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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** 2022 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:
BC1F1  (Back Cross 1 and Filial 1) plants generated were screened for resistance or susceptibility to Hessian fly (Hf) biotype L stock. Selfed seeds were collected from the Hf resistant plants to generate BC1F1:2 families. Leaves from BC1F1 parental plants were also collected and DNA has been isolated from these samples.

Additionally, 500 tetraploid wheat accessions were phenotyped against two Hf biotypes and identified ten new sources of Hessian fly resistance that can be used in future breeding programs.

HIT (Hessian fly in planta translocation) feeding assay was used to deliver insecticidal proteins including two lectins (i) Hessian fly response gene 1 (mannose-binding lectin); and (ii) Hessian fly response gene 3 (chitin-binding lectin) and 1 dipteran-specific protein (isolated from Bacillus thuringiensis) toxin, CRY11B (Pesticidal crystal protein 11B). Western blot analysis using protein-specific antibodies confirmed that the larvae ingested the toxin. Larvae fed on these toxins showed significantly delayed development as compared to Hf larvae feeding on water control plants (lacking any toxin). Thus, these toxins have detrimental effects on Hf larval development. Immunolocalization studies revealed larval midgut is the target for CRY11B toxins.

In greenbug, two biotypes (named E&K) were utilized to screen 35 wheat accessions for resistance/susceptibility to greenbug. Currently, there are 15 known lines with resistance to greenbug. This work identified four new previously unidentified tolerant lines to greenbug infestation. These lines are ‘Seneca’, ‘Molly’, reduces selection
pressure on the development of resistant biotypes which increases the durability of the deployed wheat lines. This determination of tolerance is determined by the wet weight of the plant at 17 days after infestation, live greenbug counts which are significantly lower than what is seen on susceptible plants and lower plant damage (chlorotic lesions).

Objective 2:
Genes identified in the RNA-seq experiment in greenbug identified over 1100 genes that were either up or downregulated in response to vectoring CYDV-PAV virus. Of these, four variants of Cathepsin B were up-regulated 7-12 fold. Cathepsin B upregulation has been identified as inversely related to transmission efficacy of Potato Leafroll virus in other aphids. Currently, experiments are underway to identify variations in Cathepsin B in response to other yellow dwarf virus variants.

Experiment involving dual infestation requires the use of virulent and avirulent Hf vH13 biotype stock that have visible markers for virulence (dark eyes) and avirulence (white eyes). Due to breakdown of the cold chamber in which the vH13 Hf biotype stock was stored we lost this biotype that is required for carrying out this experiment. We reached out to other USDA and university labs who are working with Hf and unfortunately, this stock is not available in any of the other labs. Therefore, we are unable to undertake these experiments. Instead, to better understand virulence and avirulence, we carried out a proteomic analysis of Hf larvae feeding on resistant and susceptible plants to provide an insight into insect proteins and what roles they are possibly playing during infestation and manipulation of the plant machinery. We are currently completing the data analysis to identify differentially expressed proteins and associated biological pathways.

To increase our understanding of the molecular interactions of wheat, greenbug, Buchnera and viruses, we continued our work on gene expression levels in viruliferous and aviruliferous greenbugs. RNA was extracted from 96 samples collected at 8 time points from 0 to 20 days after introduction to fresh host plants. 95 samples yielded 1,000,089,950,000 clean reads. These reads aligned to Schizaphis, Melanaphis, and 49 microbial genomes.
Differentially expressed genes were plotted as heatmaps separately for greenbug and microbes. A total of 127 genes were differentially expressed between viruliferous and aviruliferous greenbugs. At least 28 of them are involved in DNA binding. One gene, flightin, indicated a shift to alate production in viruliferous aphids at day 15. Additionally, 3264 genes were differentially expressed between aviruliferous biotype B and aviruliferous biotype H. The read counts decreased for gut microbes at days 15 and 20, indicating population collapse.

Objective 3: 
New sources of Hessian fly resistance must be identified due to the rapid evolution of virulence in Hessian fly populations. Ten Uniform wheat nurseries were screened against five Hessian fly biotypes. Those nurseries include three USDA nurseries, five university breeding nurseries and from two commercial nurseries. The USDA nurseries included 116 lines and the nurseries are: 1) The Uniform Eastern Soft Red Winter Wheat nursery with 31 entries; 2) The Uniform Southern Soft Red Winter Wheat nursery with 38 entries; and 3) The Uniform Bread Wheat nursery with 47 entries. The university nurseries included over 200 entries. Those nurseries screened were: 1) The Fusarium Head Blight nursery; 2) The Gulf Atlantic Wheat Nursery; 3) The Southern Universities Uniform Wheat Nursery (SUNWHEAT); 4) The Mason-Dixon Uniform Wheat Nursery, and 5) The Uniform Eastern Soft White Winter Wheat Nursery. JoMar Seed and KWS Seed are the commercial seed companies utilizing our services with 82 wheat lines screened against our Hessian fly stocks. **Accomplishments**
                                

1. Identified four wheat lines tolerant to two biotypes of Greenbug. Four wheat lines tolerant to two biotypes of Greenbug were identified. Greenbug aphids not only damage wheat by feeding, but also are vectors of diseases.  Finding new sources of resistance is important for breeders and farmers.  Hessian fly resistant wheat lines were screened against two biotypes of Greenbug and identified four tolerant lines of wheat. Tolerant wheat lines reduce the selection pressures on the insect and thereby reduce the rate at which new more virulent biotypes emerge.  Utilizing tolerance coupled with native resistance to viruses in the wheat provide breeders with germplasm that can be deployed throughout the United States wheat growing regions. 2. Identified genes differentially expressed in Greenbug carrying cereal yellow dwarf virus and Greenbugs that are not carrying the virus. Historically, control of yellow dwarf virus transmission in wheat has proved difficult.  Identification of genes within the aphid that potentially control transmission is important as a first step in the control of vectoring the virus.  These identified genes can be targeted by potential transgenic wheat lines. 3. Identified ten new sources of Hf resistance in tetraploid wheat that can be used in breeding programs. Hf damage causes severe economic losses to wheat worldwide. While planting resistant wheat cultivars is still the most effective and economical approach to manage this insect pest, development of virulent biotypes poses a threat to breakdown of plant resistance. Hence, there is urgent need to continually identify new and novel sources of Hf resistance. These new resistant wheat lines identified by our group will add to the repertoire of already available resistant cultivars and can be used by breeders and farmers to alleviate Hf damage. 4. Revealed the presence of an effector-based strategy that manipulates the host plant machinery using comparative transcriptome analysis of Hessian fly larvae feeding on host and nonhost plants. The Hf larvae expressed high levels of salivary effector genes to prevent the plant defense proteins from being toxic to the insect gut. Additionally, in the Hf larvae, genes that synthesize nutrients (such as amino acids and polyamines) are expressed at low levels allowing the larvae to utilize the nutrients derived from the host plant. Our results clearly showed how the Hf larvae manipulate the plant as per their needs and also prevent the plant defense toxins from killing the larvae, further revealing the mechanism used by larvae during feeding. This knowledge is immensely useful to researchers to target these insect mechanisms and develop molecular strategies to control this and other economically important cereal pests. 5. A cell wall degrading enzyme is produced by Hf larvae to breakdown host plant cell wall and establish susceptibility. The tiny mandibles found in the mouth parts of Hf larvae are used for puncturing the plant cell wall but cannot be utilized for chewing. Therefore, it is crucial that nutrition for the developing larva is available in a soluble form rather than solid form. We demonstrated the extra-oral secretion of  a cell wall-degrading enzyme from the virulent Hessian fly larvae into susceptible host plant cell, and its role in breakdown of plant cell wall inulin (sugar polymer) into monomers (glucose and fructose) enhancing the nutritional quality of the energy source, in addition to increased levels of amino acids and polyamines in the susceptible host that was documented by our group previously. We also demonstrated increased cell wall permeability in susceptible wheat plants, allowing these nutrients (proteins and sugars) to diffuse to the leaf surface where the virulent larvae are able to suck up the nutrients, thus establishing susceptibility. This is the first report documenting the role of a Hf cell wall degrading enzyme in establishing wheat susceptibility. This enzyme can be targeted in a transgenic approach as an additional molecular strategy for insect control. **Review Publications**
                            *1.	Subramanyam, S., Nemacheck, J.A., Xie, S., Bhide, K., Thimmapuram, J., Scofield,  S.R., Sardesai N. 2021. Comparative Hessian fly larval transcriptomics provides novel insight into host and nonhost resistance. International Journal of Molecular Sciences. 22(21): 11498. https://doi.org/10.3390/ijms222111498*
                                *Subramanyam, S., Sardesai, N., Babu, C.R. 2022. Cenchrus setigerus Vahl. secretes root agglutinins to promote colonization by plant growth-promoting rhizobacteria. Brazilian Journal of Botany. 45: 819-832.  https://doi.org/10.1007/s40415-022-00794-4.*
                                *Crane, C.E., Nemacheck, J.A., Subramanyam, C.F., Williams, S.N., Goodwin, S.B. 2022. SLAG: A program for seeded local assembly in complex genomes. Molecular Ecology Resources. 22:596–616. https://doi.org/10.1111/1755-0998.13580.*


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