regional scientific meetings. As studies are completed, the results are published in peer-reviewed journals (see products). What do you plan to do during the next reporting period to accomplish the goals? Charcoal rot and Fusarium stalk rot assays on transgenic lines will commence this year in late summer/early fall. Additional insect and anthracnose bioassays will be performed on the lignin biosynthesis overexpression lines. The lines that display the greatest differences in response to the insect or pathogen will be characterized as described in Objectives 4 and 5 to discover biological pathways involved in plant resistances.
Impacts
What was accomplished under these goals?
For Objective 2, field studies were repeated in 2014 using the near isogenic wild type, bmr6, and bmr12 lines, and the results were similar to the results from the prior years. Caterpillar-caused leaf damage was consistently reduced on the bmr6 plants compared to wild type plants from endemic populations. bmr12 plants showed intermediate levels of leaf damage compared to wild-type and bmr6. European corn borer stalk damage incidence and tunneling were lower in the bmr6 plants compared to bmr12 and wild type. Grasshopper and aphid presence and damage was also sometimes lower for the bmr lines compared to wild-type. In the laboratory, plants overexpressing the phenylalanine ammonium lyase (PAL) gene were evaluated with fall armyworms and corn earworms, there were incidences of increased consumption of the overexpression lines, but there were no significant differences in insect weight. Together these data suggest that PAL overexpression may interfere with nutrient intake of the insects. The effect of greenbugs on transgenic plant overexpression of a MYB factor that regulates lignin synthesis were evaluated and compared to normal plants. Feeding preference studies indicated the greenbugs were preferred to feed on normal sorghum leaf sections instead of MYB leaves, but the differences were not significant for any time point. For Objective 3, bmr6, bmr12 and wild-type lines inoculated with charcoal rot pathogen. bmr12 lines had significantly smaller diseases than the corresponding wild-type line (P ≤ 0.05), which indicate greater resistance. A leaf assay was developed for anthracnose to accomplish milestones associated with research on this pathogen. The 9 transgenic lines were evaluated for resistance against Colletotrichum sublineolum, the causal agent of sorghum anthracnose in the greenhouse, and all exhibited a susceptible reaction to the pathogen. For Objective 4, chemical analysis of the bmr6 and bmr12 pith tissue showed higher levels of phenolic glycosides than in wild type, were present in the fractions with the greatest inhibitory activity against corn earworms and fall armyworms. For Objective 5, RNAseq was performed on MYB, CCoAOMT, bmr6, bmr12 and wild-type lines to identify genes and potential pathways impacted by lignin modification, which may alter insect and pathogen resistance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Funnell-Harris, D. L., Sattler, S. E. and Pedersen, J. F. 2014. Response of Fusarium thapsinum to sorghum brown midrib lines and to phenolic metabolites. Plant Disease 98: 1300-1308. 10.1094/PDIS-09-13-0980-RE
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Dowd, P. F., and Sattler, S. E. 2015. Helicoverpa zea (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Lepidoptera Noctuidae) responses to Sorghum bicolor (Poales: Poaceae) tissues from lowered lignin lines. Journal of Insect Science 15(1): DOI http://dx.doi.org/1093/jisesa/ieu162.
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Kumar, A.A., Gorthy, S., Sharma, H.C., Huang, Y., Sharma, R., Reddy, B.V. 2014. Understanding genetic control of biotic stress resistance in sorghum for applied breeding. In: Wang, Y-H, Upadhyaya, H.D., Kole, C., editors. Genetics, Genomics and Breeding of Sorghum. Chapter 9. Boca Raton, FL: CRC Press. p. 198-225.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Dowd, P.F., Johnson, E.T., Sattler, S.E. Popcorn and sorghum studies by the USDA "Ag Lab" in 2013. Abstract presented at Central Illinois Irrigated Growers Association. Havana, IL, February 13, 2014.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Funnell-Harris, D. L. and Sattler, S. E. Response of Fusarium thapsinum to sorghum brown midrib lines and to phenolic metabolites. Abstract presented at the National American Phytopathological Society Meeting, Minneapolis, MN, August 9 13, 2014.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Funnell-Harris, D. L. and Sattler, S. E. Response of Fusarium thapsinum to sorghum brown midrib lines and to phenolic metabolites. Abstract presented at the North Central Division, American Phytopathological Society Meeting, Madison, WI, June 11 13, 2014.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Sattler, S. E. The impacts of lignin modifications on fungal pathogen and insect interactions in sorghum. Abstract presented at NIFA-AFRI Sustainable Bioenergy Annual PD Meeting, Arlington, VA, October 29-31, 2014
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Progress 02/01/13 to 01/31/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems: Co-PI Jeff Pedersen retired 12/31/2011, but this change did not affect planned experiments. Differences in susceptibility between the plants lines have been extremely difficult to detected in the greenbug seedling tests. In attempts to observe greater differences in greenbug susceptibility, older plants will be tested next year. What opportunities for training and professional development has the project provided? Four undergraduate students and one graduate student have been trained in scientific techniques and experimental design through experiments designed and funded by this project. A post-doc was hired in early 2014, whose scientific training will continue through her work on Objectives 4 and 5. The scientific meetings (section below) attended by the PD and Co-PIs provided opportunities for professional development and scientific networking. How have the results been disseminated to communities of interest? Three peer-viewed journal articles were submitted for publication and six presentations at national and international scientific meetings were given, which acknowledged USDA-NIFA as the funding source for this research. Sattler – Plant & Animal Genomes Conference, San Diego, CA. 2014; Meeting of Cochran Fellows on Sweet Sorghum. Tifton, GA. 2013; Sorghum Improvement Conference of North America. Lubbock, TX. 2013; U.S.-Korea Conference. East Rutherford, NJ. 2013. Huang -- Plant & Animal Genomes Conference, San Diego, CA 2014. Dowd -- Entomological Society of America Conference, Austin, TX 2014. What do you plan to do during the next reporting period to accomplish the goals? For Objective 1, lead transgenic lines will be identified for cinnamate-4 hydroxylase (C4H). For Objectives 2 and 3, 4CL, HCT, caffeoyl CoA O-methyltransferase CCoAOMT, F5H, COMT and CAD overexpression lines will be screened for susceptibility to the fungi and insects used in the project. A later plant growth stage will be used for the greenbug assays due to the lack of distinguishable differences between normal sorghum and lignin pathway modified lines in previous assays. For Objective 4, metabolic profiling will be performed to identify compounds whose levels are changed in bmr6, bmr12 and Myb overexpression lines, all of which have shown increased resistance to the insect pest or fungal pathogen relative to normal sorghum in Objectives 2 and 3. For Objective 5, gene profiling will be performed to identify genes whose expression is changed in same lines examined in Objective 4.
Impacts
What was accomplished under these goals?
For Objective 1, the lead transgenic events (the plants with highest expression level of the engineered lignin gene) were identified for the over-expression of 6 lignin pathway genes: 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl CoA:shikimate/quinate transferase (HCT), caffeoyl CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD). These transgenic lines will be screened to determine if they affect fungal infection or insect feeding in Objectives 2 and 3. For Objective 2, in the field studies, bmr6 plants had significantly less insect damage due to leaf feeding caterpillars or stalk damage due to European corn borers than bmr12 or normal sorghum plants. In laboratory studies, corn earworm and fall armyworm caterpillars fed significantly less on bmr6 leaves compared to those fed normal or bmr12 leaves, and bmr6 stalk tissue was more toxic to these insects than bmr12 or normal sorghum stalks. In seedling assays, the level of greenbug (aphid) damage was not significantly different among the bmr and normal lines tested. For Objective 3, in the greenhouse studies, 8 transgenic sorghum lines from Objective 1 were evaluated for resistance to the fungal pathogen Colletotrichum sublineolum, the causal agent of anthracnose. On the inoculated leaves, several transgenic overexpression lines exhibited significantly fewer infection-spreading fruiting bodies as compared to inoculated normal leaves. These results show that anthracnose was less able to infect the transgenic sorghum lines compared to normal sorghum lines. The myb transgenic lines were inoculated with Fusarium thapsinum, a causal agent of stalk rot disease, and the damage caused by the pathogen was used to measure susceptibility. The damage was more severe on normal plants than the Myb-overexpressing plants, but the level of damage was not significantly differently, which indicated that overexpression of this gene did not significantly impact the disease. bmr sorghum lines were exposed to Macrophomina phaseolina, the causal agent of charcoal rot disease. Preliminary results indicated the bmr lines were not more susceptible or more resistant to this fungal pathogen compared to normal sorghum. For Objective 4, phenolic compounds were analyzed from sorghum plants following fungal infection, and some of the pathway-related compounds were significantly higher in bmr6 and myb over-expression lines as compared to normal sorghum lines. These compounds may be responsible for the increased resistance to insects or fungi observed. Some of the elevated compounds from this pathway were also shown to inhibit fungal growth in laboratory tests. For Objective 5, several new bmr lines were shown to be altered in the expression of lignin pathway genes, which indicate these mutants likely affect lignin synthesis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Sattler, S., and D. Funnell-Harris, 2013 Modifying lignin to improve bioenergy feedstocks: strengthening the barrier against pathogens? Frontiers in Plant Science 4.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Funnell-Harris, D., S. Sattler, and J. Pedersen (submitted) Response of fusarium thapsinum to sorghum brown midrib lines and to phenolic metabolites. Plant Disease.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Dowd P.F, D.L. Funnell-Harris and S.E. Sattler (Under revision) Field effects of altered lignin lines of sorghum (Sorghum bicolor) on representative insect and disease pests. Euphytica.
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Progress 02/01/12 to 01/31/13
Outputs
OUTPUTS: For specific objective 1, transgenic plants overexpressing 6 genes in the sorghum lignin pathway: 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl CoA:shikimate/quinate transferase (HCT), caffeoyl CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD) were generated through Agrobacterium-mediated transformation of RTx430 sorghum variety. In the next 3 months, antibodies against these gene products will be used to identify transgenic plants with the highest levels of overexpression. For specific objective 2, the transgenic lines overexpressing three lignin biosynthesis pathway genes; phenylalanine ammonia lyase (PAL), p-coumarate 3-hydroxylase (C3H) and cinnamyl CoA reductase (CRR) and the SbMyb68 gene encoding the MYB transcription factor, were evaluated for their susceptibility to greenbug feeding using the previously developed bioassay with the virulent greenbug biotype E. All transgenic sorghum lines and control RTx430 exhibited a similar response to greenbug feeding. Damage symptoms developed quickly after co-culturing for 3 days with the insects. Most seedlings died by ten days exposure to the greenbugs. The transgenic line showed a similar level of susceptibility to greenbugs as sorghum variety RTx430 (control), which was used to develop the transgenic lines. Thus, overexpressing these genes did not significantly increase tolerance to greenbug infestation. For specific objective 3, we conducted and completed stalk rot assays to determine whether the bmr plants were altered in their susceptibility to the sorghum stalk pathogen Macrophomina phaseolina. The length of lesion at the infection site was used to measure disease severity in the three different genetic backgrounds BTx623, RTx430 and Redlan. In all cases, the lesion lengths on bmr6 and bmr12 plants were the same or shorter than those on normal (wild-type; WT) plants in RTx430 and Redlan backgrounds. Six novel bmr mutants in the BTx623 background were also screened and compared to bmr6, bmr12 and WT plants in this background. No significant differences in lesion lengths were found between these six mutants and the corresponding WT. Lesion lengths of bmr6 RTx430 and bmr6 BTx623 plants were significantly shorter than in the corresponding WT plants. In the bmr6 RTx430 plants, the lesion lengths were not significantly different between M. phaseolina infected plants and the negative control plants, which indicated that the bmr6 plants were resistant to this pathogen. In addition, we have determined that bmr6 plants in the RTx430 and BTx623 backgrounds exhibit increased resistance to another stalk rot pathogen Fusarium thapsinum. We are establishing conditions for transformation of F. thapsinum with green fluorescent protein (GFP) marker to follow disease progression within the plant. These outcomes have been presented at the American Society of Plant Biologist Midwest Sectional Meeting, American Phytopathological Society Meeting and Entomological Society of America Meeting. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Modifying lignin content and composition of plant cell walls (biomass) is currently a major target for increasing the conversion of plant biomass into biofuels. In addition, lignin is also synthesized in response to pathogen or insect attack, which suggests that lignin could play a significant role in plant defense. The lignin-modified lines analyzed did not display increased susceptibility to the fungal pathogens or insects tested in this project. Therefore, these modifications could be used to sustainably improve sorghum for bioenergy uses with minimal potential impact to its susceptibility to the diseases and pests tested.
Publications
- Sattler, S.E., Palmer, N.A., Saballos, A., Greene, A.M., Xin, Z.G., Sarath, G., Vermerris, W., and Pedersen, J.F. (2012). Identification and Characterization of Four Missense Mutations in Brown midrib 12 (Bmr12), the Caffeic O-Methyltranferase (COMT) of Sorghum. Bioenergy Research 5, 855-865.
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Progress 02/01/11 to 01/31/12
Outputs
OUTPUTS: For specific objective 1, we assembled T-DNA transformation vectors for transgenic overexpression of the genes encoding 7 enzymes in the sorghum monolignol pathway: cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl CoA:shikimate/quinate transferase (HCT), caffeoyl CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD) in R Tx430 sorghum. The Agrobacterium-mediated transformations for each transgenic cassette have been completed, and a minimum of 10 independent events were generated from each transformation. Transgenic lines overexpressing SbMyb68, phenylalanine ammonia lyase (PAL), p-coumarate 3-hydroxylase (C3H) and cinnamyl CoA reductase (CRR) were evaluated for the impact on biomass composition and total energy content. For specific objective 2, the effects of bmr2, bmr6, bmr12 and 6 novel bmr mutants on the susceptibility of sorghum seedlings to greenbug biotype E were assayed. These greenbug assays indicated the 6 novel bmr mutants were significantly more sensitive to this biotype relative to WT. For specific objective 2, the effects of bmr6 and bmr12 on the susceptibility of sorghum to corn earworm and fall armyworm were determined. First instar larvae of the corn earworm and fall armyworm were caged with sorghum leaf sections from WT, bmr6 and bmr12 plants. In addition, larvae were fed artificial diets containing 6 phenolic compounds, because previously published studies indicated bmr6 and bmr12 mutant lines contained altered concentrations of these phenolics. These feeding assays indicated that there were slight but significant differences in feeding rates between the different lines. In general, the bmr6 had less feeding damage than the wild type plants, which was not always associated with significantly reduce larval weights. The phenolic amendments to the artificial diet had no significant effect on the larval weights. The larvae were fed the stalk pith from sorghum plants. Both corn earworms and fall armyworms showed significantly higher mortality on bmr6 and bmr12 pith relative to WT pith from plants of the same age. For specific objective 3, the effects of bmr2, bmr6, bmr12 and 6 novel bmr mutants on the susceptibility of sorghum to fungal pathogens associated with stalk rot and anthracnose were assayed. The stalk rot assay was performed by toothpick inoculation of Fusarium thapsinum to the peduncle. The assay indicated the lesions associated with the fungal infection had significantly reduced lengths in bmr6 and bmr12 relative to WT as previously reported. In addition, three novel bmr mutants showed reduced lesion lengths relative to WT indicating increased resistance to the fungal pathogen. Sorghum leaves were inoculated with the foliar fungal pathogen Colletotrichum sublineolum (anthracnose) and the infected foliar area was scored based on previously published methods. Overall, none of the bmr lines showed increased susceptibility relative to WT, but two novel bmr lines showed reduced foliar area infected relative to WT. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Modifying lignin content and composition currently is a major target for bioenergy feedstock improvement for both cellulosic and thermal bioenergy conversion. We have constructed overexpression constructs for each step in monolignol biosynthesis, the pathway responsible for synthesizing the subunits of the lignin polymer. These lines will be useful tools to manipulate lignin content and composition in sorghum for bioenergy feedstock development. Our assays indicate that bmr6 and bmr12 do not increase susceptibility of sorghum to the fungal pathogens or insects tested. Thus, bmr6 and bmr12 should be useful to reduce lignin for bioenergy sorghum without impacting disease or pest pressures.
Publications
- No publications reported this period
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