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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** 2023 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**
                                

This is the final report for this project which terminated in July of 2023.  The replacement project, 5020-2100-001-000D, was certified by OSQR on 07/05/2023.
Objective 1 
Sub-objective 1a. Identify germplasm accessions, from wheat and related species, that confer resistance to Hessian fly
Discovered SNP (Single Nucleotide Polymorphism) markers linked to Hessian fly (HF) resistance (H) gene using GBS (genotype-by-sequencing) from seven homozygous resistant and two homozygous susceptible pasta wheat durum accessions to HF biotype (designated as ‘L’). Identified parental pairs showing greater than 5000 SNPs for crossing and constructing homozygous mapping population. Generated BC1F2 (Back Cross 1 and Filial 2) plants and screened for resistance or susceptibility to HF biotype L. Developed KASP (Kompetetive Allele-Specific Polymerase Chain Reaction) markers using the SNP information. Currently, the BC1F2 plants along with the parent wheat lines are being genotyped using KASP markers to map the location of the H gene and identify molecular markers linked to the gene. Further, the HF-resistant BC1F2 lines are currently being self-crossed to make homozygous resistant lines.
Identified fourteen new HF-resistant durum pasta wheat cultivars by phenotyping over 800 accessions to HF biotypes (‘L’ and ‘vH13’) that are valuable to breeders for use in breeding programs to develop resistance to HF. Documented in a peer-reviewed publication.
Identified three temperature-independent HF-resistant pasta durum wheat cultivars. These accessions maintained 100% resistance at the increased temperature of 30°C that offer valuable resources to breeders for developing elite lines, in addition to serving as candidates for use in geographical locations with higher temperatures. Documented the research in a peer-reviewed publication.

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.
Developed an innovative strategy, Hessian fly in planta translocation (HIT) assay, to successfully feed the defense proteins/toxins to HF and determine their effects on insect development. Employed HIT approach to feed the HF larvae with wheat defense proteins encoded by Hessian fly response gene 1 - a mannose binding lectin; Hessian fly response gene 3 - a chitin-binding lectin and toxins including CRY11B (crystal protein 11B), a pesticidal dipteran-specific toxin isolated from Bacillus thuringiensis. Determined that HF larvae fed on these toxins/defense proteins have detrimental effects on larval growth. Revealed, using immunolocalization studies, the larval midgut is the target for these toxins. These insecticidal and antifeedant proteins are ideal targets for developing alternate molecular strategies to complement native resistance for controlling these important cereal pests.
Sub-Objective 1c: Identify and evaluate germplasm accessions that confer resistance to wheat against greenbug. Wheat cultivars were screened against both Bird Cherry Oat Aphid (Rhopalosiphum padi) and Greenbug (GB, Schizaphus graminum). Over 50 lines were screened against each species. Eleven lines that had previously shown resistance to HF confer resistance or tolerance to the R. padi population.  One HF resistant line (H15) appears to confer strong resistance to Biotypes (E &K) of GB similar to CL17949 (Gb4) resistance. Four lines showing tolerance to GB were identified that can be used for future breeding programs.
Objective 2
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.
Identified thousands of differentially expressed genes (DEGs) in nonhost Brachypodium distachyon (Bd) that shared resemblances with DEGs identified in both the resistant (6000) and susceptible (4500) host wheat in response to HF larval attack. Revealed resemblance of Bd with resistant wheat plants with respect to increased expression of genes encoding defense-related proteins including lectins, proteinase inhibitors, proteases, kinases, and dirigent proteins indicating an early defense strategy to prevent the larvae from establishing permanent feeding sites. Demonstrated resemblance of Bd to the susceptible wheat plants by suppressing cell-wall associated and photosynthetic pathway genes as well as activating the expression of susceptibility-associated genes including Mds-1 gene (encodes a small heat-shock protein), amino acids and polyamine biosynthetic pathways which allows the larvae to reprogram the host machinery and improve the nutrient value of the plant benefitting insect development. Documented nonhost Bd genome having a phenotypic and molecular response that is intermediate to the resistant and susceptible host wheat and this model system has the potential to be used towards better understanding of plant-HF interactions. Documented this work in a peer-reviewed publication. 
Characterized another model genome, Kitaake rice for phenotypic and molecular response to HF larval attack. Identified similarities of rice (nonhost) response with that of resistant wheat plants. Identified and characterized the Ald1 gene, that is significantly upregulated in rice as possible early defense strategy. Generated Ald1 CRISPR genome-edited mutants and overexpression lines, as collaborative research (NACA agreement) with Ohio cooperator. Currently, these mutants are being phenotyped to elucidate the role of Ald1 in defense against HF. Documented use of rice as a surrogate genome in a peer-reviewed publication and was cited as one of the ‘Top Research Accomplishments’ in ARS Science Report (2021).
Discovered around 6000 DEGs in virulent HF larvae feeding on susceptible host wheat plants that include genes triggering changes in developmental pathways and proteins that detoxify plant defense molecules, and process nutrients derived from the plant that are beneficial to the developing larvae. Discovered several larval genes encoding effector proteins that are currently being characterized to determine their role in virulence leading to plant susceptibility. Documented in a peer-reviewed publication.
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.
Completed proteome (total protein) analysis of HF biotype L feeding on resistant (avirulent larvae) and susceptible (virulent larvae) wheat at 1 and 3 days after egg hatch. Identified a total of 4,400 proteins in virulent and avirulent HF larvae. Revealed 531 and 1,389 differentially expressed proteins (DEPs) in avirulent and virulent HF larvae, respectively, as compared to the neonate (newly hatched) larvae (that are not feeding and used as the control). Identified a higher number and expression of DEPs involved in stress response, detoxification, cell wall degradation, and amino acid metabolism in the virulent larvae as compared to avirulent HF larvae. Identified a total of 25 DEPs as putative salivary gland secreted effector proteins unique to the virulent HF larvae, that are known to reprogram the host plant physiology, and were unique to the virulent HF larvae.  Currently, a few candidate effector proteins are being used in downstream functional as well as protein-protein interaction studies to determine their role in HF virulence to wheat. 
Sub-objective 2c. Increased understanding of the molecular basis of the quadratrophic interactions between wheat, greenbug, Buchnera and viruses. We measured the effect of Cereal Yellow Dwarf Virus (CYDV) on gene expression in GB.  We discovered a set of 127 genes with significant differences in gene expression in viruliferous (carrying virus) vs. aviruliferous (without virus) GB. A new transcriptome was completed based on the gene expression differences. Significantly upregulated genes in response to virus status included a lysosomal-trafficking regulator, peptidylprolylisomerase, RNA helicase, and two secreted effector proteins. Endosymbiotic gene expression also dropped dramatically with increased feeding time on virus carrying wheat.  In order to better understand the relationship between the endosymbionts and GB, we have begun a project to understand the endosymbiotic microbiome in response to five agriculturally important species (Wheat, Sorghum, Barley, Rye and Aegilops). The 16s and 18s rDNA region was amplified to understand microbiome composition changes for these species. 

Objective 3
Screened breeder-provided wheat lines from seven nurseries for resistance to five HF biotypes (‘B’, ‘C’, ‘D’, ‘O’ and ‘L’) each year and provided the data to the breeders. This year, these included three USDA nurseries: (i) Uniform Eastern Soft Red Winter Wheat Nursery (28 lines); (ii) Uniform Southern Soft Red Winter Wheat Nursery (37 lines); and (iii) Uniform Bread Wheat Nursery (34 lines). The four University nurseries included: (i) SUN Wheat Nursery (80 lines); (ii) Mason Dixon Uniform Wheat Nursery (67 lines); (iii) Gulf Atlantic Wheat Nursery (59 lines); and (iv) Fusarium Head Blight Nursery (63 lines). Private companies soliciting screening against the above-mentioned HF biotypes included: (i) Jo-Mar Seed (21 lines); KWS (59 lines); and Gro-Pro Seed (76 lines). Additionally, 19 wheat lines from University of Georgia were screened against two HF biotypes (‘L’ and ‘vH13’). Evaluated the effectiveness of the 38 known H genes for protection of wheat against HF field populations collected from Louisiana. Currently, evaluating the effectiveness of these H genes against HF field populations from Texas. **Accomplishments**
                                

1. Identification of putative effector genes in greenbug. Effector proteins are molecules that help the greenbug invade and successfully feed on its host plant by reducing wheat defense responses. ARS researchers in West Lafayette, Indiana, identified two gene encoding effectors that are secreted by greenbugs while feeding. These effector proteins allow the aphid to feed on the wheat more efficiently allowing for an increase in weight and fecundity and, thus induces greater aphid production and a more rapid need for the aphid to move onto fresh plants as the wheat begins to die. This more rapid movement of greenbug onto other wheat plants, increases transmission of the virus. 2. The first characterization of E chromosomes. Hessian fly, like many other flies, has stable cell line (somatic or S) chromosomes and eliminated (E) chromosomes that are only in the germ line. What is known is that removal of the E chromosomes results in sterilization, thus they must provide a unique function. Little else is known about the E chromosomes, so ARS researchers in West Lafayette, Indiana, investigated their properties and origin. A retrotransposon – WORF was identified only on the E chromosomes, as well as a few unique sequences, whose function remains unknown.  Ultimately, it appears that the E chromosomes are derived from S chromosomes by the acquisition or conversion of sequences around the centromere of each chromosome. While the function of these unique sequences is currently unknown, they do present a novel target for insecticides as they have only been found in Hessian fly to date. 3. New Hessian fly-resistant durum pasta wheat cultivars for effective insect management. The Hessian fly is an economically important pest of wheat in US, causing severe losses in yields annually. Deployment of resistant wheat cultivars harboring Hessian fly resistance genes (H) is still the most effective and economical strategy to manage this insect pest. However, the Hessian fly populations adapt to overcome the newly deployed resistance genes within a few years of release. There is currently a severe Hessian fly outbreak in wheat fields in LA, GA, FL, TX, and NC. ARS researchers at West Lafayette, Indiana, screened wheat accessions for resistance against two Hessian fly biotypes (‘L’ and ‘vH13’). Fourteen durum pasta wheat lines were resistant to both biotypes. These new Hessian fly-resistant pasta wheat cultivars offer valuable resources to breeders for use in breeding programs to develop elite wheat lines for durable resistance to Hessian fly and efficient crop management. 4. Hessian fly-resistant wheat cultivars ideal for use in regions with elevated temperatures. Global warming and environmental changes pose a risk for breeders, because elevated temperature weakens host plant defense and immunity against insects causing normally resistant plants to become susceptible. ARS researchers at West Lafayette, Indiana, identified three pasta durum wheat accessions that maintained 100% resistance to the highly virulent Hessian fly biotype L at the increased temperature of 30°C (normal growth temperature is 18°C). These three new durum wheat accessions offer resources to breeders for developing elite lines, in addition to serving as potential candidates for use by farmers in geographical locations with higher environmental temperatures, that still provide robust resistance to Hessian fly. **Review Publications**
                            *Subramanyam, S., Nemacheck, J.A. 2023. New sources of Hessian fly resistance in Triticum turgidum wheat lines from Asia and Europe. Genetic Resources and Crop Evolution. 70:1341-1347. https://doi.org/10.1007/s10722-023-01566-z.*
                                *Crane, Y.M., Crane, C.F., Cambron, S.E., Springmeyer, L.J., Schemerhorn, B.J. 2023. Molecular characterization of eliminated chromosomes in Hessian Fly (Mayetiola destructor (Say)). Chromosome Research (2023). 31(1). Article 3.  https://doi.org/10.1007/s10577-023-09718-8*
                                *Nemacheck, J.A., Flynn, R.D., Zapf, K., Subramanyam, S. 2023. Identification of new sources of Hessian fly resistance in tetraploid wheat. Crop Science. 63(3):1354-1363. https://doi.org/10.1002/csc2.20917.*


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