2007 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 In the initial 5 months of this project, full-length sequences of both the cDNA and genomic clones of 4 additional putative parasitism genes (GrCLE1, GrCLE4, Gr33E05, and GrVAP1) were identified from the Golden Nematode (GN). Extensive DNA sequence and regular DNA gel blot analyses did not identify pathotype-specific polymorphisms useful for diagnostic marker development. However, we found that the GrCLE4 gene is organized as tandem repeats in the genome of the GN. Genomic DNA fragments of the intergenic regions between repeats of the GrCLE4 gene were cloned and analyzed. Initial sequence analysis revealed extensive polymorphisms in these intergenic regions. Detailed sequence analysis was conducted to identify potential pathotype-specific polymorphisms. The 4 parasitism genes were predicted to encode secretory proteins and found to be expressed exclusively within the esophageal gland cells of the nematode, suggesting that they may be important for modulating nematode parasitism. We evaluated potato clones developed by the Cornell University Breeding Program for GN resistance. Ro1 resistance was identified in 757 of 1053 clones and Ro2 resistance was identified in 182 of 381 clones. In addition, the advanced clone NY 140 was confirmed to be resistant to Ro2. We have developed 3 cDNA libraries from aphid head tissue, gut tissue and whole bodies. These have been screened with a yeast 2 hybrid system to identify aphid proteins that bind luteovirus and polerovirus structural proteins. We have 7 candidate aphid proteins that may regulate the transmission of viruses. Concurrently, we are comparing the total proteomes from aphid genotypes derived from the same parents, but that differ in their ability to transmit viruses as an alternative means to identify aphid proteins that regulate the transmission of viruses. To date we have identified over 20 candidate proteins that differ among vector and nonvector populations. Towards our goal of developing transgenic potato resistant to Potato virus Y, 6 cultivars were evaluated for their ability to regenerate plants from transformed cells, 4 were selected for further study. We have successfully transformed one variety of potato with a mutated version of a pepper or potato gene that is required for the virus to replicate. Preliminary data suggest that overexpression of the gene results in resistance to virus infection. Over 18,000 potato tubers collected from 16 states have been tested for PVY infection. More than 2500 positive samples were identified. Ongoing molecular and biological analysis of the viruses will provide a third year of data on the genetic diversity and geographic distribution of PVY strains affecting the U.S. seed potato crop. These data will be used to make recommendations to the potato industry regarding disease management and distribution of seed stocks. 4.Accomplishments Functional similarity of GN CLE proteins as plant peptide signals. Phytonematodes secret a variety of secretory proteins and these secretions play direct roles in nematode parasitism. Two major types of CLE-like genes (GrCLE1 and GrCLE4) that have sequence similarity as Arabidopsis CLE genes were cloned from the GN. Studies of overexpression of GrCLE genes in tomato hairy roots and in vitro peptide application assays provided additional evidence that Golden Nematode CLE proteins may function as ligand mimics of plant peptide signals to facilitate nematode parasitism. A better understanding of this molecular mimicry will provide new targets for developing novel resistance in potato. 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. Small changes in the Potato leafroll virus (PLRV) capsid protein alter the survival and transmission of the virus. PLRV causes a yield limiting disease in potato and control options are limited. The development of novel control measures is dependent upon a better understanding of how the virus causes disease and spreads in nature. Our study identified features of the virus structural protein that regulates its ability to form stable virus particles necessary for plant infection and identified other regions that are required for the virus to be transmitted by aphids to other plants. These protein features provide targets for new disease control strategies that are aimed at preventing virus movement within the plant or preventing aphids from transmitting the disease and infecting new plants. 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 None. 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 1 Review Publications Ali, A., Li, H., Schneider, W.L., Sherman, D.J., Gray, S.M., Smith, D., Roossinck, M.J. 2006. Analysis of genetic bottlenecks during horizontal transmission of cucumber mosaic virus. Journal of Virology. 80:8345-8350 Liu, F., Wang, X., Xie, Y., Gray, S.M., Zhou, G., Gao, B. 2007. A chinese isolate of barley yellow dwarf virus-pav represents a third distinct species within the pav serotype. Archives of Virology. 152:1365-1373 Kaplan, I.`., Lee, L., Ripoli, D., Palukaitis, P., Gildow, F., Gray, S.M. 2007. Point mutations in the potato leafroll virus major capsid protein alter virion stability and aphid transmission. Journal of General Virology. 88:1821-1830.