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 Crop Production and Pest Control Research: West Lafayette, IN
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ARS Home Midwest Area West Lafayette, Indiana Crop Production and Pest Control Research Research





- Schemerhorn, Brandi
- Subramanyam, Subhashree
- 2023
- 2022
- 2021
- 2020
- 2019
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)

Research Project: Molecular Characterization of Host-Insect Interactions in Cereal Crops **Location:
                            Crop Production and Pest Control Research** 2019 Annual Report **Objectives**
                                

Objective 1:  Identify new sources of resistance to Hessian fly and aphids in cereal crops for use in breeding programs to reduce damage from these pests and associated pathogens.
Sub-objective 1a. Identify germplasm accessions, from wheat and related species, that confer resistance to Hessian fly.
Sub-objective 1b. Characterize effectiveness against Hessian fly of insecticidal and antifeedant proteins from wheat and other plant sources for potential use as transgenic resistance to pyramid with and protect native resistance loci.
Sub-objective 1c. Identify and evaluate germplasm accessions that confer resistance to wheat against greenbug. 

Objective 2:  Characterize and evaluate plant-pest interactions at the molecular level in cereals to improve methods of control for insect pests of wheat. 
Sub-objective 2a. Compare Hessian fly-wheat and greenbug-wheat interactions among different cereals/grasses to identify genes consistently associated with resistance, susceptibility, virulence and avirulence.
Sub-objective 2b. Investigate timing and composition of overlapping resistance and susceptibility responses when both virulent and avirulent Hessian fly larvae inhabit the same wheat plant, as can happen in field infestations.
Sub-objective 2c. Increased understanding of the molecular basis of the quadratrophic interactions between wheat, greenbug, Buchnera and viruses. 

Objective 3:  Evaluate germplasm and regional insect populations to assist cereal breeders in selecting effective sources of resistance for their breeding programs. 
Sub-objective 3a. Evaluation of wheat breeding lines in regional uniform nursery tests. **Approach**
                                

Objective 1. Resistance phenotypes in new and under-utilized resistant wheat lines will be characterized using genotype-by-sequencing. 
Objective 2.  The Hessian fly-wheat and greenbug-wheat interactions among different cereals/grasses will be used to identify genes consistently associated with resistance, susceptibility, virulence and avirulence. This will be accomplished by analyzing Illumina HiSeq time-course data of resistant, tolerant and susceptible wheat and the Hessian fly on those hosts. Genes of interest will be verified by quantitative real-time polymerase chain reaction (qRT-PCR). We will characterize the induction of susceptibility also known as obviation.  Transcript profiling and qRT-PCR will quantify the abundance of transcripts leading to biomarker genes for compatible and incompatible interactions at a variety of timepoints. In wheat, gene expression differences when infested with aphids carrying barley yellow dwarf virus (BYDV) will be characterized by whole genome mRNA profiles using high throughput sequencing and qRT-PCR. Selected genes of interest  that are significantly upregulated or down regulated in both the aphid and wheat will be examined further. 
Objective 3. To assist cereal breeders in selecting effective sources of resistance, we will evaluate germplasm and regional insect populations. New sources of germplasm containing resistance to Hessian fly will be identified using traditional screening procedures in a greenhouse setting. A variety of insect populations will be used to determine resistance and susceptibility of available wheat lines. The efficacy of resistance Rgene intervention will be assessed by comparing the change in frequency of phenotypic resistance to historical data. **Progress Report**
                                

Objective 1: Identify new sources of resistance to Hessian fly and aphids in cereal crops for use in breeding programs to reduce damage from these pests and associated pathogens.

Sub-objective 1a. Identify germplasm accessions, from wheat and related species, that confer resistance to Hessian fly. Prepared a manuscript and submitted for peer review documenting data generated from phenotypic response of 41 Triticum durum (tetraploid) wheat accessions to infestation by two different Hessian fly (biotype L and vH9) stocks. Identified 5 homozygous resistant and 2 homozygous susceptible durum wheat lines as candidates for undertaking GBS (genotype-by-sequencing) project to make parental pairs with very high number (~5000) of polymorphic markers to be further used to create a mapping population. Due to limited stock of seeds available for undertaking any further experiments with the desired resistant and susceptible tetraploid lines, additional plants were grown and harvested the seeds.  Currently, growing the selected wheat lines for harvesting plant tissue for DNA isolations. 

Sub-objective 1b. Characterize effectiveness against Hessian fly of insecticidal and antifeedant proteins from wheat and other plant sources for potential use as transgenic resistance to pyramid with and protect native resistance loci. We developed and improved the HIT (Hessian fly in planta translocation) feeding assay in preliminary experiments with Hessian fly-susceptible wheat (‘Newton’) plants using varying concentrations of HFR-1 (Hessian fly responsive) and Cry11B Bt (Bacillus thuringiensis) proteins. We have determined the most effective concentrations of each protein and have determined our ideal time-point for data collection. We have observed significant reduction in virulent Hessian fly larval length when fed on these antinutrient proteins as compared to the control (water) plants confirming putative antifeedant properties. Currently, a feeding assay set up is planned to procure data for statistical analysis using larger sets of plants at the ideal conditions optimized, as well as taking photomicrographs for documentation in a publication. Additionally, further work is being planned to undertake in situ hybridization studies to determine target sites for these proteins in the midgut of virulent Hessian fly larvae. Discovering the putative role of these antifeedant proteins are critical in their usage as potential transgenes for resistance to complement native resistance in wheat to Hessian fly.  

Sub-objective 1c. Identify and evaluate germplasm accessions that confer resistance to wheat against greenbug.  Wheat cultivars that have shown resistance to Hessian fly in our previous experiments as well as new germplasm available through a collaborator from Purdue have been collected and in some cases, increased. R. padi from 3 separate populations have been screened on Molly, Newton and Iris for preliminary results. The wheat was seeded at a rate of 22 seeds in randomized half-rows. Susceptible wheat ‘Custer’ was planted in ‘check’ rows at the ends and middle of each flat to check for even infestation. TAM 110 was as a reference for full resistance. Plants were scored as resistant or susceptible. While only one replicate was undertaken, we know that this setup will work in additional screening efforts.
 
Objective 2: Characterize and evaluate plant-pest interactions at the molecular level in cereals to improve methods of control for insect pests of wheat. 

Sub-objective 2a. Compare Hessian fly-wheat and greenbug-wheat interactions among different cereals/grasses to identify genes consistently associated with resistance, susceptibility, virulence and avirulence. Published a manuscript in a peer-reviewed journal documenting molecular responses of nonhost Brachypodium distachyon transcriptome to Hessian fly larval feeding by identifying over 2000 differentially expressed genes (DEGs) using RNA-Sequencing (RNA-Seq), and comparing and contrasting transcriptional response with those observed in host wheat. Documented conclusively the suitability of B. distachyon genome as surrogate model to functionally characterize Hessian fly-resistance and -susceptibility wheat candidate genes. An additional manuscript is currently in review documenting the utility of using diploid wheat accessions as a surrogate for functional genomic studies of hexaploid (bread) wheat. Further characterization with Hessian fly-responsive biomarker genes revealed similarities between resistant diploid and hexaploid wheats at the molecular level.  We have also successfully concluded analyses of RNA-Seq data generated from avirulent and virulent Hessian fly larvae feeding on host wheat and larvae feeding on nonhost B. distachyon and discovered DEGs and associated biological pathways. DEGs include genes encoding salivary gland effector proteins, amino acids, glycoside hydrolases, CYP450s, carboxyl esterases, and proteinase inhibitors. At present, these genes are being validated by qRT-PCR for further characterization. A manuscript is being prepared documenting this work.

Sub-objective 2b. Investigate timing and composition of overlapping resistance and susceptibility responses when both virulent and avirulent Hessian fly larvae inhabit the same wheat plant, as can happen in field infestations.  Currently, planning an ideal experiment for undertaking dual infestation with virulent and avirulent Hessian fly biotypes and optimizing methodologies including designing primers, PCR parameters, and DNA isolations that will enable clear detection of virulent and avirulent larvae on the same plant that is crucial for a valid obviation experiment. Additionally, mining the previously conducted RNA-Seq and Affymetrix transcript time-course expression data obtained from compatible (susceptible plant) and incompatible (resistant plant) interactions to identify Hessian fly-responsive biomarker genes that can be used for downstream transcript profiling by qRT-PCR. 

Sub-objective 2c. Increased understanding of the molecular basis of the quadratrophic interactions between wheat, greenbug, Buchnera and viruses.
Currently, we are planning experiments to inoculate greenbug with BYDV-PAV/SGV and BYDV-MAV in order to begin mRNA collection from aphid and wheat.

Objective 3. Evaluate germplasm and regional insect populations to assist cereal breeders in selecting effective sources of resistance for their breeding programs. 

Sub-objective 3a. Evaluation of wheat breeding lines in regional uniform nursery tests. Evaluated Hessian fly-infested wheat lines from three nurseries. These include Uniform Eastern Soft Red Winter Wheat (USERWW), Uniform Southern Soft Red Winter Wheat (USSRWW), and Uniform Bread Wheat Trial (UBWT) nurseries. Screened a total of 35 wheat lines against 5 Hessian fly biotypes from both USERWW and USSRWW nurseries, and 46 lines against 5 biotypes from UBWT nursery. Screening data from USSERWWN and USSRWWN are documented at https://ars-usda.app.box.com/v/UniformEasternSouthern. Evaluated wheat lines infested with 5 Hessian fly biotypes from 5 nurseries managed by different cooperators including Gulf Atlantic Wheat (52), Fusarium Head Blight (50), Mason-Dixon Wheat (68), Uniform Eastern Soft White Winter Wheat (20), Southern Universities Wheat (91) and Virginia State Wheat Trial (148) nurseries. Data from Virginia State Wheat Trial nursery are posted online at https://www.pubs.ext.vt.edu/SPES/SPES-46/SPES-46.html.  Screened phenotypic responses of 77 wheat lines against 5 Hessian fly.  Screening results with respect to a number of resistant or susceptible wheat lines have been provided to respective nurseries for dissemination to breeders for undertaking further research. **Accomplishments**
                                

1. Brachypodium distachyon identified as potential surrogate model for functional characterization of Hessian fly-responsive defense genes in wheat. ARS researchers in West Lafayette, Indiana, have characterized the molecular response of Brachypodium distachyon (Bd) plants, a model grass genome and nonhost to Hessian fly, following larval attack.  The study clearly documented that Bd plants exhibit responses intermediate between resistant and susceptible host wheat. Resembling the resistant wheat host, Bd plants produce insecticidal defense proteins that does not allow the larvae to complete their development. Resembling the susceptible wheat host Bd plants also showed increase in expression of susceptibility-associated genes thus allowing the larvae to survive up to 41 days longer than the dying larvae feeding on resistant host wheat.  The scientists have prepared a model depicting the molecular mechanism during interaction of Bd with Hessian fly and documented the work in a high-impact peer reviewed journal. This work confirms the suitability of utilizing this nonhost genome to study and characterize the function of candidate Hessian fly-responsive genes that will allow for efficient management of this insect pest and thereby prolong the durability of wheat cultivars. The same approach can also be used by other researchers to manage other devastating insect pests of wheat, and other important cereal crops that are of economic importance to the farmers. 2. Development of novel targeted population sequencing in Hessian fly. Hessian fly is a gall midge that historically has been a devastating pest of wheat. Hessian fly larvae do not move after they have begun to feed, and their feeding on wheat seedlings reprograms the shoot apex to stop normal development.  Instead, the wheat tissues at the feeding site become abnormally permeable, and the larvae lap up exuded nutrients.  Since secreted salivary proteins are the first intimate molecular contact between the larvae and the wheat host, researchers in West Lafayette, Indiana, looked at proteins from salivary glands in Hessian fly to find possible candidates that reprogram the wheat allowing for the fly to overcome the wheat defenses.  We also developed a for surveying populations allowing for the assessment of 52 different proteins in 48 individuals at one time. This accomplishment provides cheaper and more efficient methods for surveying HF populations and will be valuable to wheat breeders and researchers by allowing a rapid assessment of extant fly populations for potential hazards for new wheat deployment. **Review Publications**
                            *Subramanyam, S., Nemacheck, J.A., Hargarten, A., Sardesai, N., Williams, C.E., Schemerhorn, B.J. 2019. Multiple molecular defense strategies in Brachypodium distachyon surmount Hessian fly (Mayetiola destructor) larvae-induced susceptibility for plant survival. Scientific Reports. 9:2596. https://doi.org/10.1038/s41598-019-39615-2.*


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