2009 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 (GN), a quarantined pest of agricultural importance, secretes parasitism proteins during the interaction with its host plant. The identification of genes encoding these secreted proteins is necessary for discovering diagnostic molecular markers and developing novel strategies for GN control. In addition to ten parasitism genes that were cloned previously, we have isolated two additional parasitism genes from GN. Analyses showed that these two genes are similar between nematode pathotypes and not useful for diagnostic marker development. We have conducted a detailed functional characterization of all twelve parasitism genes using molecular biology and functional genomics approaches. Preliminary results indicated that all these genes play a critical role in nematode infection and parasitism of the host plant. In particular, the functional characterization of four genes that encode CLE peptides determined that these peptides can functionally mimic plant CLE peptides. Plant CLE peptides have diverse roles in plant growth and development and our findings suggest that molecular mimicry of plant CLE peptide may be an important mechanism for GN to parasitize the host root. A better understanding of this peptide mimicry may identify plant targets for developing novel nematode control strategies. The use of nematode resistant potato varieties is the most effective and environmentally-sound means for GN control. We have also evaluated potato clones developed from a variety of potato breeding programs. Ro1 resistance was identified in 457 of 632 clones.
Luteoviruses, which include the Yellow dwarf and Potato leafroll viruses are transmitted by aphid vectors. These viruses are recognized by aphid proteins that allow the virus to move 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 and proteomic studies of aphid genotypes that differ in vectoring capacity identified over 50 proteins that were differentially expressed between vectors and nonvectors. Several of these proteins can be associated with gut or salivary gland barriers and are likely to be tissue specific proteins. Mass spectrometry has identified a majority of the proteins into functional groups that include endocytosis, trafficking to endosomes and peroxisoms, as well as transcription and translation. Ongoing molecular and biological analysis of potato virus Y (PVY) isolates collected during a survey of the U.S. seed potato crop has identified a variant that is detected by serological tests used by state and federal regulator agencies as a necrotic isolate, but using more comprehensive diagnostics this variant was found to belong to the ordinary strain of PVY. An alternative testing procedure was proposed to avoid misidentification that has caused suspension of international shipments of potato. Greenhouse testing of potato transformed with a genetically modified version of a pepper or potato gene that is required for PVY to infect plants confirmed the plants are resistant to multiple strains of PVY.
Secreted proteins encoded by nematode parasitism genes play critical roles in nematode infection and parasitism of host plant. We have cloned four parasitism genes (Gr-Cle) from GN that encode secreted proteins with similarity to plant CLE peptides. Plant CLE peptides have diverse roles in plant growth and development. Our in-depth studies have demonstrated that nematode-produced proteins encoded by these Gr-CLE genes function similarly as plant CLE peptides, revealing an extraordinary example of molecular mimicry that may have evolved to facilitate the parasitic success of GN. Understanding the mechanism of this molecular mimicry may identify plant targets for developing novel forms of nematode resistance in potatoes.
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Lu, S., Tian, D., Borchardt Wier, H., Wang, X. 2008. Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis. Molecular and Biochemical Parasitology. 162:1-15.
Cavatorta, J., Savage, A., Yeam, I., Gray, S.M., Jahn, M. 2008. Positive Darwinian selection at single amino acid sites conferring plant virus resistance. Journal of Molecular Evolution. 67(5):551-559.
Peter, K., Gildow, F., Palukaitis, P., Gray, S.M. 2009. The C-terminus of the Polerovirus P5 readthrough domain limits virus infection to the phloem. Journal of Virology. 83(11):5419-5429.
Hu, X., Meacham, T., Ewing, L., Gray, S.M., Karasev, A.V. 2009. A novel recombinant strain of Potato virus Y allows identification of a new viral genetic determinant of vein necrosis in tobacco. Virus Genes. 143(1):68-76.