Progress 09/01/16 to 08/31/22
Outputs
Target Audience:The primary focus group of this project has been National Sorghum Producers, United Sorghum Check-off and Nebraska Grain Sorghum Producers. Presentations were made duringGenomic Sciences Program Annual Principal Investigator (PI) Meeting, Plant Health (American Phytopathological Society national meetings), Sorghum Improvement Conference of North America and AUSSORGM. Presentations were also made at the following universities: University of Nebraska, Kansas State University and University of Southern Queensland. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Post doctoral associate-Dr. Khasin has had further professional training under the guidance of the co-PIs of this grant. They have broadened their analytical skills and been provided further training in manuscript preparation and publication. Additionally, Dr. Khasin has received further training in the field of Plant Pathology, informally and by participation in virtual meetings and seminars. Dr. Khasin now has broad training in microbiology, plant pathology, as well as bioinformatics. They have demonstrated adept abilities in design of in planta experiments with different diseases, including proper experimental design. They have in turn provided training to technologists and undergraduate students. Dr. Khasin would be a valuable asset to research programs involving broad and interacting topics. Technologist-Ms. Bernhardson has received extensive training culturing and maintaining plants for experimental conditions, in designing and conducting greenhouse and field experiments, culturing and maintenance of microorganisms, taking detailed and accurate measurements, and preparation of documents for analyses.Ms. Bernhardson completed on-line training in handling infectious agents as well as several other safety issues relevant to operations in a laboratory. Ms. Bernhardson became an invaluable addition and guide for training undergraduate students in the safe and correct uses of experimental equipment and conducting experimental techniques. Since completion of her work for this grant, Ms. Bernhardson has obtained a permanent technician position with USDA-ARS, Lincoln, NE. Undergraduate thesis-Mr. Kilts conducted his undergraduate thesis under this project, fulfilling requirements for graduation from Universitu of Nebraska with a major in chemistry. Mr. Kilts grew plants in the greenhouse and field, extracted biochemicals from leaves, and performed High Performance Liquid Chromotography (HPLC) on the extracts . This involved reading the literature and maximizing the extraction and HPLC protocols for our equipment. Because of his high skill level with analytical equipment, he was hired by another researcher within USDA-ARS as a Term Biological Science Technologist. Undergraduate honors thesis-Mr. Van Roy conducted his undergraduate honors thesis in this project, fulfilling requirements to graduate with honors from UNL in microbiology and mathematics. This project required him to conduct metagenomic analyses of microorganisms growing in field-grown biomass sorghums to determine whether aspects of plant microbiomes were associated with increasedresistance to stalk pathogens. Mr. Van Roy has since been accepted into the MD/Ph.D. program at University of Nebraska's Medical Center. How have the results been disseminated to communities of interest?During the lifetime of this grant, presentations were made during the Genomic Sciences Program Annual Principal Investigator (PI) Meeting, Plant Health (American Phytopathological Society national meetings), Sorghum Improvement Conference of North America and AUSSORGM. Presentations were also made at the following universities: University of Nebraska, Kansas State University and University of Southern Queensland. During the past year of the grant, in-person presentations were limited due to CDC-imposed COVID restrictions. Nonetheless, Dr. Funnell-Harris made the following presentations: Deanna L. Funnell-Harris (2021) Modifying cell wall phenylpropanoids of small grains for increased usability and disease and drought resistance. University of Nebraska, Department of Plant Pathology Seminar Series. Maya Khasin, Patrick M. O'Neill, Lois F. Bernhardson, Nathan Palmer, Erin Scully, Gautam Sarath, Scott E. Sattlerand Deanna L. Funnell-Harris. 2022.Wound and defense induced cell wall phenylpropanoids in sweet sorghums with differing responses to stalk pathogens. Presented at Plant Health 2022, Aug. 6 - 10, Pittsburgh, PA. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Obj. 1a: 1) Plants (brown midrib (bmr)-6, bmr12 and wild-type) were inoculated under well-watered and water-limited conditions in a greenhouse with two stalk pathogens, Fusarium thapsinum and Macrophomina phaseolina. 2) Lesions were measured 0, 3 and 13 days after inoculation. Fungal growth in asymptomatic stalk tissues also was assessed. Other measurements of the plant (days to anthesis, plant heights, stalk diameters, panicle lengths) also were made. 3) Mean lesion lengths were analyzed by analysis of variance (ANOVA). It was found that reduced lignin line bmr12was more resistant to two sorghum stalk pathogensunder drought conditions than the near-isogenic wild-type line or the same line grown under adequate water. There was evidence that this may be due to increased endophytic (asymptomatic) growth of the pathogens in the bmr12 stalks. 4) These results indicated that modifications in the plant cell wall may alter responses to pathogens by altering defense-related pathways. Obj. 1b: 1) Lines (a total of 11) overexpressing genes in the sorghum monolignol biosynthesis pathway were screened for responses to sorghum stalk pathgoens, F. thapsinum and M. phaseolina. TheSbMyb60 and SbCCoAOMT oveerexpression lines were assessed under drought and well-watered conditions for responses to F. thapsinum and M. phaseolina through disease development; these results are currently being analyzed.2) Lesions were measured 18 days after inoculation. Other measurements of the plant (days to anthesis, plant heights, stalk diameters, panicle lengths) also were made.3) Mean lesion lengths were analyzed by ANOVA. 4)Lines overexpressing the sorghum Myb transcription factor SbMyb60, that controls expression of genes for enzymes in the monolignol biosynthesis pathway, and lines overexpressing the enzymecaffeoyl coenzyme A 3-O-methyl transferase (SbCCoAOMT) had increased resistance to two stalk pathogens in greenhouse conditions, as compared with the wild-type recipient line, but only SbMyb60 overexression lines were more resistant under field conditions. Obj. 1c, d: 1) Lines with the stay-green trait (post-flowering nonsenescence), high levels of the cyanogenic glycoside, dhurrin, and previously observed field resistance, were screened for responses to the pathogens, F. thapsinum and M. phaseolinain the greenhouse. Lines also were screened for post-flowering non-senescence and dhurrin in both the greenhouse and field. A subset of lines were screened for responses to F. thapsinum in field conditions. 2) Lesion lengths were collected, dhurrin levels were determined using high pressure liquid chromotography, and senescence ratings were performed. 3) Mean lesion lengths, dhurrin levels and senescence ratings were each analyzed by ANOVA; measurements were compared using Pearson Correlations. Two lines with relatively low levels of dhurrin at flowering, and increased resistance,were identified. These lines were grown subsequently under well-watered and drought conditions in a greenhouse, inoculated with the same two pathogens. These data are being analyzed.4) The two lines exhibiting resistance to the two pathogens have great potential for development of breeding lines with increased resistance to the two pathogens but with reduced dhurrinin the biomass for forages or bioenergy uses. Objective 2: Obj. 2a: 1) Phenylpropanoids and phytohormones were analyzed in the low lignin line,bmr12, another low lignin line bmr6 and the near-isogenic wild-type line in plants subjected to drought and well-watered conditions during disease development when inoculated with F. thapsinum or M. phaseolina. 2) Metabolites were analyzed using GC/MA.Metabolite analysis was performed using the R programming environment. 3) Results were consistent withalterations in the composition of the secondary cell wall affecting plant immunity by influencing phenolic composition. The bmr12 plants inoculated with the pathogens had increased levels of salicylic acidand jasmonic acid across both watering conditions and significantly reduced lesion sizes under water limitation compared to adequate watering. 4) These results suggested that drought may prime induction of pathogen resistance. Obj. 2b: 1) Two lines overexpressing SbMyb and the wild-type recipient line were grown in a greenhouse and stalks were inoculated with F. thapsinum at flowering. Plant tissues were collected 18 days after inoculation.2) These tissues are available for analysis ofphenylpropanoid and phytohormone using liquid chromotography/mass spectometry (LC/MS) (not yet performed). 3) Analyses will be performed in the R environment and will be compared with lesion and RNA-sequencing data. Obj. 2c and 2d: 1) Plant tissues were collected for two stay-green lines (post-flowering senescent) and a senescent line from plants inoculated F. thapsinum, grown under drought and well-watered conditions, during disease development following inoculation. 2) These tissues are available for analysis ofphenylpropanoid and phytohormone analyses using liquid chromotography/mass spectometry (LC/MS) (not yet performed). 3) Analyses will be performed in the R environment and will be compared with lesion and RNA-sequencing data. Objective3: This objective was replaced with global gene analysis using RNA-Sequencing Objective 4: Obj. 4a: 1) Plants were inoculated under well-watered and water-limited conditions in a greenhouse with two stalk pathogens, F. thapsinum and M. phaseolina. Plant tissues were collected (lesions and 3-cm distant from lesions)0, 3 and 13 days after inoculation. RNA was extracted from tissues. Reverse transcription quantitative PCR was conducted to confirm results of RNA-sequencing. 2)The purified RNA samples were quantified and sent to the University of Nebraska Medical Center Genomics Core Facility (https://www.unmc.edu/vcr/cores/vcrcores/ genomics/index.html) for furtherprocessing. 3)Reads were pseudoaligned to the Sorghumbicolor genome (v3.1) The qPCR analysis indicated agreement with RNA-Seq findings. 4)Alterations in the composition of the secondary cell wall affect immunity by influencing phenolic composition and phytohormone signaling, leading to the action of defense pathways. Some of these pathways appear to be activated or enhanced by drought. Secondary metabolite biosynthesis and modification in salicylic acidand jasmonic acid signal transduction may be involved in priming a stronger defense response in water-limited bmr12 plants. Obj. 4b: 1) Two lines overexpressing SbMyb and the wild-type recipient line were grown in a greenhouse and stalks were inoculated with F. thapsinum at flowering. Plant tissues were collected 18 days after inoculation. RNA was extracted from the tissues. 2)The purified RNA samples were quantified and sent to the University of Nebraska Medical Center Genomics Core Facilityfor further processing. 3)Reads were pseudoaligned to the Sorghumbicolor genome. Results are currently being analyzed.4)The goal isto identify the genes, gene networks and metabolic pathways involved in the increased resistance to stalk pathogens in SbMyb overexpression lines. Obj. 4c, 4d: 1) Plant tissues were collected for two stay-green lines (post-flowering senescent) and a senescent line from plants inoculated F. thapsinum, grown under drought and well-watered conditions, at 0, 3 and 13 days following inoculation. RNA was extracted from the tissues.2)The purified RNA samples were quantified and sent to the University of Nebraska Medical Center Genomics Core Facilityfor further processing. 3)Reads were pseudoaligned to the Sorghumbicolor genome. Results are currently being analyzed.4)The goal isto identify the genes, gene networks and metabolic pathways involved in the increased resistance to F. thapsinum in the stay-green lines grown under water-limited condisions as compared with the senescent line.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Funnell-Harris, D., ONeill, P., Sattler, S., Gries, T, Berhow, M. and Pedersen, J. 2017. Response of sorghum stalk pathogens to brown midrib plants and soluble phenolic extracts from near isogenic lines. Eur. J. Plant Pathol. 148:941953.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Khasin, M., Bernhardson, L., ONeill, P., Palmer, N., Scully, E., Sattler, S. and Funnell-Harris, D. 2021. Pathogen and drought stress affect cell wall and phytohormone signaling to shape host responses in a sorghum COMT bmr12 mutant. BMC Plant Biology 21: 390.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Funnell-Harris, D. L., Sattler, S. E., ONeill, P. M., Toy, J. J., Bernhardson, L. F., Kilts, Mark, R., Khasin, M. 2022. Association of dhurrin levels and post-flowering non-senescence with resistance to stalk rot pathogens in Sorghum bicolor. Eur. J. Plant Pathol. 163: 237-264.
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
Funnell-Harris, D. L., Sattler, S. E., Toy, J. J., ONeill1, P. M., and Bernhardson, L. F. Response of sorghum lines carrying recently identified brown midrib (bmr) mutations to stalk rot pathogens and water deficit. Plant Pathology (accepted with minor revision 11-16-2022)
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
Funnell-Harris, D. L., Sattler, S. E., ONeill, P. M., Gries, T., Ge, Z. and Nersesian, N. Effects of altering three steps of monolignol biosynthesis on sorghum responses to stalk pathogens and water deficit. Plant Disease (accepted with minor revision 12-6-2022)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Khasin, M., Bernhardson, L. F., ONeill, P. M., Palmer, N. A., Scully, E. D., Sattler, S. E., Sarath, G. and Funnell-Harris, D. L. Phenylpropanoids following wounding and infection of sweet sorghum lines differing in responses to stalk pathogens. Phytopathology (Submitted 12-7-2022)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Khasin, M., ONeill1, P. M., Bernhardson, L. F., Palmer, N., Scully, E., Sarath, G., Sattler, S. E. and Funnell-Harris, D. L. Cell wall phenylpropanoids involved in wounding and defense in sweet sorghum lines differing in responses to stalk pathogens. Presented at Plant Health 2022, Pittsburgh, PA, August 6 - 10, 2022
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Due to COVID-19, meetings were cancelled, postponed or presented virtually. Unfortunately, interactions with important commodity groups such as Nebraska Grain Sorghum Producers, National Sorghum Producers and United Sorghum Check-off were limited. These presentations were made locally, nationally and internationally: Dr. Funnell-Harris presented the invited seminar, "Modifying cell wall phenylpropanoids of small grains for increased usability and disease and drought resistance" during the Complex Biosystems Seminar Series, Sept. 3, 2020 through University of Nebraska (UNL). The target audience was UNL faculty, students, researchers and extension and local and regional USDA scientists. Dr. Funnell-Harris presented the invited talk, "The cell wall and signaling during defense responses in sorghum lines exhibiting resistance to stalk rot pathogens and drought" during Sorghum Improvement Conference of North America, Oct. 30, 2020. The target audience was sorghum researchers, producers, commodity boards, industry and National Program Leaders. Dr. Khasin and Dr. Funnell-Harris presented the accepted poster, "Overexpression of a phenylpropanoid master regulator impacts responses to stalk rots" during Plant Health 2021, Aug. 6, 2021. The target audience was state, national and international researchers and industry. Dr. Funnell-Harris has been invited to present "Effects of cell wall phenylpropanoid modifications on responses of small grains to disease and drought" during the Department of Plant Pathology 2021 Fall Seminar Series, UNL, Oct. 25, 2021. The target audience is UNL faculty, students, researchers and extension, and local and regional USDA scientists. Changes/Problems:At the time this proposal was written, the sensitivity of RNA-Seq to environmental conditions and to plant wounding was not yet reported. We found that performing this technique on tissues that had been collected in multiple assays performed throughout the year resulted in being unable to identify gene differences among the treatments. Apparently even slight difference in temperature, light intensities and daylength, and even the wounding caused by collecting tissues, can induce genes, masking the genetic responses to the intended treatments. Although we found ways of analyzing the RNA-Seq results, it became harder and harder to find impactful venues to publish the results of these studies. The pathology results for the experiments originally proposed in the grant, are still essential to the research. However, we are including other experiments to follow-up on these results, including highly controlled single repetition RNA-Seq experiments, enzyme activity assays, imagining experiments and microscopy. Because of the reduced costs of RNA-Seq since the original writing of the grant proposal, we are able to include these experiments. In this way, we will be able to publish a greater number of manuscripts then originally proposed. What opportunities for training and professional development has the project provided?Post doctoral associate-Dr. Khasin has had further professional training under the guidance of the co-PIs of this grant. They have broadened their analytical skills and been provided further training in manuscript preparation and publication. Additionally, Dr. Khasin has received further training in the field of Plant Pathology, informally and by participation in virtual meetings and seminars. Dr. Khasin now has broad training in microbiology, plant pathology, as well as bioinformatics. They have demonstrated adept abilities in design of in planta experiments with different diseases, including proper experimental design. They have in turn provided training to technologists and undergraduate students. Dr. Khasin would be a valuable asset to research programs involving broad and interacting topics. Technologist-Ms. Bernhardson has received extensive training culturing and maintaining plants for experimental conditions, in designing and conducting greenhouse and field experiments, culturing and maintenance of microorganisms, taking detailed and accurate measurements, and preparation of documents for analyses. In spite of the restrictions imposed due to COVID-19, Ms. Bernhardson completed on-line training in handling infectious agents as well as several other safety issues relevant to operations in a laboratory. Ms. Bernhardson has become an invaluable addition and guide for training undergraduate students in the safe and correct uses of experimental equipment and conducting experimental techniques. Ms. Bernhardson would now be wholly qualified to independently manage a small laboratory. Undergraduate thesis-Mr. Kilts conducted his undergraduate thesis under this project, fulfilling requirements for graduation from UNL with a major in chemistry. Mr. Kilts grew plants in the greenhouse and field, extracted biochemicals from leaves, and performed High Performance Liquid Chromotography (HPLC) on the extracts . This involved reading the literature and maximizing the extraction and HPLC protocols for our equipment. Because of his high skill level with analytical equipment, he was hired by another researcher within USDA-ARS as a Term Biological Science Technologist. Undergraduate students-due to restrictions imposed by COVID-19, we necessarily reduced the number of undergraduate students participating in this grant research. During the past year, we had a total of four undergraduate students assisting with various aspects including field and greenhouse cultivation of plants, culturing of microorganisms and inoculation and scoring of plants. These students have been provided hands-on training in basic plant biology, microbiology and molecular biology techniques, as well as laboratory, greenhouse and field safety. How have the results been disseminated to communities of interest?Due to COVID-19, meetings were cancelled, postponed or presented virtually. Unfortunately, interactions with important commodity groups such as Nebraska Grain Sorghum producers, National Sorghum Producers and United Sorghum Check-off were limited. These presentations were made locally, nationally and internationally: Dr. Funnell-Harris presented the invited seminar, "Modifying cell wall phenylpropanoids of small grains for increased usability and disease and drought resistance" during the Complex Biosystems Seminar Series, Sept. 3, 2020 through University of Nebraska (UNL). The target audience was UNL faculty, students, researchers and extension and local and regional USDA scientists. Dr. Funnell-Harris presented the invited talk, "The cell wall and signaling during defense responses in sorghum lines exhibiting resistance to stalk rot pathogens and drought" during Sorghum Improvement Conference of North America, Oct. 30, 2020. The target audience was sorghum researchers, producers, commodity boards, industry and National Program Leaders. Dr. Khasin and Dr. Funnell-Harris presented the accepted poster, "Overexpression of a phenylpropanoid master regulator impacts responses to stalk rots" during Plant Health 2021, Aug. 6, 2021. The target audience was state, national and international researchers and industry. Dr. Funnell-Harris has been invited to present "Effects of cell wall phenylpropanoid modifications on responses of small grains to disease and drought" during the Department of Plant Pathology 2021 Fall Seminar Series, UNL, Oct. 25, 2021. The target audience is UNL faculty, students, researchers and extension, and local and regional USDA scientists. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Low lignin lines: Greenhouse experiments are completed. High lignin lines: Completion of follow-up experiments with seedling root infections. & D. We will conduct a single repetition with a subset of treatments to collect tissues specifically for RNA-Seq to account for high variabilities in temperatures, light intensities, flowering times, growth habits, etc. when conducting multiple assays year-round. Objective 2: Metabolic analyses using GC/MS or Near Infrared Spectroscopy (NIRS) will be performed as follow-up experiments for RNA-Seq results for high lignin lines (B) or Stay-green/field resistant lines (C & D) Objective 3: N/A Objective 4: RNA-Seq analyses will be completed (high lignin lines; B) and performed (Stay-green/field resistance; C & D) The following manuscripts will be prepared and submitted: Altered susceptibility to Fusarium thapsinum pathogenic or endophytic growth in stalks of sorghum COMT mutant bmr12 Overexpression of a phenylpropanoid master regulator impacts responses to stalk rots in sorghum & D. Association of post-flowering non-senescence (Stay-green) and dhurrin levels with resistance to stalk rot pathogens in Sorghum bicolor (due to be submitted by 9/30/2021; target journal: European Journal of Plant Pathology). Regulation of genomic and metabolic pathways in non-senescent (Stay-green) and senescent sorghum lines subjected to infection with two stalk rotting pathogens and drought.
Impacts
What was accomplished under these goals?
Impact: A major component of plant cell walls is lignin that provides rigidity and protects against pathogens. The goals of this grant were to investigate bioenergy sorghums with resistance to stalk rot pathogens and drought, and to determine the genes and biochemical pathways involved in defense. Stalk rotting fungi reduce biomass and their destructiveness increases during drought. The sorghum diseases charcoal rot and Fusarium stalk rot result in lodging, further reducing biomass yield by impairing harvest. The brown midrib (bmr) mutants, with less lignin, either exhibit no change in response to stalk pathogens, or are more resistant than the wild-type. One bmr line was more resistant to stalk pathogens but only during drought. We examined gene and biochemical activity to identify traits and compounds that contribute to this unique resistance. We showed that alterations in the cell wall affected responses to diseases due to altered compounds and plant hormone signaling, leading to defense strategies that may be activated or enhanced by drought. We previously developed lines with increased enzyme activity in lignin synthesis. Lines overexpressing SbMyb60 that controls most genes in lignin synthesis, were more resistant to Fusarium stalk rot disease. Analysis of gene activity showed alterations in plant hormone signaling and protein processing and turnover. Another set of lines overexpressing a key enzyme in lignin synthesis was more resistant to a stalk rot pathogen, but only under drought. We used greenhouse and field screens to identify four more lines with increased resistance to stalk pathogens. One line has low levels of a toxic compound commonly found in stalk rot resistant sorghum (dhurrin); this low dhurrin line is a good candidate for breeding biomass sorghum with animal feed end-uses. We demonstrated that pathways involved in plant traits, and how they interact with the environment, determine how quickly plants respond to diseases under stress. We found several lines that we are using to identify key genes or biochemical pathways that enhance stalk rot and drought resistance for bioenergy sorghum. Following a 10-week lockdown in 2020, the building within which Dr. Funnell-Harris' laboratory is housed was limited to 25% occupancy. This is still in effect, reducing personnel to performing this grant research. Nonetheless, Dr. Funnell-Harris, and the employees supported by this funding, have made significant research progress toward completion of the goals of this grant. Objective 1: Low lignin (brown midrib; bmr) lines -Manuscript describing genes and pathways associated with increased resistance to stalk pathogens under drought in the bmr12 line is in press. -Progress towards a second manuscript describing genes and pathways associated with endophytic (asymptomatic) colonization observed in bmr12: Colonization experiments with the Fusarium stalk rot pathogen under conditions of drought or adequate water are completed. Extent of fungal colonization in stalks and effects on growth were determined. Logistic regression was used to determine whether plant genotype and watering condition influence colonization. Soon after infection, bmr12 has less disease than the wild-type, while later, infection in bmr12 was similar. Plant materials to perform global gene expression and metabolite analyses, and enzyme assays, were collected. B. High lignin lines 1 & 2) Lines overexpressing either SbMyb60, that controls genes in lignin biosynthesis or a key enzyme (SbCCoAOMT) in the pathway, were infected with stalk pathogens. The SbMyb60 lines infected with the Fusarium stalk rot fungus were more resistant under both well-watered and drought conditions. The following are being performed: Mature plants under well-watered and drought conditions are inoculated and uninoculated plants are included to determine effects of wounding. Seedling roots are inoculated with a stalk pathogen under adequate water and drought. Imaging technology (Rhizovision Explorer) is used to assess effects on growth. Plants are collected for biochemical analyses. 3. Logistic regression is being used to determine whether plant genotype and watering condition influence colonization and root morphology. 4. The overexpressed gene, pathogen and watering condition appear to affect root branching in high lignin lines. C.-D. Lines exhibiting "Stay-green" (stays green after flowering) and other resistance traits. -Manuscript describing screens of lines for responses to pathogens, dhurrin levels and Stay-green will be submitted by Sept. 30, 2021. Four lines exhibiting resistant to pathogens in greenhouse and field conditions were identified. -For a second manuscript, greenhouse inoculations during drought and adequate watering were performed. Progress: 1. Using four stalk rot resistant lines, infections with pathogens under drought and adequate water were performed, then disease was monitored. The experiment is completed. Follow-up experiments: i. Stalk rot resistant and susceptible lines are inoculated with a pathogen under well-watered and drought conditions in a greenhouse, and tissues collected for RNA-Seq and other analyses; and ii. Stalk rot resistant and susceptible lines are inoculated with a pathogen in the field and tissues collected for RNA-Seq and other analyses. 2. Lesion lengths and physical features of stalks and grain heads were measured and flowering times and daylengths were recorded. 3. A mixed linear model was fitted to lesion data. These, as well as physical traits and daylengths will be used in correlation analyses with gene expression and metabolomics results. 4. Two stay-green lines had the lowest disease under well-watered and drought conditions. Key genes and biochemical pathways involved in defense will be identified. Objective 2: 1. Metabolites from the phenylpropanoid pathway, including lignin synthesis, and plant hormones were surveyed in wild-type and bmr12 (low lignin) plants infected with the Fusarium stalk rot pathogen, under adequate water and drought. 2. Plant hormone analysis was conducted at University of Nebraska. Phenylpropanoid metabolites were analyzed in our laboratory. 3. Metabolite analysis was performed in R. 4. Low lignin plants had higher levels of defense signaling molecules and were altered in phenylpropanoid metabolites. These may prime a stronger defense response when plants are subjected to drought. This manuscript is in press (BMC Plant Biology). Objective 3: Analysis of gene expression using RNA-Seq, supported with metabolite analyses, RT-qPCR and enzyme activity assays, replaced high-throughput gene expression. Objective 4: Plant materials are chosen for RNA-Seq based on responses of lines to pathogen and watering treatments. The objective is completed for low lignin (A) lines (manuscript in press) and being performed for high lignin (B) and Stay-green and field resistant (C & D) lines. Plant tissues are processed to obtain RNA, which is sent to the University of Nebraska Medical Center's Genomics Core Facility for further processing. Unfortunately, processing has been profoundly delayed due to necessarily circumventing services and supplies for COVID-19 testing. Analysis of gene coexpression was performed using WGCNA. Genes were correlated with plant physiological traits (from Objective 1) and metabolites (from Objective 2) measured on the same plants. RT-qPCR validation of expression of phenylpropanoid genes was performed to confirm patterns in RNA-Seq data. For the low lignin lines, we Demonstrated that the defense response included plant hormone signaling, cell wall synthesis genes, and genes encoding components of protein processing. Alterations in cell wall composition in bmr12 affect general immunity by influencing cell wall phenylpropanoid composition and plant hormone signaling, leading to a faster defense response. Some defense pathways appear to be activated or enhanced by drought.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
Khasin, M., Bernhardson, L. F., ONeill, P. M., Palmer, N. A., Scully, E. D., Sattler, S. E. and Funnell-Harris, D. L. Pathogen and drought stress affect cell wall and phytohormone signaling to shape host responses in a sorghum COMT bmr12 mutant. BMC Plant Biology (In press).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Khasin, M., Bernhardson, L. F., O'Neill, P. M., Scully, E. D., Tetreault, H. M., Sattler, S. E. and Funnell-Harris, D. L. 2021. Overexpression of a phenylpropanoid master regulator impacts responses to stalk rots. Presented at Plant Health 2021 virtual conference, August 2-6, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Funnell-Harris, D. L. Modifying cell wall phenylpropanoids of small grains for increased usability and disease and drought resistance. Presented during Complex Biosystems Seminar Series, University of Nebraska, Sept. 3, 2020.
|
Progress 09/01/19 to 08/31/20
Outputs
Target Audience:Dr. Maya Khasin presented research supported by this NIFA grant to the University of Nebraska, Plant Pathology Seminar series, titled "Transcriptomics responses of sorghum lines to stalk pathogens", 2-3-2020. Dr. Funnell-Harris was invited to speak at the Sorghum Improvement Conference of North America (SICNA), Dallas, TX, March 23 - 25, 2020,to provide updates on this NIFA grant research (see http://sicna.net/docs/2020_Agenda.pdf). However,our employer, USDA-Agricultural Research Service, cancelled all non-essentialtravelfor March 11, 2020 onward and the meeting was subsequently cancelled on March 13, 2020. The meeting has been rescheduled tenatively for October 26 - 30, 2020 with the invitation extended. Dr. Maya Khasin and Dr. Funnell-Harris presented research supported by this grant in the virutual "Plant Health 2020" meeting (https://www.apsnet.org/meetings/annual/planthealth2020/Pages/default.aspx), Aug 3 -7, 2020,in posters titled "Transcriptional response of sweet sorghum lines varying in resistance to stalk rot pathogens" and "Association of post-flowering non-senescence (Stay-green) anddhurrin levels in response to stalk rot pathogens in Sorghum bicolor," respectively. Poster content was available to all registrants for the on-line meeting. The meeting included academic, industry,goverment and NGO researchers and extension specialists, nationally and internationally. Dr. Funnell-Harris is invited to present the seminar titled "Modifying cell wall phenylpropanoids of small grains for increased usability and disease and drought resistance" for the Complex Biosystems Seminar Series, University of Nebraska on Sept. 3, 2020. Dr. Funnell-Harris has had an open invitation to share sorghum research with the Nebraska Grain Sorghum Board (www.nebraskasorghum.org). Normally, such meetings have been scheduled for Spring, but this year the Sorghum Board's business meetings were virtual and did not include presentations due to concerns with COVID19. Dr. Funnell-Harris and Dr. Maya Khasin have been primarily teleworking since March 26, 2020, due to concerns of COVID19. To my knowledge, sorghum field days and other sorghum-specific events (such as SICNA) and other meetings and events of larger-scope have been cancelled or postponed. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Post doctoral associate-Dr. Maya Khasin has had further professional training under the guidance of the co-PIs of this grant. She has furthered her analytical skills, trained in manuscript preparation, guidance in presentation of her research during University Seminars and at professional meetings. Additionally, Dr. Khasin has recieved informal training in the field of Plant Pathology, expanding her research scope. She has demonstrated adept abilities to develop in planta experiments with different diseases, including proper experimental design. She has in turn provided training to technologists and undergraduate and graduate students. Technologist-Ms. Lois Bernhardson has received extensive training culturing and maintaining plants for experimental conditions,in designing and conducting greenhouse and field experiments, culturing and maintenance of microorganisms, taking detailed and accurate measurements and preparation of documents for analyses. Additionally, Ms. Bernhardson has become an invaluable addition and guide for training undergraduate students in the safe and correct uses of experimental equipment and conducting experimental techniques. Ms. Bernhardson would now be wholly qualified to independentlymanage a small laboratory. Undergraduate thesis-Mr. Mark Kilts conducted his undergraduate thesis under this project, fulfilling requirements for graduation from University of Nebraska's Department of Chemistry. Mr. Kilts performed a project in which he grew plants in the greenhouse and the field, and harvested tissues for High Performance Liquid Chromotography (HPLC) to determine dhurrin levels in 14 plant lines. This involved reading the literature and maximizing the extraction and HPLC protocols for our equipment. Because of his high skill level with analytical equipment, he has been given another assignment in our laboratory to analyze grain starches (unrelated to the current project). Undergraduate and high school students-an additional three undergraduate and one high school students were trained in basic plant biology, microbiology and molecular biology techniques, including greenhouse and field cultivation and maintenance of plants, preparation and culturing of microorganisms, DNA extraction and PCR molecular identification techniques, and electronic data base management. How have the results been disseminated to communities of interest?Dr. Maya Khasin presented research supported by this NIFA grant to the University of Nebraska, Plant Pathology Seminar series, titled "Transcriptomics responses of sorghum lines to stalk pathogens", 2-3-2020. Dr. Funnell-Harris was invited to speak at the Sorghum Improvement Conference of North America (SICNA), Dallas, TX, March 23 - 25, 2020,to provide updates on this NIFA grant research (see http://sicna.net/docs/2020_Agenda.pdf). However,our employer, USDA-Agricultural Research Service, cancelled all non-essentialtravelfor March 11, 2020 onward and the meeting was subsequently cancelled on March 13, 2020. The meeting has been rescheduled tenatively for October 26 - 30, 2020 with the invitation extended. Dr. Maya Khasin and Dr. Funnell-Harris presented research supported by this grant in the virutual "Plant Health 2020" meeting (https://www.apsnet.org/meetings/annual/planthealth2020/Pages/default.aspx), Aug 3 -7, 2020,in posters titled "Transcriptional response of sweet sorghum lines varying in resistance to stalk rot pathogens" and "Association of post-flowering non-senescence (Stay-green) anddhurrin levels in response to stalk rot pathogens in Sorghum bicolor," respectively. Poster content was available to all registrants for the on-line meeting. The meeting included academic, industry,goverment and NGO researchers and extension specialists, nationally and internationally. Dr. Funnell-Harris is invited to present the seminar titled "Modifying cell wall phenylpropanoids of small grains for increased usability and disease and drought resistance" for the Complex Biosystems Seminar Series, University of Nebraska on Sept. 3, 2020. Dr. Funnell-Harris has had an open invitation to share sorghum research with the Nebraska Grain Sorghum Board (www.nebraskasorghum.org). Normally, such meetings have been scheduled for Spring, but this year the Sorghum Board's business meetings were virtual and did not include presentations due to concerns with COVID19. Dr. Funnell-Harris and Dr. Maya Khasin have been primarily teleworking since March 26, 2020, due to concerns of COVID19. To my knowledge, sorghum field days and other sorghum-specific events (such as SICNA) and other meetings and events of larger-scope have been cancelled or postponed. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: 1. Completion of endophyte assessments in the greenhouse. Completion of gene expression and phenylpropanoid metabolite analyses. Drafting of manuscript. 2. Completion of analyses of senescent/nonsenescent resistant lines. Completion and submission of manuscript. 3. Completion of drought assays of senescent/nonsenescent resistant lines. Completion of gene expression and phenylpropanoid metabolite analyses. Drafting of manuscript. Objective 2: Conduct phenolic metabolite analyses as needed to identify potential genes or gene networks involved in increased resistance to stalk pathogens under drought conditions in endophyte infected lines and senescent/nonsenescent resistant lines. Objective 3: Conduct real time RTqPCR as needed to confirm potential genes involved in increased resistance to stalk pathogens under drought conditions in endophyte infected lines and senescent/nonsenescent resistant lines. Objective 4: 1. Conduct global gene expression analyses on endophyte colonized plant tissues; draft manuscript. 2. Conduct global gene expression analyses on senescent/nonsenescent resistant lines infected with stalk pathogens under drought conditions. Draft manuscript.
Impacts
What was accomplished under these goals?
Impact: A major component of the plant cell wall is the resilient phenolic polymer, lignin, which can protect against pathogens. Stalk rotting fungi reduce biomass yield, and their destructiveness increases under drought. The stalk rot pathogens used in this study cause the sorghum diseases charcoal rot, and Fusarium stalk rot that can cause lodging, reducing biomass and impairing harvest. We have shown that brown midrib mutants, with less lignin, either exhibit no change in response to stalk pathogens, or the mutant plants are more resistant. The bmr12 line, more resistant to stalk pathogens under drought is a valuable tool to identify genes, traits and compounds that contribute to resistance. We showed that alterations in the plant cell wall can affect plant responses to diseases due to changes in compounds and in hormone signaling occurring throughout the plant, leading to the action of plant defense strategies that may also be activated or enhanced by drought. We have developed lines with increased enzyme activity in the lignin synthesis pathway. Lines overexpressing a factor (SbMyb60) that controls genes in lignin synthesis were more resistant when infected by the Fusarium stalk rot pathogen under drought. We also have identified four more lines with increased resistance to stalk pathogens. One line has low levels of the toxic compound, dhurrin, thus it could be used in breeding for biomass sorghum with animal feed end-uses. We have demonstrated that pathways involved in plant traits, and how they interact with the environment, are involved in determining how quickly plants respond to diseases under stressful conditions. Objective 1: A).Brown midrib (low lignin) lines-endophytic growth The experiment proposed in the original grant did not yield adequate tissues. We have developed an alternate protocol to assess extent of asymptomatic growth in stalks. Seedlings are inoculated at the base of the stalk,water-deficit treatments are applied on half the plants,then asymptomatic stalk tissues with the fungus are isolated for further analyses. Lesion length data are measured, when visible. If not visible, response was positive if fungus was recovered from asymptomatic tissue. Logistic regression will be used to determine whether genotype and watering condition influence asymptomatic growth. Using this procedure, adequate tissues can be obtained for transcriptional, phenolic metabolite and microscopic analyses. B) High lignin lines Transgenic lines constitutively expressing either a factor (SbMyb60) that regulates genes for enzymes in lignin biosynthesis or an enzyme (SbCCoAOMT) central to the pathway were screened under well-watered and water deficit conditions for responses to Fusarium stalk rot and charcoal rot. The results revealed that the SbMyb60 lines inoculated with the Fusarium stalk rot fungus were more resistant to the disease under both well-watered and water-deficit conditions. Overexpression lines and the wild-type line were inoculated with either the Fusarium stalk rot or the charcoal rot pathogen. Lesions were measured at 0, 3 and 13 days after inoculation. Analysis of variance (ANOVA) on lesions with main affects being overexpression construct, inoculum, water treatment and timepoint. The results demonstrated that there was significantly less disease on SbMyb60 overexpression lines under both well-watered and water-deficit conditions inoculated with either pathogen. There were no differences between wild-type and SbCCoAOMT overexpression lines under all conditions. The SbMyb60 overexpression lines have increased resistance to stalk rot pathogens under both ideal conditions and drought stress. Our finding will provide valuable information on genes and pathways associated with increased resistance under multiple biotic and abiotic stresses that could be used in breeding for stalk rot resistant lines under drought. C) & D) Responses of drought tolerant and sensitive lines, and lines reported to be resistant to stalk rot pathogens Four lines with increased resistance to both Fusarium stalk rot and charcoal rot were identified. Lesion lengths were determined, dhurrin levels were determined using High Performance Liquid Chromatography (HPLC0 and senescence levels were scored on a 1 (nonsenescent) to 5 (completely senesced) scale. ANOVA was performed with lesion length the only fixed effect and main effects being repetition, replications within repetition, genotype, and inoculum. Dhurrin assays showed that PI267379 had significantly less dhurrin than all other lines while PI533882 and PI534053 had levels similar to PI534133 and a check. PI534133 was non-senescent while the other three resistant lines had low senescence. This study identified a line with improved resistance to stalk pathogens and low levels of dhurrin. Line PI267379 will prove invaluable for breeding for biomass with resistance to stalk pathogens and low dhurrin levels for uses as animal feed and bioenergy. Objective 2: 1) Phenylpropanoid metabolism and phytohormone signaling were surveyed in wild-type and the brown midrib mutant, bmr12, inoculated with the Fusarium stalk rot pathogen or the control under well-watered and water deficit. 2) Phytohormone analysis was conducted at the University of Nebraska. Phenolic analysis was conducted in our laboratory. 3) Metabolite analysis was performed in the R programming environment. The bmr12 plants were correlated with decreased levels of hydroxycinnamic acids. They had elevated levels of salicylic acid (SA), and jasmonic acid (JA). Pathogen-infected Wild-type plants had higher concentrations of soluble syringic acid compared to controls, but not in bmr12 plants. The bmr12 plants had significantly elevated levels of sinapic acid, contrary to a previous model. 4) The bmr12 plants had elevated levels of plant defense signaling molecules, SA and JA, and an altered hydroxycinnamic acid profile. The bmr12 mutation may provide information to identify biochemical pathways that can be modified for development of stalk rot- and drought-resistant sorghum biomass. Objective 3: Global gene expression has replaced high-throughput gene expression. Objective 4: Genes were identified by response to lesion formation at 13 DAI. For RNA, freeze-dried and ground plant tissue samples were extracted then sent to the University of Nebraska for further processing. A consensus network was constructed for gene expression across the three DAI. Signed networks were constructed by DAI. Module-trait Pearson correlation was calculated and adjusted for false discovery rates. KEGG enrichment in modules identified through WGCNA was calculated. Transcription factor enrichment was calculated. Putative priming and smaller lesion-associated genes were closely associated with bmr12. Priming genes cluster with sinapic acid and Fusarium stalk rot inoculation. Smaller lesion and susceptibility genes also cluster with the water deficit treatment, and bmr12. Across all three DAI, genes associated with shorter lesions were positively correlated with bmr12 and controls. Altering cell wall structure enables the study of metabolic and transcriptomic alterations on drought and disease response.The bmr12 mutation impacted genes involved in signal transduction, RNA and protein processing and turnover, and transcription and translation.Patterns of cell wall biosynthetic genes and regulatory pathways that respond to both drought and disease indicates that the cell wall connects defense, growth, and development. Drought may prime bmr12 plants for disease resistance through activation of defense pathways. Activation of pathways associated with priming may be a way to increase disease resistance in crops while providing reduced lignin lines for bioenergy or forage production.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Khasin, M., Bernhardson, L. F., ONeill, P. M., Palmer, N. A., Scully, E. D., Sattler, S. E. and Funnell-Harris, D. L. Pathogen and drought stress affect cell walls and phytohormone signaling to shape host responses in a sorghum COMT bmr12 mutant. Target journal: BMC Plant Science
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Khasin, M., ONeill, P. M., Sattler, S. E. and Funnell-Harris, D. L. 2020. Transcriptional response of sweet sorghum lines varying in resistance to stalk rot pathogens. Plant Health 2020, virtual, Aug. 3 - 7, 2020 (poster).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Funnell-Harris, D. L., Sattler, S. E., O'Neill, P. M., Bernhardson, L. F., Kilts, M. and Khasin, M. Association of post-flowering non-senescence (Stay-green) and dhurrin levels in response to stalk rot pathogens in Sorghum bicolor. Plant Health 2020, virtual, Aug 3 - 7, 2020.
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Progress 09/01/18 to 08/31/19
Outputs
Target Audience:Presentations at University of Nebraska (UNL), Department of Plant Pathology (9-17-2018; invited talk; Lincoln NE) and Plant Science Initiative (10-12-2018; accepted poster; Nebraska City, NE): Dr. M. Khasin presented research supporting extensive crosstalk between metabolic pathways in sorghum undergoing pathogen and abiotic stress to researchers and extension faculty from Nebraska and Arizona. 2019 Genomic Sciences Program Annual PI meeting (2-26-2019) invited plenary session speaker and accepted poster: Dr. Funnell-Harris presented scope of research into resistance of biomass for bioenergy production to stalk pathogens to a diverse audience of scientists. Established collaboration with Dr. T. Jamannfrom University of Illionois. Nebraska Grain Sorghum Board: on 5-6-2019, Dr. Funnell-Harris and Dr. S. Sattler met with director, Mr. N. Blum, to discuss general research directions and goals. On 6-5-2019, Dr. Funnell-Harris presented research to board members as well as UNL faculty, extension and students. Received funding (~$4,000) to analyze waxy (low amylose) hybrids. Invited presentations at Australian Summer Grains Conference 2019, AusSoRGM (Gold Coast; 8-9 and 8-11-2019) and 2019 Centre for Crop Health Seminar Series, University of Southern Queensland (USQ, Toowoomba; 8-12-2019): Dr. D. Funnell-Harris presented research on effects of drought and stalk pathogens on fitness, physiological traits and gene expression in sorghum biomass production. The audiences included Australian, Brazilian, Canadian, Irish, Indian and U. S. researchers, students, extension specialists, producers,and commodity board and industryrepresentatives. The visit to USQ included interactions with collaborator Dr. A. Sparks. Plant Health 2019, 8-3 through 8-7-2019, Cleveland, OH: Dr. Khasin and Dr. Funnell-Harris presented aspects of grant-funded research in accepted talk and poster, respectively, regarding responses of sorghum lines modified in lignin biosynthesis. The conference, the annual meeting of the American Phytopathological Society, attracts national and international researchers, extension specialists and industry researchers and representatives. Changes/Problems:Objective 4: We identified two sorghum genotypes, one highly susceptible and one highly resistant, to bothMacrophomina phaseolinaandFusarium thapsinuminoculation. We are currently conducting global gene analyses of these interactions as well. Although not specifically identified in the original proposal, it is highly relevant to this research and should provide complementary findings. What opportunities for training and professional development has the project provided?A total of 5 undergraduate students worked on this project during the past year. Five of the students (University of Nebraska (UNL), Nebraska Wesleyan, and Concordia UniversityNebraska) were informally trained to perform different aspects of the grant proposal experiments including: culturing and maintaining plants in the greenhouse, preparing materials to perform greenhouse and field inoculations, conducting inoculations, proper collection and preservation of plant materials for downstream analysis, preparation of materials for extraction of metabolites and RNA, prepartion of culture media and culturing of fungal isolates (i. e. sterile technique). One of these five students, Mr. M. Kilts, also had a formal agreement through UNL to perform a specific summer project in which a protocol for detection of the cyanogenic glucoside, dhurrin was detected using HPLC. Dr. M. Khasin has conducted full-time research on this project, performing bioinformatic analyses on thelow lignin lines and a second project involving lines identified to be resistant and susceptible to both stalk pathogens. How have the results been disseminated to communities of interest?Presentations at University of Nebraska (UNL), Department of Plant Pathology (9-17-2018; invited talk; Lincoln NE) and Plant Science Initiative (10-12-2018; accepted poster; Nebraska City, NE): Dr. M. Khasin presented research supporting extensive crosstalk between metabolic pathways in sorghum undergoing pathogen and abiotic stress to researchers and extension faculty from Nebraska and Arizona. 2019 Genomic Sciences Program Annual PI meeting (2-26-2019) invited plenary session speaker and accepted poster: Dr. Funnell-Harris presented scope of research into resistance of biomass for bioenergy production to stalk pathogens to a diverse audience of scientists. Established collaboration with Dr. T. Jamannfrom University of Illionois. Nebraska Grain Sorghum Board: on 5-6-2019, Dr. Funnell-Harris and Dr. S. Sattler met with director, Mr. N. Blum, to discuss general research directions and goals. On 6-5-2019, Dr. Funnell-Harris presented research to board members as well as UNL faculty, extension and students. Received funding (~$4,000) to analyze waxy (low amylose) hybrids. Invited presentations at Australian Summer Grains Conference 2019, AusSoRGM (Gold Coast; 8-9 and 8-11-2019) and 2019 Centre for Crop Health Seminar Series, University of Southern Queensland (USQ, Toowoomba; 8-12-2019): Dr. D. Funnell-Harris presented research on effects of drought and stalk pathogens on fitness, physiological traits and gene expression in sorghum biomass production. The audiences included Australian, Brazilian, Canadian, Irish, Indianand U. S. researchers, students, extension specialists, producers,and commodity board and industryrepresentatives. The visit to USQ included interactions with collaborator Dr. A. Sparks. Plant Health 2019, 8-3 through 8-7-2019, Cleveland, OH: Dr. Khasin and Dr. Funnell-Harris presented aspects of grant-funded research in accepted talk and poster, respectively, regarding responses of sorghum lines modified in lignin biosynthesis. The conference, the annual meeting of the American Phytopathological Society, attracts national and international researchers, extension specialists and industry researchers and representatives. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: -Complete bioassays with overexpression lines and field-resistance lines. -Complete manuscripts on field-resistance lines, including recent data on dhurrin levels (a trait associated with pathogen resistance) Objective 2: -Conduct phenolic and hormone analyses with overexpression lines Objective 3: -As needed, conduct any RTqPCR to support findings from metabolic and global gene expression analysis of overexpression lines. Objective 4: -Conduct global gene expression analysis of overexpression lines. -Preparation and submission of paper with overexpression lines, inoculated with stalk pathogens under well-watered and water-limited conditions.
Impacts
What was accomplished under these goals?
Impact: Because sorghum is naturally drought-resistant, it is an ideal bioenergy crop for marginal lands that are not normally used for food production. The first line of defense in the plant cell against pathogens is the cell wall surrounding its contents. The primary cell wall is primarily composed of sugar polymers while the secondary cell wall is composed of the more resilient phenolic polymer, lignin. Lignin isrequired for plant fitness. Stalk rotting fungi reduce biomass yield, and the destructiveness of these diseases increase under drought conditions. The stalk rot pathogens utilized in this study were Macrophomina phaseolina, that causes charcoal rot, and Fusarium thapsinum, that causes Fusarium stalk rot.Previous work has shown that mutations inbrown midrib (bmr) genes, which result in lower lignin levels, either result in no change in response to stalk pathogens, or the mutant plants are even more resistant to these pathogens. For example, the mutant line, bmr12, is more resistant to stalk pathogens under drought conditions. This observation is counter to previously-held ideas of the role of lignin in resistance to pathogen or environmental stresses. The bmr12 line is proving to be a valuable tool to identify genes, physiological traits and metabolic networks that contribute to this resistance. We previously had developed lines withincreased enzyme activity in the lignin pathway using a transgenic approach. Lines overexpressing a transcription factor (SbMyb60) and four different enzymes in lignin biosynthesis (SbPAL, Bmr2, SbC3H and SbCCoAOMT) were screened in the greenhouse for resistance to stalk pathogens. The SbMyb60 and SbCCoAOMT lines were also screened under field conditions. The SbMyb60 lines exhibited increased resistance under both conditions. We also screened eleven lines that have previously exhibited field resistance to stalk pathogens and identified four that have increased resistance to stalk pathogens under greenhouse conditions.Our recent results have demonstrated that pathways involved in plant physiological traits, and how theyinteract with the environment, are hugely involved in determining how quickly plants respond to pathogens under stressful conditions. Objective 1: 1) Major activities: Responses to drought in low lignin lines were completed. Responses to drought in lignin overexpression lines were delayed due to loss of two repetitions during the government shut-down; six of eight neededrepetitions are completed. Lines previously showing field resistance were screened with both pathogens in the greenhouse and 4 resistant lines have been identified. Assessment of responses under drought has commenced. 2) Data collected: lesion lengths, endophytic growth, soil moisture levels and physiological responses. 3) Summary statistics and results: A general linear model fit the data.At 13 days after inoculation (DAI), when comparingMacrophomina-inoculated plants under well-watered conditions, bmr6 had significantly smaller lesions than wild-type. When considering between water treatments with the same inoculum and plant genotype, it was found that for bmr12 inoculated with each pathogen, the plants under water-limitation conditions had significantly shorter lesions than when inoculatedunder well-watered conditions. 4) Key outcomes: -Thebmr12line is resistant to Fusariumand?Macrophomina under drought, providing a valuable tool to identify genes or pathways involved in this increased resistance. -The SbMyb60 overexpression lines exhibit resistance to both pathogens under well-watered conditions in the greenhouse; andtoFusariumin the field. -Four lines previously exhibiting field resistance were resistant to both pathogens under greenhouse conditions. Objective 2: 1) Major activities: phenolics and plant hormones were assessed forbmr12and wild-type plants inoculated with Fusarium, 3 DAI. 2) Data collected: compounds and amounts were recorded. 3)Summary statistics and results:Correlation analysis revealed metabolites and phytohormones wereassociated with plant genotype, lesion size, and physiology. 4)Key outcomes: -Plants with bmr12had greater amounts of several plant hormones involved in defencethan wild-type. Objective 3:As indicated in previous reports, global gene expression has replaced high-throughput gene expression. Objective 4: 1) Major activities: RNASeq and analyses were performed on select low lignin and wild-type tissues inoculated with pathogens under well-watered and water-limited conditions. 2) Data collected: Sequences of expressed genes and comparisons with known genes in databases. 3) Summary statistics and results:WGCNA analysis identified coexpression modules for which module-trait and gene-trait correlations were calculated. KOBAS was then used to calculate KEGG pathway enrichment. PlantRegMap was used to analyze transcription factor relationships.The transcriptomic survey implicated crosstalk between disease responses, the cell wall, and circadian rhythm, particularly regarding lignin subunits and plant hormones. 4) Key outcomes: -Drought may prime a defense response in bmr12plants. -Flavonoid biosynthetic enzymes may bridge drought tolerance with reduced lesionsin bmr12?; these genes may be potential targets for increasing resistance to stalk pathogens under drought.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Funnell-Harris, D., Khasin, M. and Sattler, S. 2019. Resistance to Stalk Pathogens for Bioenergy Sorghum. 2019 Genomic Sciences Annual PI Meeting. In: US Department of Energy 2019 Genomic Sciences Annual PI Meeting Abstract Book, Feb. 24 - 27, 2019, Tysons, VA.
Funnell-Harris, D., Sattler, S., Khasin, M. and Prom, L. 2019. Responses of sorghum improved for end-uses to pathogens. Australian Summer Grains Conference 2019. July 8 - 10, 2019, Gold Coast, Australia. https://www.dropbox.com/s/hgx9untyh5u1agb/ASGC_2019_Program_Speaker%20Consent.pdf?dl=0
Funnell-Harris, D. L., Sattler, S. E., Khasin, M. and Wegulo, S. N. 2019. Responses of sorghum and wheat modified in phenylpropanoid metabolism to fungal pathogens. 2019 Centre for Crop Health Seminar Series, University of Southern Queensland. July 12, 2019, Toowoomba, Australia.
Funnell-Harris, D. L., Sattler, S. E., O'Neill, P. M., Gries, T. and Tetreault, H. M. 2019. Response of sorghum enhanced in monolignol biosynthesis to stalk rot pathogens. Plant Health 2019. American Phytopathological Society Annual Meeting. Aug. 3 - 7, 2019. Cleveland, OH.
Khasin, M., Bernhardson, L. F., Patrick, P. M., Palmer, N. A., Funnell-Harris, D. L. and Sattler, S. E. Drought stress shapes host responses to pathogen infection. Plant Health 2019. American Phytopathological Society Annual Meeting. Aug. 3 - 7, 2019. Cleveland, OH.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Funnell-Harris, D. L., Sattler, S. E., O'Neill, P. M., Gries, T., Tetreault, H. M. and Clemente, T. E. 2019. Response of sorghum lines overexpressing monolignol biosynthesis genes to stalk pathogens. Plant Disease DOI: 10.1094/PDIS-09-18-1622-RE
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:Presentations of recent research for this proposal were made to scientists and researchers at the following venues: -Dr. D. Funnell-Harris was invited to present recent research, including that on bioenergy sorghum, to the Departments of Agronomy and Horticulture at the University of Nebraska, Lincoln, Sept. 15, 2018. The audience included faculty, research associates and students and was streamed to other University locations (https://agronomy.unl.edu/responses-sorghum-and-wheat-modified-increased-usability-pathogens). -2018 Genomic Science Program Annual Principal Investigator Meeting (Feb. 25 - 28, 2018)-attended by numerous scientists, including those undertaking genomic investigations of bioenergy feedstocks. Dr. D. Funnell-Harris presented recent results and interacted with numerous scientists in many disciplines involved with genomics and bioenergy. -The American Society of Plant Biologists (Montreal, Quebec, July 14 - 18, 2018)-Dr. M. Khasin (Postdoctoral associate) presented recent research results and interacted with numerous scientists from the U. S., Canada and internationally. These researchers included those in bioinformatics, pathology, biochemistry, etc., all related to the plant sciences. -International Congress of Plant Pathology (Boston, MA, July 29 - Aug. 3, 2018)-Dr. Funnell-Harris attended this internationally renowned meeting, presenting recent research and interacting with plant pathologists with expertise in bioinformatics, biochemistry, molecular biology, etc. Additionally, Dr. Funnell-Harris attended the Sorghum Improvement Conference of North America meeting (St. Louis, Jan. 28 - 31, 2018) and had valuable discussions with scientists in academia, USDA-ARS and NGOs (e. g. Danforth Center), researchers in industry, including biofuels, and producers and directors on state and national sorghum boards, including National Sorghum Producers and Sorghum Checkoff. Changes/Problems:Objective 1: Bioassays are continuing as expected. Because of the relativelyreduced density of tissues in peduncles as compared with other areas of the stalk, additional repetitions are being performed to ensure adequate materials are available for later analyses. Objective 2: Metabolomic analyses (GC-MS)on phenolics will be performed in-house. Other analyses, such as for plant hormone levels or flavonoids, will need to beconducted atappropriate facilities (for fee). Objective 3: As indicated in previous report, high throughput gene expression analysis is unlikely to be performed. However, RT-qPCRwill be performed in-house as needed to further elucidate expression levels of individual genes or genes from gene networks. Objective 4: RNAseq analyses will proceed as expected. Materials for RNA-seq will be chosen on the basisof results from bioassays in Obj. 1. Global gene expression will be the driver in determining which metabolic analyses will be performed, whether gene expression will enhance results, and what other types of analyses (e. g. protein levels, biochemical) would be informative. What opportunities for training and professional development has the project provided?As of Jan. 1, 2018, we hired a post-doctoral research molecular biologist with expertise in bioinformatics involving signalling in algae. Since Dr. Khasin has joined the laboratory, she has been trained in the basics of plant-microbe interactions. Dr. Khasin is enjoying conducting RNAseq with a complex system involving bothbiotic and abiotic stresses. We also have hired a term technologist who is assisting Dr. Khasin with conducting the biological assays and with sample preparation. Ms. Bernhardson plans to attend graduate school in future and is obtaining extensive experiences that will help her achieve that goal. Additionally, we have trained three undergraduate students,two pre-medical and one pre-pharmacy. One of the pre-medical students is conducting his honor thesis with us. The pre-pharmacy student has strong training in physical chemistry but is expanding his knowledge-base in biology by studying in my laboratory. How have the results been disseminated to communities of interest?Presentations of recent research for this proposal were made to scientists and researchers at the following venues: -Dr. D. Funnell-Harris was invited to present recent research, including that on bioenergy sorghum, to the Departments of Agronomy and Horticulture at the University of Nebraska, Lincoln, Sept. 15, 2017. The audience included faculty, research associates and students and was streamed to other University locations (https://agronomy.unl.edu/responses-sorghum-and-wheat-modified-increased-usability-pathogens). -2018 Genomic Science Program Annual Principal Investigator Meeting (Feb. 25 - 28, 2018)-attended by numerous scientists, including those undertaking genomic investigations of bioenergy feedstocks. Dr. D. Funnell-Harris presented recent results and interacted with numerous scientists in many disciplines involved with genomics and bioenergy. -The American Society of Plant Biologists (Montreal, Quebec, July 14 - 18, 2018)-Dr. M. Khasin (Postdoctoral associate) presented recent research results and interacted with numerous scientists from the U. S., Canada and internationally. These researchers included those in bioinformatics, pathology, biochemistry, etc., all related to the plant sciences. -International Congress of Plant Pathology (Boston, MA, July 29 - Aug. 3, 2018)-Dr. Funnell-Harris attended this internationally renowned meeting, presenting recent research and interacting with plant pathologists with expertise in bioinformatics, biochemistry, molecular biology, etc. Additionally, Dr. Funnell-Harris attended the Sorghum Improvement Conference of North America meeting (St. Louis, Jan. 28 - 31, 2018) and had valuable discussions with scientists in academia, USDA-ARS and NGOs (e. g. Danforth Center), researchers in industry, including biofuels, and producers and directors on state and national sorghum boards, including National Sorghum Producers and Sorghum Checkoff. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: -Complete time-course bioassays with low lignin (bmr) lines and overexpression lines. -Choose from among Staygreen lines and field-resistant lines(those demonstrating best resistance) and begintime course assays under well-watered and reduced water conditions. -Prepare manuscript on Staygreen and field-resistant lines (analysis of dhurrin levels, Staygreen trait, and disease responses). Objective 2: -Conduct and complete analyses of phenolics (GC-MS; in-house), hormones (University of Nebraska) and flavonoids (University of Missouri) on bmr12 and wild-type plants. -Use results from global gene expression from bmr6 and wild-type to determine which analyses would be most fruitful. -Initiate metabolomic analyses of overexpression lines as compared with wild-type, inoculated with two stalk pathogens, under drought stress and well-watered conditions, based on results from global gene expression. Objective 3: For the low lignin lines or the overexpression lines, if global gene expression and/or metabolomics need confirmation, the RT-qPCR will be conducted on select genes in-house. Objective 4: -Completion of analyses of global gene expression, metabolomics and biochemical analysesfor bmr12 and wild-type lines, under pathogen and drought stress following early inoculation; complete and submit manuscript. -Continue analyses of bmr6 and wild-type and determine which metabolic/gene expression/protein level analyses/biochemical analyses, etc. would be needed to clarify or confirm RNAseq results. Prepare and submit manuscript -Use results from bioassays to inform what materials from among low lignin or overexpression lines will be analyzed for global gene expression.
Impacts
What was accomplished under these goals?
Impact: Because sorghum is a naturally drought-resistant crops, itis an ideal bioenergy crop for marginal lands that are not normally used for food production. Reducing lignin increases the conversion efficiency of biomass into sugars, but lignin is important for plant defenses against pathogens. Stalk rot fungi reduce biomass yield, and the destructiveness of these diseases increase under drought conditions. Mutations inbrown midrib(bmr) genes, which result in lower lignin levels, change the susceptibility of sorghum to these stalk rot diseases. Two bmr lines and the normal line were tested for susceptibility to Fusarium stalk rot and charcoal rot diseases under adequate water and reduced water conditions. Neither bmr line showed increased susceptibility under the two water conditions, but one line, calledbmr12, was actually more resistant than the normal line under reduced water. The source of increased resistance in bmr12 is currently being investigated as a potential tool for identification of molecular markers for resistance to these diseases under water stress in sorghum. Objective 1: 1) Major activities completed: completion of greenhouse experiments inoculating reduced lignin (bmr) lines with the stalk rot pathogens, Fusarium thapsinum and Macrophomina phaseolina. A total of 14 repetitions were needed to obtain adequate results and materials for later experiments. Greenhouse experiments with overexpression lines (SbMyb60and SbCCoAOMT), Staygreen lines and lines exhibiting field resistance to stalk pathogens are ongoing. 2) Time courses were conducted for bmr lines and overexpression lines. Mean lesion lengths and fungal endophytic growth were determined at 0, 3 and 13 days after inoculation (dai).Staygreen lines and field-resistant lines are being screened for responses to the two pathogens. Mean lesion lengths will be determined. 3) Overall, there were significant differences in mean lesion lengths resulting on two bmrlines (bmr6andbmr12) at 13 dai with each fungus under adequate water or water deficit conditions as compared to wildtype plants with these treatments. In particular, bmr6 plants under adequate water and bothbmr6and bmr12 plants under reduced water had significantly smaller mean lesion lengths than wild-type 13 days after inoculations with M. phaseolina. No significant differences were apparent after inoculations with F. thapsinum under adequate water, but bothbmr lines had significantly smaller mean lesion lengths than wildtype under reduced water condition. Interestingly, bmr12 plants had significantly smaller mean lesion lengths under water deficit than under adequate water when inoculated with either pathogen, counter to expected response to stalk pathogens under water stress. Across both water conditions, bmr12plants had reduced F. thapsinumsurvival within lesions. At 3-cm beyond the lesion border, there was reduced pathogen survival in both bmr6 (F. thapsinumandM. phaseolina) andbmr12(M. phaseolina) plants as compared with wild-type. 4) These results suggested that reduced survival of the pathogens within bmr6 and bmr12stalks is likely due to changes induced by impaired monolignol biosynthesis. Objective 2: 1) Collection of peduncle materials for GC-MS of phenolic metabolites for each treatment and at each time point, and assessment of plant hormones and flavonoids by an independent laboratory. Since peduncle materials are less dense and in less abundance than stalk or leaf materials, we are conducting test runs to determine the minimum amount of material needed for detection of metabolites. 2) Mean lesion lengths will be determined. Amounts of phenolic metabolites from monolignol biosynthesis will be determined. 3) No results to report. 4) No conclusions at this time. Objective 3) As indicated in previous report, global gene expression will replace high-throughput gene expression (Fluidigm). If RNAseq results inform us that quantitative PCR on individual genes or genes within gene networks would be valuable for validation of results, it will then be performed within our laboratory. Objective 4) 1) RNAseq analyses has been performed using bmr6, bmr12 and wild-type plant tissues inoculated with either fungus or the control, 3 dai under well-watered and water-deficit conditions. At this time, analyses has focused onbmr12 and wild-type tissues. 2) Approximately 10 X 106 reads from eachof4 replicates of factorial (plant genotype X water treatment X inoculum treatment) (total of 72 samples) for 3 dai tissues. Further analyses have focused onbmr12and wild-type tissues. 3) The key findings are: -RNAseq samples cluster according to genotype. -Under well-watered conditions,bmr12exhibits an increased abundance of genes related to chlorophyll biosynthesis, central metabolism, carbon fixation and dhurrin biosynthesis. -Under drought conditions,bmr12 appears to attenuate brassinosteroid (BR) and abscisic acid (ABA) signaling pathways. -Under drought conditions, differences betweenbmr12and wild-type are driven by redox-active genes, flavonoids/phenylpropanoids and chitinases. -The majority of differentially expressed genes within a given lesion was not differentially expressed in all pathogen-, genotype- or watering-conditions. -Transcriptional differences between lesion-treatments are related to chromatin remodeling, cell wall biosynthesis and remodeling, and flavonoid/ phenylpropanoid, phytohormone, carbohydrate/sugar signalling, nodulin or redox-active enzymes. 4)bmr12(impaired in monolignol biosynthesis) has a unique transcriptome compared with the wild-type under drought conditions.bmr12seems to be more relatively drought tolerant, with fewer differentially expressed genes, and is more resistant to the two stalk pathogens under drought. Brassinosteroid, ABA, salicylic acid, and phenylpropanoid/flavonoid pathways are altered relative to the wild-type. The monolignol biosynthesis pathway appears to be an important target to engineer both drought and pathogen tolerance. Manuscript is in preparation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Funnell-Harris, D. L., O'Neill, P. M. and Sattler, S. E. 2018. Field response of near-isogenic brown midrib sorghum lines to fusarium stalk rot, and response of wildtype lines to controlled water deficit. Plant Pathology 67: 1474-1482. doi: 10.1111/ppa.12863
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Funnell-Harris, D., ONeill, P., Sattler, S. Khasin, M. and Scully, E. 2018. Resistance to stalk pathogens for bioenergy sorghum. Abstract presented at the 2018 Genomic Sciences Program Annual Principal Investigator Meeting, Tysons, VA, Feb. 25 28, 2018. (accepted poster).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Funnell-Harris, D. L., O'Neill, P. M. and Sattler, S. E. 2018. Field response of near-isogenic brown midrib sorghum lines to Fusarium thapsinum and effects of controlled water deficit on stalk rot diseases. Abstract presented at the 2018 International Congress of Plant Pathology, Boston, MA., July 29 - Aug. 3, 2018 (accepted poster)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Khasin, M., Bernhardson, L. F., O'Neill, P. M., Gries, T. L., Sattler, S. E. and Funnell-Harris, D. L. 2018. Putative transcriptional mechanisms of resistance to simultaneous drought and stalk rot pathogens in Sorghum bicolor bred for bioenergy. Abstract presented at the Plant Biology Annual Meeting, Montreal, Quebec, Canada, July 14 - 18, 2018 (accepted poster)
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Progress 09/01/16 to 08/31/17
Outputs
Target Audience:The two major target audiences were researchers in bioenergy and plant pathology, and representatives of Nebraska sorghum producers. These were accomplished by attending the following conferences and meetings: -2017 Genomic Sciences Program Annual Principal Investigator (PI) Meeting-oral (invited) and poster (accepted) presentations (2/5-2/8) -2017 American Phytopathological Society Annual meeting, San Antonio, TX, Aug. 5 - 9. Oral and poster presentations (accepted). -Invited to present research for University of Nebraska, Agronomy and Horticulture Dept. Seminar Series (9/15) -Invited to present current and future research on sorghum to Nebraska Grain Sorghum Board, Lincoln, NE(3/17/2017) Changes/Problems:Objective 1, sub-objective 1a-Because of recent availability of germplasm, near-isogenicbmr2line hasbeen included in the pathology assessments. Objective 2: Recent experience with high-throughput targetted gene expression (Fluidigm) has lead to the conclusion that the most cost effective research approach is to conduct small-scale global gene expression on a subset of materials, then, based on analyses of these results, choose genes that are responsive to pathogens and/or drought conditions. Then RTqPCR will be conducted using primers designed to target these genes. Objective 4: Analyses of global gene expression will be conducted earlier on in this research than originally proposed. Results from this analyses, as well as results from biological assessments, should rapidly result in another manuscript. What opportunities for training and professional development has the project provided?Four undergraduate students have been trained to take soil moisture measurements, apply correct water treatments, inoculate plants, and properly collect, process and storeplant tissuesfor future experiments. How have the results been disseminated to communities of interest?Summary of proposed research, results leading to hypotheses development and preliminary results were presented in the following venues: -2017 Genomic Sciences Program Annual Principal Investigator (PI) Meeting, Arlington, VA-oral (invited) and poster (accepted) presentations (2/5-2/8) -2017 American Phytopathological Society Annual meeting, San Antonio, TX, Aug. 5 - 9. Oral and poster presentations (accepted). -Invited to present research for University of Nebraska, Lincoln, Agronomy and Horticulture Dept. Seminar Series (9/15) -Invited to present current and future research on sorghum to Nebraska Grain Sorghum Board, Lincoln, NE(3/17/2017) What do you plan to do during the next reporting period to accomplish the goals?Objective 1, subobjective 1a-complete pathology assays with bmr6, bmr12, bmr2 and wild-type lines; subobjective 1b-complete pathology assays with high lignin transgenic lines; sub-objective 1c-begin pathology assays with post-flowering non-senescent lines. Publish results from biological assessments of bmr and high lignin transgenic lines. (Manuscript was submitted 9-24-2017). Objective 2: targetted gene expression analyses of genes, based on RNA-Seq results, to identify potential sources of markers for disease or drought resistance. Objective 3: Choose subset of plant materials, based on biological assessments for response to pathogens, timepoints and water conditions, for metabolite profiling. Objective 4: Choose subset of plant materials, based on biological assessments for responses to pathogens, timepoints and water conditions, for RNA extraction, library preparation and RNAseq analyses.
Impacts
What was accomplished under these goals?
Objective 1, subobjective 1a-Six repetitions of near-isogenic brown midrib (bmr)-6, bmr12 and wild-type lines inoculated with two stalk pathogens under water deficit and adequate water conditions have been completed. The plants were inoculated with Fusarium thapsinum (Fusarium stalk rot pathogen), Macrophomina phaseolina (charcoal rot pathogen) or broth (no pathogen) control. Peduncle (the upper part of the stalks) tissues were taken from the inoculation site or the edge of the lesion, and 3 cm from the inoculation site or the edge of the lesion, at the time of inoculation (controls for each treatment), at3 days after inoculation (dai) and at 13 dai. Preliminary analyses of six (out of nine) repetitions have provided intriguing results in that the plant responses to each pathogen is different. For M. phaseolina-inoculations, the greatest effects occured at the 3-day time point, which is when the lesion begins to become visible. For both bmr12 and wild-type, these initial lesions were larger under water deficit conditions than respective lines under adequate water, while bmr6 was relatively unaffected by the two water regimes. When inoculations were performed with F. thapsinum, the greatest effects were observed at day 13 (estimated time of rapid lesion expansion). In this case, bmr6 and bmr12 plants had significantly smaller mean lesions under water deficit than wild-type plants under the same conditions. Objective 1, subobjectives 1c and 1d-previously identified stalk pathogen resistant plants were grown in the field to determine if these lines have post-flowering non-senescence, a trait associated with stalk rot resistance. Objectives 2 through 4: materials were collected,processed and preparedto begin analyses of targetted gene expression, and global metabolites and gene expression.
Publications
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