Source: PURDUE UNIVERSITY submitted to
MOLECULAR GENETIC INTERACTIONS OF WHEAT RESISTANCE AND HESSIAN FLY AVIRULENCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0216641
Grant No.
2009-35302-05262
Project No.
IND011462G1
Proposal No.
2008-03994
Multistate No.
(N/A)
Program Code
51.2B
Project Start Date
Jan 15, 2009
Project End Date
Jan 14, 2011
Grant Year
2009
Project Director
Stuart, J.
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
ENTOMOLOGY
Non Technical Summary
Many agricultural insect pests are controlled using cultivars that carry single insect resistance (R) genes that are bred into the plants using conventional breeding methods. This method of pest control eliminates the negative environmental impacts and the high costs associated with chemical pesticides. However, the efficacy of any single R gene has a limited durability. This problem is caused by "virulence" mutations in insect avirulence (Avr) genes that arise and spread in the pest populations. These mutations permit the pests to overcome the resistance conferred by the R genes. A complete lack of knowledge regarding the structure and function of Avr genes and virulence mutations in insects compounds this problem. To date, no insect Avr gene has been molecularly characterized. Moreover, the manner in which insect Avr gene products physically interact with plant R gene products is also unknown because a corresponding insect-Avr/plant-R gene-pair has not to be isolated. This research will remedy this situation by cloning both an insect Avr gene and its corresponding plant R gene. The Avr gene, named vH9 will be cloned from the Hessian fly, the world's most important insect pest of wheat. The corresponding Hessian fly R gene (H9) will be cloned from wheat. H9 is still an effective R gene in much of the U.S. However, virulence mutations in vH9 have already been detected in Hessian fly populations in both the laboratory and the field. Importantly, previous work has already genetically mapped vH9 within a relatively small (84-kb) segment of sequenced Hessian fly DNA. vH9 will be identified among the genes that reside in this segment by comparing their structures and expression in individuals isolated from genetic-mapping and field-collected populations. Gene discovery and allelic comparisons within this segment of the Hessian fly genome will be made using DNA amplification, RNA amplification, DNA-RNA hybridization, and DNA sequencing. The Hessian fly susceptible form of the H9 R gene has also been isolated and sequenced. This knowledge will be used to isolate the resistant form of H9 from CI 17714 wheat plants, which carry this allele. DNA amplification, and DNA hybridization techniques will be used to discover the resistant H9 allele in small fragments of CI 17714 genomic DNA. Those same fragments will then be transformed into the genomes of susceptible wheat plants to confirm that they confer H9 resistance to the Hessian fly. Three short-term outcomes of this investigation are expected: 1) the ability to predict the structures of the H9 and vH9 gene products, 2) molecular diagnostics for Hessian fly H9-virulence in the field, and 3) the ability to examine the interactions between the products of a plant R gene and a corresponding insect Avr gene for the first time. Results are expected to be immediately transferable to other plant pest problems. The long-term outcome of this investigation is expected to be the ability to design a more durable form of pest management.
Animal Health Component
(N/A)
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110108030%
2113110104020%
2113110113010%
2111549108020%
2011549108010%
2011549104010%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1549 - Wheat, general/other; 3110 - Insects;

Field Of Science
1080 - Genetics; 1130 - Entomology and acarology; 1040 - Molecular biology;
Goals / Objectives
Plant resistance to many plant pathogenic fungi and bacteria is elicited by the interactions between the products of avirulence (Avr) genes in plant pathogens and the products of cultivar specific resistance (R) genes in plants. These molecular interactions trigger biochemical responses in the plants that kill the invading parasitic organisms. Genetic studies indicate that similar gene-for-gene interactions exist between certain plant parasitic insects and their host plants. Until now however, it has not been possible to definitively test this hypothesis because an insect Avr gene and a corresponding plant R gene have not been molecularly identified. To remedy this problem, this research focuses on the interaction between wheat (Triticum spp.) and its most important insect pest, the Hessian fly (Mayetiola destructor). This specific interaction was chosen because of its agricultural importance, its genetic tractability, and the preliminary work that has made it possible to molecularly isolate a corresponding Avr-R gene pair from this specific interaction. The research has two major objectives: 1) To discover the virulence and avirulence alleles of Hessian fly avirulence gene vH9, and 2) to discover the corresponding Hessian fly H9 resistance allele in wheat. The objectives will be pursued simultaneously and the completion of both is expected within two years. This investigation will definitively test and clarify the existence of gene-for-gene interactions between plant parasitic insect pests and their plant hosts. Therefore, this knowledge will be immediately transferable to similar interactions between other economically important insect pests and important crop plants. With these discoveries it will be possible to predict the structures of the products of both genes and examine their interactions both in vitro and in vivo. The development of the first molecular diagnostics for Hessian fly virulent genotypes able to overcome resistance in wheat is also anticipated. Understanding the interaction between H9 and vH9 is expected to permit engineered plant resistance that is less vulnerable to the evolution of virulent insect genotypes. The long-term outcome of this study will therefore improve the sustainability of an economically important and environmentally benign method of pest control.
Project Methods
The first objective is to discover the virulence and avirulence alleles of Hessian fly Avr gene vH9. Map-based methods have positioned vH9 within an 84-kb segment of sequenced genomic DNA. To discover the genes that exist within this sequence, gene prediction software will be applied. Structural and transcriptional analyses will then be performed to determine which of the predicted genes best corresponds to vH9 by evaluating three expectations: 1) vH9 should encode a secreted protein. 2) vH9 should be expressed in the larval salivary gland. And 3) in comparison to the vH9 avirulence allele, the vH9 virulence allele should contain mutations that cause either mistranscription or mistranslation. The structure and transcription of the predicted genes isolated from laboratory purified H9-virulent and H9-avirulent Hessian fly strains will be compared in these analyses. Structural comparisons of the predicted genes in these strains will use the polymerase chain reaction (PCR) and DNA sequencing. Transcription analyses of these strains will use reverse transcription-PCR (RT-PCR), quantitative real time RT-PCR (qRT-PCR), and in situ hybridization. To test the functionality of predicted alleles, association mapping will be performed using both recombinant inbred lines and field-collected populations. Individuals from these populations will be genotyped as H9-virulent or H9-avirulent in testcrosses. The DNA sequences of the predicted genes of each testcrossed individual will then be examined. This will determine whether the predicted H9-virulence alleles are strictly associated with virulence and whether the predicted H9-avirulence alleles are strictly associated with H9-avirulence, as expected. The analysis will thereby definitively identify the virulence and avirulence alleles of vH9. The second objective is to discover the H9 resistance allele in wheat. The H9 susceptibility allele has already been sequenced. A cosmid library will be made using genomic DNA isolated from seedlings of the original H9 resistance allele donor (CI 17714). This library will be screened for clones containing the H9 resistance allele using PCR primers and radio-labelled probes that will be developed using the DNA sequence of the H9 susceptible allele as template. PCR screening will follow a 2-dimensional colony pooling method. Cosmids positive for the H9 gene will be sequenced. To verify that this sequence is that of the H9 resistance allele, Hessian fly susceptible wheat plants will be co-transformed with the cosmids and an herbicide resistance gene, bar. Ten to 20 transformed lines will then be screened against H9-avirulent Hessian fly larvae. Transformed plants will also be microscopically examined for a characteristic H9 resistance morphology at 6 h, 54 h, 78 h, and 126 h post-infestation. The larvae are expected to die on the transformed plants and the plants are expected to display the characteristic H9 resistance morphology if the H9 resistance allele has been identified. Indisputable evidence that the H9 resistance allele has been cloned from the original donor is expected.

Progress 01/15/09 to 01/14/11

Outputs
OUTPUTS: Many agricultural insect pests are controlled using cultivars that carry single insect resistance (R) genes that are bred into the plants using conventional breeding methods. This method of pest control eliminates the negative environmental impacts and the high costs associated with chemical pesticides. However, the efficacy of any single R gene has a limited durability. This research has advanced knowledge pertaining to the durability of R gene mediated resistance, particularly with respect to plant resistance conferred against insects. The focus of the investigation was the Hessian fly (Mayetiola destructor) and its interaction with its host plant, wheat (Triticum spp.). The Hessian fly is the world's most important insect pest of wheat. Its genetic tractability makes it possible to investigate the genetic basis of resistance in plants to insects, and the ability of insects to overcome that resistance. On susceptible wheat seedlings, Hessian fly larvae produce a gall where the larvae feed and develop. Plant galling stunts the seedlings and prevents them from producing grain. On resistant cultivars, Hessian fly larvae are neither able to induce a gall nor live on the plant. Resistant seedlings develop normally and yield grain. Thirty-three different resistance genes in wheat, named H1 through H32 and Hdic, condition resistance to the Hessian fly. Each gene can act independently to condition resistance to the Great Plains (GP) strain of the Hessian fly. The objectives of the investigation were to discover the structure of a single R gene (H9) and the mutations in the Hessian fly strains that are permit the insect to survive on plants carrying H9. The results of this investigation have been disseminated in publications, presented as invited lectures at national and international meetings, presented as invited seminars, and presented as talks and posters at national meetings. Invited lectures include presentations at the 2009, 2010, and 2011 National Meetings for the Entomological Society of America, the January 2011 Plant and Animal Genome Workshop, the 2010 International Plant Resistance to Insects Workshop, and the 2009 Third Annual Arthropod Genomics Symposium. Further dissemination of the results of this investigation will be forthcoming, including an invitation to publish a review in the Annual Review of Phytopathology. PARTICIPANTS: Dr. Rajat Aggarwal, a postdoc in the laboratory of Dr. Stuart (Dept. of Entomology, Purdue University), participated in the physical and genetic mapping of BAC clones on the chromosomes of the Hessian fly. Thiago Benatti, graduate student in the laboratory of Dr. Stuart (Dept. of Entomology, Purdue University), participated in the physical and genetic mapping of BAC clones on the chromosomes of the Hessian fly. Dr. Ming Shun-Chen (USDA-ARS, Dept. of Entomology, Kansas State University) participated in the physical mapping of BAC clones on the chromosomes of the Hessian fly. Dr. Brandon Schemerhorn (USDA-ARS, Dept. of Entomology, Purdue University) participated BAC-end sequencing and the discovery of molecular genetic markers in the sequence. Dr. Brett Tyler, Professor, Virgina Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg VA 24061 Chaoyang Zhoa, , graduate student in the laboratory of Dr. Stuart (Dept. of Entomology, Purdue University), participated in the physical and genetic mapping of BAC clones on the chromosomes of the Hessian fly. TARGET AUDIENCES: Wheat growers, wheat breeders, entomologists, molecular insect scientists, plant pathologists, plant geneticists. PROJECT MODIFICATIONS: Attempts to clone wheat R gene H9 were complicated by the size of the gene. While cloning H9 remained a goal, we shifted our attention to the closely linked R gene Hdic. We found that Hdic was smaller, and thus more easily cloned. On going efforts to identify the Avr gene that corresponds to Hdic are expected to provide the first cognate plant-insect R-Avr gene pair.

Impacts
The first arthropod avirulence (Avr) genes were discovered. These are effector protein encoding genes. This result is consistent with the hypothesis that plant parasitic insects, like biotrophic plant pathogenic microorganisms, have a gene-for-gene relationship with their host plants. This knowledge has enabled the development of DNA-based diagnostics for the presence of H9 and H13 virulence in field populations of the Hessian fly. Like the effectors of filamentous plant pathogens, Hessian fly effectors contain RXLR motifs that bind phosphatidylinositol-3-phosphate (PI3P), and enable the effector to pass through the plant cell membrane. Lipid-binding assays and cell-entry assays demonstrated that the vH13 effector's RXLR motif specifically binds PI3P and that this binding mediates cell entry. Double stranded RNA interference (RNAi) was shown to knock down gene expression in the Hessian fly. RNAi will now become an additional tool that enables the functional analysis of Hessian fly genes. This is expected to contribute to our understanding of plant gall induction and development. Experimental matings performed while investigating the genetics of the Hessian fly-wheat interaction revealed chromosome inversions that determine whether Hessian fly females produce female families or male families. Their discovery is consistent with sex-chromosome evolution theory and suggests that organisms that have post-zygotic sex determining mechanisms may be inclined to evolve into species composed of unisexual families. The first Hessian fly R gene (Hdic) in wheat was discovered. Hdic was genetically mapped near H9 near the telomere of wheat chromosome arm 1AS. Hdic was cloned using a map-based cloning approach, and Bobwhite (susceptible) wheat transformation experiments demonstrated that the cloned gene was the Hdic resistance allele. This was confirmed by knockdown of the Hdic in its original source (wheat WGRC44). Hdic encodes a protein that contains both a nucleotide-binding site and a leucine rich repeat. The mechanics of Hessian fly feeding and gall formation were elucidated using scanning and transmission electron microscopy. Results demonstrated that resistance is manifested by localized cell death, the fortification of the cell wall, and an elaboration of the endoplasmic reticulum-Golgi complex. The fitness costs associated with Hessian fly strains that are able to live on plants carrying R genes H9 and H13 were measured. This indicated that insects lacking vH9 and vH13 Avr gene encoded effectors are less fit than the insects that produce those effectors. A lowered fitness may act to maintain the vH9 and vH13 Avr genes in Hessian fly populations where the H9 and H13 R genes are not deployed. In summation, the wheat-Hessian fly interaction has remarkable similarities to fungal- and oomycete-plant interactions. Similarities were observed in Avr structure, R gene structure, fitness, and the mechanics of feeding and plant resistance. This suggests that methods may be developed to interfere with these common features to control both plant parasitic insects and filamentous plant pathogens.

Publications

  • Zhang, H., K.M. Anderson, J.J. Stuart, S. Cambron, and M.O. Harris. 2011. A reproductive fitness cost associated with Hessian fly (Diptera: Cecidomyiidae) virulence to gene-for-gene resistance. Journal of Economic Entomology (in press).
  • Anderson, K.M., Q. Kang, J. Reber, and M.O. Harris. 2011. No fitness cost for wheat's H-gene mediated resistance to Hessian fly (Diptera: Cecidomyiidae). Journal of Economic Entomology (in press).
  • Xu, S.S., C.G. Chu, M.O. Harris and C.E. Williams. 2011. Comparative analysis of genetic background in eight near-isogenic wheat lines with different H genes conferring resistance to Hessian fly. Genome 54: 81-89.
  • Harris, M.O., T.P Freeman, K.G. Anderson, J.A. Moore, S.A. Payne, K.M. Anderson, and O. Rohfritsch. 2010. H gene-mediated resistance to Hessian fly exhibits features of penetration resistance to fungi. Phytopathology 100: 279-289.
  • Liu X.M., Williams C.E., Nemacheck J.A., Wang H.Y., Subramanyam S., Zheng C., and Chen, M.S. (2010) Reactive Oxygen Species Are Involved in Plant Defense against a Gall Midge. Plant Physiology. 152: 985-999.
  • Yu, G.T., X. Cai, M.O. Harris, Y. Gu, M. Luo, and S.S. Xu. 2010. Development and validation of molecular markers closely linked to H32 for resistance to Hessian fly in wheat. Crop Science 50: 1325-1332.
  • Behura, SK, RH Shukle, JJ Stuart. 2010. Assessment of Structural Variation and Molecular Mapping of Insertion Sites of Desmar-like Elements in the Hessian Fly Genome. Insect Mol. Biol. 19:707-715.
  • Chen, M-S, X Liu, Z Xang, HX Zhao, RH Shukle, JJ Stuart, S Hulbert. 2010. Unusual conservation among genes encoding small secreted salivary gland proteins from a gall midge. BMC Evolutionary Biology 10, 296 (2010).


Progress 01/15/09 to 01/14/10

Outputs
OUTPUTS: The long-term goal of the project is to understand how mutations in Avirulence (Avr) genes in the HF genome enable the insect to overcome the resistance conferred by specific HF resistance (R) genes in wheat. Toward that goal, this project focuses on two objectives: 1) Discover the mutations that enable the insect to overcome the resistance conferred by the H9 R gene, and 2) discover the H9 R gene resistance allele. Mutations that enable the Hessian fly to overcome the resistance conferred by the H9 R gene were first positioned on a physical map of the Hessian fly genome. This map was consisted of Hessian fly bacterial artificial chromosomes (BACs) whose positions on the chromosomes were determined using fluorescence in situ hybridization to the insect's polytene chromosomes. Both ends of each of the BAC clones had also been determined so that DNA markers could be rapidly discovered in the physical map. Using the map and BAC-end sequences, the mutations causing virulence to H9 were located within 170 kb of the Hessian fly genome near the telomere of the short arm of chromosome X1. Two BACs were discovered to cover this region, and were then sequenced to identify DNA polymorphisms that exist between H9-virulent and H9-avirulent individuals. The sequence was also annotated to reveal putative genes within the sequence. Male Hessian flies were collected from fields in South Carolina, Georgia, Alabama, Louisiana, and North Dakota and genotyped as H9-virulent and H9-avirulent in laboratory bioassays. The genotyped individuals were then used to refine the position of the mutations causing H9-virulence. Mutations that were completely associated with H9-virulence were positioned in a single gene, named vH9. The putative amino acid sequence of vH9 was determined from the DNA sequence. The temporal and spatial expression of vH9 was determined using reverse transcription (RT) PCR. The length of the putative H9 resistance allele complicated our attempts to clone the gene. However, a closely linked HF R gene (Hdic) was cloned and transformed into three independent HF susceptible plants. Transformation was associated with Hdic resistance. The T1 offspring of one resistant plant segregated for Hdic resistance to HF. The T1 offspring of the remaining transformed plants are currently being tested. Results of this work have been disseminated in peer-reviewed publications and in posters and talks presented at scientific meetings. Additional publications of are in preparation. PARTICIPANTS: Dr. Ming Shun-Chen (USDA-ARS, Dept. of Entomology, Kansas State University) participated in the physical mapping of BAC clones on the chromosomes of the Hessian fly. Dr. Rajat Aggarwal, a postdoc in the laboratory of Dr. Stuart (Dept. of Entomology, Purdue University), participated in the physical and genetic mapping of BAC clones on the chromosomes of the Hessian fly. Thiago Benatti, graduate student in the laboratory of Dr. Stuart (Dept. of Entomology, Purdue University), participated in the physical and genetic mapping of BAC clones on the chromosomes of the Hessian fly. Chaoyang Zhoa, , graduate student in the laboratory of Dr. Stuart (Dept. of Entomology, Purdue University), participated in the physical and genetic mapping of BAC clones on the chromosomes of the Hessian fly. TARGET AUDIENCES: Wheat growers, wheat breeders, entomologists, molecular insect scientists. PROJECT MODIFICATIONS: The length of the putative H9 resistance allele complicated our attempts to clone the gene. However, a closely linked HF R gene (Hdic) was cloned. To obtain a cognate R gene Avr gene pair, we have begun to develop the mapping populations that will permit us to identify the Avr gene that corresponds to Hdic.

Impacts
Results of this investigation demonstrate that plant parasitic insects use effector proteins in their interactions with their host plants. These proteins interfere with the plant's basal immune system, but also interact with the products of plant resistance genes to stimulate effector-triggered immunity. The interaction between the effectors and resistance gene products is the basis of the gene-for-gene interaction observed between plant pathogens and their host plants. Results confirm that certain insects, like the Hessian fly, also have a gene-for-gene interaction with their hosts.

Publications

  • Benatti, T, FH Valicente, R Aggarwal, C Zhao, JG Walling, M-S Chen, SE Cambron, BJ Schemerhorn, JJ Stuart. 2010. A neo-sex-chromosome that drives post-zygotic sex determination in the Hessian fly (Mayetiola destructor). Genetics 184:769-777.
  • Aggarwal, R, TR Benatti, N Gill, C Zhao, M-S Chen, BJ Schemerhorn, JP Fellers, JJ Stuart. 2009. A BAC-based physical map of the Hessian fly genome anchored to polytene chromosomes. BMC Genomics 10:293.