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ARS Home » Midwest Area » West Lafayette, Indiana » Crop Production and Pest Control Research » Research » Research Project #434610

Research Project: Molecular Characterization of Host-Insect Interactions in Cereal Crops

Location: Crop Production and Pest Control Research

2021 Annual 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. 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.

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
Sub-ojective 1a: Identifying new sources of native resistance is crucial to counter the devastating effects of virulent Hessian fly biotypes. Crosses were made between a polymorphic resistant and susceptible durum (pasta) wheat parental pair to generate the F1 progeny. These plants were screened with Biotype L Hessian fly. The resistant and susceptible F1 plants were then subsequently used to make crosses and generated BC1F1 plants. Currently, the BC1F1 plants are being grown to harvest the BC1F1:2 families. Genotype sequencing undertaken on resistant and susceptible parents, in the previous year, has identified several polymorphic markers that can be used in marker-assisted selection. The sequences from these markers are being currently analyzed so as to develop flanking Hessian fly resistance molecular markers that can be used to screen the BC1F2 mapping population. Sub-objective 1b: Insecticidal and antifeedant proteins can be used in alternate transgenic strategy to complement native resistance. Since the Hessian fly cannot be reared on artificial diet, we have developed an innovative strategy called as Hessian fly in planta translocation (HIT) assay to feed these toxins to the insect while they are still feeding on the plant and determine their effects on insect development. Currently, we are using the HIT assay to screen several toxic proteins, in addition to the ones identified in previous years. We are also optimizing the midgut sectioning and localization protocol to determine the targets for these insecticidal proteins within the larval midgut. Sub-objective 1c: Twenty Hessian fly resistant wheat lines were screened against greenbug Biotype E and Biotype K populations. To date, we have identified one line (H15) that presents a strong resistance response similar to CL17959 (GB4) resistance. Eleven other Hessian fly resistant wheat lines have presented a tolerance response with greenbug similar to our documented Hessian fly tolerant response. We are currently screening these lines further to determine if the weight and number of seed in the wheat head are comparable to what is seen in an uninfested plant. This type of intermediate resistance response is determined by a high wet weight of the plant at 17 days after infestation, live greenbug counts that are significantly lower than is seen on known susceptible greenbug lines, and lower chlorotic lesions. Final analysis and manuscript are in progress. Sub-objective 2a: RNAseq was performed on two biotypes of greenbug carrying cereal yellow dwarf virus. The gene expression data was compared to non-virus carrying greenbugs. A total of 127 genes were identified as either being up or down regulated compared to non-virus carrying counterparts. A substantial portion of these genes are associated with DNA or RNA binding in the insect. One gene identified had a plausible immune function, interferon-inducible double-stranded RNA-dependent protein kinase. Additionally, it has been reported that different biotypes have different susceptibility to viruses or viral sub-types in greenbugs. In this work, it was determined that biotype H, overall, had a higher gene responsiveness (upregulation) than seen in biotype B. Sub-objective 2b: Experiments involving dual infestation requires the use of virulent and avirulent vH13 Hessian fly stocks that have visible marker for dark (virulence) and white (avirulence) eyes, respectively. Overheating of one of the cold chambers has resulted in the loss of white eye Hessian fly stocks. Our attempts to procure more of these fly stocks from other USDA labs working with Hessian fly were also unsuccessful as they lack this fly type. Therefore, we are unable to perform this experiment. Instead to better understand virulence and avirulence we have undertaken proteomic analysis of Hessian fly feeding on resistant and susceptible plants to provide insight into insect proteins and what functions they are possibly playing during larval infestation and manipulation of plant machinery. Sub-objective 2c: In an effort to better understand the molecular interactions of wheat, greenbug, Buchnera and the viruses, we measured the effect of Cereal Yellow-Dwarf Virus (CYDV) on gene expression in the vector, the greenbug. RNA was sampled from two biotypes with and without CYDV. A set of 127 genes differed significantly between the virus-free and viruliferous greenbugs. Many of these were DNA-binding transcription factors or retrotransposon-like proteins. In terms of the endosymbiont Buchnera, the overall population counts collapsed between days 10 and 15, particularly on the plants developing virus symptoms. A paper has been submitted detailing these results. Objective 3: There is continual need to identify new sources of Hessian fly resistance in wheat fields due to limited genetic sources of native resistance in wheat. We have evaluated Hessian fly-infested wheat lines from two nurseries. These include Uniform Southern Soft Red Winter Wheat (USSRWW), and Uniform Eastern Soft Red Winter Wheat (UESRWW) nurseries. A total of 46 wheat lines were screened against HF biotypes B, C, D, and L for the USSRWW and 34 wheat lines for UESRWW. Evaluated 163 wheat lines infested with five Hessian fly biotypes from two nurseries managed by cooperators.

1. Greenbug tolerant and resistant wheat lines identified. Work screening extant lines of Hessian fly resistant wheats has revealed at least one resistant and 11 tolerant wheat lines against two greenbug biotypes.

2. Establishment of rice plants as an important tool for functional genomic studies to understand and manage plant disease resistance. The genetic complexity of the common bread wheat, which contains three different genomes, has been extremely challenging to researchers for carrying out studies to conclusively pinpoint the role of numerous insect/pest-responsive genes that are potentially involved in disease resistance. ARS researchers at West Lafayette, Indiana, demonstrate that this problem can be overcome by using less complex model genomes, such as rice. Rice exhibits nonhost resistance to Hessian fly. Larvae feeding on rice seedlings do not develop beyond the first-instar stage and infested plants show comparable leaf lengths to uninfested control plants resembling the phenotypic response observed in host resistant wheat, Further, similar to wheat, the rice plants show limited and transient wall permeability indicating minimal damage caused to the cell wall by larval probing. Therefore, the larvae are unable to establish permanent feeding sites identical to the responses observed in resistant host wheat. In nonhost rice, at the molecular level, expression of Hessian fly-responsive resistance-associated biomarker genes was also similar to resistant wheat plants. Since rice resembles bread wheat in its responses to Hessian fly infestation at the physical and molecular level, we have now demonstrated that rice plants are suitable for downstream functional studies of genes that can help us better understand how wheat defends against this and other destructive insect pests. Similar strategy can be employed by other plant researchers to characterize genes to further understanding of other plant-pest/pathogen interactions and develop effective mitigation strategies that complement native resistance.

Review Publications
Subramanyam, S.N., Nemacheck, J.A., Bernal-Crespo, V., Sardesai, N. 2021. Insect-derived extra-oral GH32 plays a role in susceptibility of wheat to Hessian fly. Scientific Reports. 11, 2081.
Subramanyam, S., Nemacheck, J.A. 2021. Initiation of compatible wheat-Hessian fly interactions triggers the expression of a novel UDP-glycosyltransferase, MdesUGT1, in virulent Hessian fly larvae. Arthropod-Plant Interactions (2021). 15:363-374
Subramanyam, S.N., Nemacheck, J.A. 2021. Nonhost Kitaake rice displays phenotypic characteristics of host resistant wheat and molecular biomarkers of both resistant and susceptible wheat in response to feeding by Hessian fly larvae. Journal of Plant Interactions. 16(1):156-165.
Roe, K.E., Schemerhorn, B.J. 2021. Wheat yield in a tolerant winter wheat line infested by Hessian fly (Mayetiola destructor). Advances in Entomology. 9(2):70-84.