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United States Department of Agriculture

Agricultural Research Service

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2008 Annual Report

1a. Objectives (from AD-416)
The long-term objective of this project is to develop an improved understanding of how endemic and emerging pathotypes of nematodes and viruses become established and are subsequently maintained in potato and grain crops. This knowledge is critical to the development of effective and sustainable control strategies, and obtaining this knowledge has become more imperative due to recent events. The emergence of new pathotypes of the golden potato cyst nematode (GN, Globodera rostochiensis), has raised new concerns about the ability of scientists and regulatory agencies to detect the nematode and about the continued effectiveness of current quarantine and management strategies. Similarly, national surveys of Potato virus Y (PVY) in seed potato production areas indicate an increase in the genetic diversity of PVY and emergence of necrotic forms of the virus. All of these findings are restricting interstate and international movement of potatoes. Over the next 5 years we will focus on the following objectives: Objective 1: Improved detection and characterization of emerging pathotypes of the GN and emerging strains of PVY. Sub-objective 1.A. Develop molecular markers that differentiate pathotypes of the GN based on divergence in nematode parasitism gene sequences. Sub-objective 1.B. Determine the geographic and genetic distribution of PVY strains affecting the U.S. potato crop and develop improved diagnostic assays. Objective 2: Identification and characterization of genes regulating the pathogenicity and transmission of viruses and nematodes affecting potato, and viruses affecting small grains. Sub-objective 2.A. Identify and characterize nematode parasitism genes. Sub-objective 2.B. Identify and characterize aphid and virus genes regulating virus transmission. Objective 3: Development of industry and consumer acceptable potato genotypes that express novel or improved resistance to virus and nematode pathogens.

1b. Approach (from AD-416)
Genetic diversity and diagnostic studies will identify differences in nematode parasitism genes that may be involved in pathogenicity or virulence/avirulence of the nematode. This information will be used to develop molecular diagnostic tools that will distinguish these two pathotypes. Serological, biological, and molecular characteristics of PVY isolates representative of each state, potato variety and production area will be used to group virus isolates and develop improved diagnostic assays to detect the strains of the virus that are potentially the most economically damaging in terms of yield and trade. Functional studies of pathogenicity and transmission genes will focus on secretory proteins encoded by parasitism genes expressed within the nematode’s esophageal gland cells known to be the principal molecular signals regulating both pathogenicity and virulence/avirulence of the nematode; as well as on aphid genes expressed in gut and salivary tissues whose products interact with specific domains on the two virus structural proteins. Genomic and proteomic based technologies will be employed to identify and characterize nematode and aphid proteins, and determine their functional role in the host-parasite/pathogen interaction. The development of nematode resistant potato varieties will focus on conventional breeding practices to transfer known nematode resistance genes to new germplasm with improved horticultural traits. Transgenic technologies will be used to isolate potato genes required by viral pathogens for replication, modify these potato genes and re-introduce them into accepted potato varieties so the altered forms no longer support virus replication.

3. Progress Report
Five genes (Gr-33E05, Gr-VAP1, Gr-SKP1, Gr-7E05, and Gr-Ubi1) were characterized from both pathotypes (Ro1 and Ro2) of the golden nematode (GN) and discovered to be involved in parasitism. DNA sequence analysis showed the genes to be similar between the pathotypes and not useful for diagnostic marker development. Gr-33E05, Gr-VAP1, Gr-SKP1 and Gr-Ubi1 were found to be expressed exclusively within the esophageal gland cells of the nematode. Additionally, the functional characterization of another parasitism gene (Gr-CM1) was completed and discovered to use a unique method of expression known as alternative splicing. Additionally, we have conducted detailed functional characterization of Gr-CLE genes. The Gr-CLE genes cloned from the GN encode novel CLE peptides that contain multiple CLE domains. In planta overexpression studies and in vitro peptide application assays suggest that this novel class of nematode CLEs has functional similarity as plant CLE signaling peptides and highlight an important role for ligand mimicry in plant parasitism by the golden nematode. We have also evaluated potato clones developed by the Cornell University Breeding Program and other potato breeding programs. Ro1 resistance was identified in 334 of 482 clones and Ro2 resistance was identified in 43 of 60 clones. Yellow dwarf viruses, the most important virus diseases of cereal crops worldwide, are transmitted by aphid vectors; a process that requires the virus be recognized by aphid proteins that then transport virus through gut and salivary cells. These aphid proteins are unknown; however, their identification is critical to the development of molecular targeting strategies to control virus spread. Genetic studies of aphid genotypes that differ in vectoring capacity indicated that transmission of each virus is controlled by different proteins at the gut and salivary tissues. The total protein content of vector and nonvector genotypes was compared using a technique called Two-dimensional Difference Gel Electrophoresis. Over 40 proteins were identified that are differentially expressed between vectors and nonvectors. Distinct proteins were also found associated with gut or salivary gland barriers. The identification of these proteins is in progress using Mass Spectrometry. Ongoing molecular and biological analysis of over 2500 potato virus Y (PVY) isolates collected during a survey of the U.S. seed potato crop has identified numerous PVY strains not previously considered to be prevalent or widespread in North America. Necrotic isolates of PVY are now widespread in the U.S., although the common strain still predominates. These data are used to make recommendations to the potato industry regarding disease management and distribution of seed stocks. Furthermore, we have successfully transformed potato with a genetically modified version of a pepper or potato gene that is required for PVY to infect plants. Overexpression of these modified genes render some transgenic lines resistant to multiple strains of PVY. Research relates to NP 303, Component 2–Biology, Ecology, Epidemiology and Spread of Plant Pathogens and Their Relationships with Hosts & Vectors.

4. Accomplishments
1. The proteins that are produced and secreted by plant-parasitic nematodes as they feed play essential roles in nematode infection of host plants. We have isolated and functionally characterized a chorismate mutase gene that encodes a secreted protein from the golden nematode, an important pest of potato. Importantly, we have discovered that the activity of this secreted protein is controlled by a novel mechanism that has never been reported for any plant-parasitic nematodes. Furthermore, our study found that the chorismate mutase protein is linked to a novel form of nematode resistance in potato. Understanding the function of nematode secretions may lead to the development of novel nematode control strategies. NP 303, Component 2-Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement A-Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen.

2. Potato leafroll virus (PLRV) has two structural proteins that make up its outer shell. The minor protein is multifunctional and is known to play a role in spread of the virus both within plant hosts and between plants by insect vectors. Our work has determined how this protein works and has defined what parts of the protein are responsible for different functions. One end of the protein is responsible for restricting the virus to plant vascular tissues, a novel finding that is a first for virology. Furthermore, in this capacity this protein functions as a soluble rather than structure protein. When the protein is incorporated into the shell of the virus, another part of the protein is required for the virus to be transmitted by its insect vectors. Understanding how a virus moves within and between plants provides potential targets for novel control strategies. NP 303, Component 2-Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement B-Plant-Microbe-Vector Interactions.

5. Significant Activities that Support Special Target Populations

Review Publications
Mitchum,, M.G., Wang, X., Davis,, E.L. 2008. Diverse and conserved roles of CLE peptides. Current Opinion in Plant Biology. 11:75-81.

Wang, X., Replogle, A., Davis, E., Mitchum, M. 2007. The tobacco Cel7 gene promoter is auxin-responsive and locally induced in nematode feeding sites of heterologous plants. Molecular Plant Pathology. 8:423-436.

Sukno, S.A., Mccuiston, J., Wong, M., Wang, X., Thon, M.R., Hussey, R., Baum, T., Davis, E. 2007. Quantitative Detection of Double-Stranded RNA-Mediated Gene Silencing of Parasitism Genes in Heterodera glycines. Journal of Nematology. 39:145-152.

Singh,, R.P., Valkonen,, J.T., Gray, S.M., Boonham,, N., Jones,, A.C., Kerlan,, C., Schubert,, J. 2008. The naming of Potato virus Y strains infecting potato. Archives of Virology. 153(1):1-13.

Yang, X., Thannhauser, T.W., Burrows, M.E., Cox Foster, D., Gildow, F., Gray, S.M. 2008. Coupling genetics and proteomics to identify aphid proteins associated with vector-specific transmission of Polerovirus (Luteoviridae). Journal of Virology. 82:291-299.

Ramsey,, J., Wilson,, A., De Vos,, M., Sun,, Q., Tamborindinguy,, C., Winfield,, A., Mallack,, G., Smith,, D., Fenton,, B., Gray, S.M., Jander,, G. 2007. Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design. Biomed Central (BMC) Genomics. 8:423.

Peter,, K., Liang,, D., Palukaitis,, P., Gray, S.M. 2007. Small deletions in the potato leafroll virus readthrough protein affect particle morphology, aphid transmission, virus movement and accumulation. Journal of General Virology. 88:1821-1830.

Last Modified: 2/23/2016
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