2012 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.
The golden potato cyst nematode (a.k.a. golden nematode or GN) is a devastating quarantine pest that threatens the U.S. potato industry. This potato pest secretes effector proteins into root cells to manipulate host cellular processes for successful infection. ARS researchers have analyzed several GN effector genes (previously called parasitism genes) and identified sequence variations that are useful for developing diagnostic methods for nematode identification. From the identified gene sequence variations, we developed a molecular diagnostic method for identifying a new nematode species related to the GN that was recently detected in Oregon. We have conducted in-depth study on several GN effector genes and discovered a critical role these effectors play in GN parasitism. Most importantly, we found that some of these effectors act as mimics of plant signaling peptides to regulate plant developmental pathways to promote successful nematode parasitism and we are starting to discover host plant receptors that perceive these important effectors. By using the new information discovered, we are exploring novel methods for generating engineered nematode resistance in potato. The significance of this research was demonstrated by the filing of a conventional patent application in collaboration with University scientists. Collaborations with Cornell and several other university potato breeding programs have identified many potato clones including advanced clones that are resistant to GN. Several advanced potato clones such as NY140 that is resistant to both pathotypes of GN may be released as named varieties in the near future. The availability of GN resistant varieties is critical for helping maintain GN quarantine in the U.S. This year we employed a new method called Protein Interaction Reporter (PIR) technology to identify sites on the two virus capsid proteins that interact with each other and themselves to form structures necessary for the virus to be transmitted by insects. Several of the genes encoding aphid proteins we previously linked to virus transmission competency were identified in other related insects that transmit plant viruses in a manner similar to aphids. This reveals some potential common targets that could be exploited to disrupt virus transmission in several insect taxa. Infectious clones of two Yellow Dwarf virus species were developed that can be introduced into plants via agrobacterium. These allow precise changes to be made in the viruses to study the function of their genes and proteins. A transformation and regeneration system was developed for Nightshade, a valuable experimental host for luteovirus genetic studies of virus transmission and plant infection. Studies on potato virus Y focused on genetic diversity of the virus and the development of improved diagnostic assays. The major recombinant strain of PVY overtaking the common strain in the US has at least two independent evolutionary origins. Using these data, serological assays have been improved for detection of the new tuber necrotic strain emerging in the US.
Improved diagnostic method to identify tuber necrotic strains of Potato virus Y (PVY). Tuber necrotic strains of PVY are emerging in the US and have the potential to become a major quality disease issue for the US potato industry, threatening farm income and export options. In collaboration with scientists at the University of Idaho, ARS researchers at Ithaca, New York have developed new knowledge of the specificities and shortcomings of commercially available diagnostic reagents for PVY. This led to improved testing protocols that eliminate false positives and allow detection of all variants within the tuber necrotic strain of PVY. Protocols were transferred to state and federal partners that conduct product testing and regulate interstate and international commerce of potatoes.
A molecular diagnostic method for identifying a new nematode species related to GN has been developed. A new nematode species related to potato cyst nematodes was recently detected in Oregon. There was no quick and reliable method for identifying this new species. By using sequence variations identified in an effector gene, ARS researchers at Ithaca, New York have developed a molecular method that provides highly reliable and rapid identification of this Oregon nematode species. This method would be useful for monitoring the potential spread of this new potato pest within Oregon and in other potato producing states.
Cilia, M., Bereman, M., Fish, T., Maccoss, M.J., Gray, S.M. 2012. Homopteran vector biomarkers for efficient circulative plant virus transmission are conserved in multiple aphid species and the whitefly Bemisia tabaci. Journal of Integrative Agriculture. 11(2):249-262.
Chavez, J., Cilia, M., Ju, H., Weisbrod, C., Eng, J.K., Gray, S.M., Bruce, J.E. 2012. Cross-linking measurements of the potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus-plant interactions. Journal of Proteome Research. 11(5):2968-2981.
Kerlan, C., Nikolaeva, O.V., Hu, X., Meacham, T., Gray, S.M., Karasev, A. 2011. Identification of the molecular make-up of the Potato virus Y strain PVYZ: genetic typing of the PVYZ-NTN. Phytopathology. 101:1052-1060.
Yoon, J., Choi, S., Palukaitis, P., Gray, S.M. 2011. Agrobacterium-mediated infection of whole plants by yellow dwarf viruses. Virus Research. 160:428-434.
Karasev, A., Hu, X., Brown, C., Kerlan, C., Nikolaeva, O., Crosslin, J., Gray, S.M. 2011. Genetic diversity of the ordinary strain of potato virus Y (PVY) and origin of recombinant PVY strains. Phytopathology. 101:778-785.
Nikolaeva, O., Roop, D., Galvino-Costa, S., Dos Reis Figueira, A., Gray, S.M., Karasev, A. 2012. Epitope mapping for monoclonal antibodies recognizing tuber necrotic isolates of Potato virus Y. American Journal of Potato Research. 89:(2):121-128.
Mitchum, M.G., Wang, X., Wang, J., Davis, E.L. 2012. Role of nematode peptides and other small molecules in plant parasitism. Annual Review of Phytopathology. 50:175-195.
Jittayasothorn, Y., Chen, S., Wang, X., Zhong, G. 2011. Influences of Agrobacterium rhizogenes strains, plant genotypes, and tissue types on the induction of transgenic hairy roots in Vitis species. Vitis. 50(3):107-114.