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United States Department of Agriculture

Agricultural Research Service

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2010 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
The golden potato cyst nematode (GN), a quarantine pest, secretes proteins into the plant as it feeds Identification of the genes encoding these proteins will aid in the development of diagnostic molecular markers and novel control strategies. We have cloned 3 additional GN genes and identified sequence variants for each that may be useful for diagnostic marker development. An in-depth functional characterization of these cloned genes using molecular biology, biochemical, proteomics, and functional genomics approaches indicate these genes play a significant role in nematode infection and parasitism of the host plant. Most importantly, we found that GN uses molecular mimicry of plant CLE signaling peptides to parasitize the host root. We are in the process of identifying potato receptor proteins that interact with nematode-secreted CLE peptides. A further study of this molecular mimicry may identify plant targets for developing novel strategies to reduce or disrupt nematode parasitism of potato plants. The use of nematode resistant potato varieties is the most effective and environmentally-sound means for GN control. Evaluation of potato clones from several potato breeding programs identified Ro1 and Ro2 resistance in 265 of 455 and 51 of 76 clones, respectively. The Yellow dwarf and Potato leafroll viruses are transmitted by aphid vectors and are recognized by aphid proteins that regulate virus movement through gut and salivary cells. Identification of these proteins is critical to the development of strategies to control virus spread. Genetic and proteomic studies of aphid genotypes differing in vectoring ability and viruses differing in transmissibility identified aphid, virus, plant and bacterial endosymbiont proteins involved in virus transmission. These provide novel targets for developing strategies to disrupt the virus transmission process. Several of the aphid and bacterial proteins were confirmed as biomarkers for vector genotypes providing an opportunity to predict the likelihood of virus disease epidemics and to target insect control only towards vector populations. A 5 yr, $4.8 million grant was awarded to develop effective management options for Potato virus Y (PVY) in seed potatoes and to eradicate emerging tuber necrotic viruses from seed stocks. This allows for an expansion of the PVY research program and the development of an education and outreach program to assist the potato industry to more effectively recognize and control PVY. We have developed modifications to PVY testing protocols used by certification and regulatory agencies to eliminate erroneous identifications of PVY variants that have negatively impacted trade. NAPPO is currently revising their testing protocols based on our research data. Continued testing of a novel intragenic potato line resistant to PVY determined the resistance is effective under field production conditions and has no impact on plant growth and tuber yield. The intragenic strategy, whereby the transgene is derived from the target crop itself, avoids many of the concerns surrounding transgenic crops and may facilitate acceptance by the potato industry and consumers.

4. Accomplishments
1. A potato virus Y resistant potato developed using intragenic technology. PVY is the most important disease problem for the seed potato industry and emerging tuber necrotic strains of PVY threaten the quality of the commercial crop unless appropriate control strategies are developed. ARS researchers in Ithaca, NY and Aberdeen, ID in collaboration with scientists at Cornell University and the University of Wisconsin have developed and field tested an intragenic potato line that is resistant to multiple strains of Potato virus Y (PVY). Natural mutations in the plant eIF4E translation factor have been shown to confer resistance to potyviruses in many plant species, but not potato. Using knowledge about eIF4E resistance genes in other solanaceous plants, the potato eIF4E gene was modified to resemble resistance alleles from pepper. The modified potato genes were over-expressed in potato (cv Russet Burbank) and the plants were unable to support the accumulation or systemic movement of several different strains of PVY. The resistant plants grew similar to untransformed controls and produced similar numbers of tubers. PVY was not detected in plants sprouted from any of the tubers from the inoculated resistant plants. This strategy should be applicable to engineer PVY resistance in any potato cultivar as well as resistance in other crops to a number of economically important viruses. Furthermore, the ‘intragenic’ strategy, whereby the transgene is derived from the target crop itself, avoids many of the concerns surrounding transgenic crops.

2. Sensitive molecular diagnostics test developed for potato cyst nematodes. Current traditional methods for validation of the presence of potato cyst nematodes in suspected soils have relied upon morphological analysis, bioassay with susceptible potato plants, and sequencing of the ITS-region of extracted DNA from suspect cysts, all of which are time-consuming and expensive to perform. ARS Researchers in Ithaca, NY have developed highly specific and sensitive molecular (qPCR) methods for the differentiation of the two species of potato cyst nematode (PCN), which currently exist in the U.S. Our newly-developed molecular diagnostic methods offer rapid, specific, and sensitive identification of PCN species compared to traditional methods. The high degree of specificity and sensitivity of these methods should permit nematode detection in soil extracts that contain extremely low amounts of nematode DNA. These methods will provide a valuable detection tool useful in nematode regulatory and quarantine programs throughout the U.S. We have filed a conventional patent application for the techniques, and the method has been shared with USDA, APHIS, for internal validation.

Review Publications
Lu, S., Chen, S., Wang, J., Yu, H., Chronis, D., Mitchum, M.G., Wang, X. 2009. Structural and functional diversity of CLAVATA3/ESR (CLE)-like genes from the potato cyst nematode Globodera rostochiensis. Molecular Plant-Microbe Interactions. 22:1128-1142.

Benitez-Alfonso, Y., Cilia, M., San Roman, A., Thomas, C., Maule, A., Hearn, S., Jackson, D. 2009. Control of Arabidopsis meristem development by thioredoxin-dependent regulation of intercellular transport. Proceedings of the National Academy of Sciences. 106(9):3615-3620.

Cilia, M., Fish, T., Yang, X., Mclaughlin, M., Thannhauser, T.W., Gray, S.M. 2009. A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomics Studies of Aphid Proteins. Journal of Biomolecular Techniques. 20(4):201-215.

Last Modified: 2/23/2016
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