Location: Emerging Pests and Pathogens Research
2021 Annual Report
Objectives
Our long-term objective is to develop improved management strategies for the range of pathotypes of the major invasive and emerging nematode and virus pathogens affecting the United States potato crop. While the potato industry is eager to improve cultural and genetic (i.e. resistance) management strategies that can be readily translated to the field, they are also interested in developing fundamental information on emerging pathogens to ensure appropriate and timely detection and the development of novel management strategies. Over the next 5 years we will focus on the following:
Objective 1: Define the genetic diversity and evolution of PCN and virus populations, and optimize associated diagnostic assays. [NP303, C1, PS1]
Sub-Objective 1.1: Compare genome sequences of G. rostochiensis pathotypes (Ro1 and Ro2) to identify sequence variations that may be used for developing molecular diagnostic markers.
Sub-Objective 1.2: Monitor PVY strain diversity in the seed potato crop.
Sub-Objective 1.3: Characterize PVY diversity and evolution.
Objective 2: Discover and characterize genes and proteins regulating virus-vector-host and nematode-host interactions. [NP303, C2, PS2A and PS2C]
Sub-Objective 2.1: Characterize candidate effector protein-encoding genes and their associated host proteins contributing to nematode parasitism and virulence.
Sub-Objective 2.2: Characterize infection and transmission competence of PVY strains and strain combinations in potato and aphid populations.
Sub-Objective 2.3: Define the mechanism of tissue tropism of poleroviruses in plants and aphid vectors.
Objective 3: Develop virus and nematode resistant potatoes that are acceptable to the potato industry and consumers. [NP303, C3, PS3A]
Sub-Objective 3.1: Determine the resistance of potato clones and wild potato species to G. rostochiensis pathotypes.
Sub-Objective 3.2: Collaborate with potato breeders to develop genetic markers for phenotypic traits useful in the development of durable virus resistance.
Approach
In general, nematode parasites and virus diseases of potatoes cause severe crop loss and effective control measures are lacking. Nematicides effective against the Potato cyst nematodes (PCN) are no longer available and alternative control strategies for emerging pathotypes/populations have not been developed. Similarly, virus disease control strategies are completely lacking. New plant biotechnologies will provide the basis for the development of novel methods of nematode and virus control, but the success of these methods will be dependent upon a more complete understanding of the fundamental mechanisms of host-nematode and host-virus-vector interactions. One approach is to define the genetic diversity and evolution of PCN and virus populations, and optimize associated diagnostic assays. Inbreed lines of different races of PCN will be sequenced. Candidate SNPs and other variations indicated to be unique for PCN pathotypes will be further analyzed and converted into PCR-based or other types of markers and finally validated by examining a range of PCN populations. Virus populations and strains will be monitored in the potato crop to identify new recombinants and facilitate diagnostic updates. Effects of vertical and horizontal transmission on virus populations will provide information on selection pressures most important in driving the emergence of new strains. A second approach is to discover and characterize genes and proteins regulating virus-vector-host and nematode-host interactions. Stylet-secreted PCN effector proteins that manipulate multiple host cellular processes to promote successful infection will be discovered and characterized by multiple technologies to better understand the function of these effectors in nematode parasitism and virulence. Virus work with focus on the infection and transmission competence of Potato virus Y (PVY) strains to identify factors that regulate virus acquisition and transmission by aphids and synergistic/antagonistic interactions of PVY strains in plants that limit the availability of virus to aphid vectors. We will continue to investigate how potato leafroll virus protein domains direct long distance virion movement while other domains direct virus cell to cell movement of RNA-protein complexes. The third approach is to develop virus and nematode resistant potatoes that are acceptable to the potato industry and consumers. Working with breeders we will continue to use bioassays and marker assisted selection to evaluate breeding clones and wild potato accessions for nematode resistance and develop genetic markers linked to phenotypic traits caused by PVY infection such as tuber necrosis and leaf necrosis. Conceptually novel information on the population genetics of nematode, aphid and virus pests of potato crops, and on host–pathogen-vector interactions will aid in the development of new effective biologically-based disease control strategies. Improved virus and nematode diagnostics as well as genetic markers for disease resistance and disease resistant cultivars developed through conventional breeding and genetic engineering can be transferred readily to customers.
Progress Report
Sub-Objective 1.1: Compare genome sequences of potato cyst nematode (PCN) pathotypes (Ro1 and Ro2) to identify sequence variations that may be used in molecular diagnostics. The potato cyst nematode, Globodera rostochiensis (golden nematode), is a quarantine pest that poses a serious threat to the U.S. potato industry. Two pathotypes of GN, Ro1 and Ro2, are currently present in infested fields in New York. Ro1 is well controlled by resistant potato cultivars containing the H1 resistance gene. However, Ro2 can reproduce well on H1-containing potato cultivars, and control measures for this virulent pathotype are very limited. Determining the presence of Ro2 from the endemic pathotype Ro1 still relies on the traditional bioassay method that takes approximately twelve months to achieve a reliable determination. Thus, developing reliable and rapid molecular diagnostic tools for identifying different pathotypes of the golden nematode is urgently needed. Researchers in Ithaca, New York, made significant progress in analyzing sequence variations that were previously identified by whole-genome sequencing of Ro1 and Ro2 single cyst lines. They have analyzed > 200 sequence variations found between Ro1 and Ro2 populations using dCAPS (derived cleaved amplified polymorphic sequences) and PCR assays and have identified four sequence variations that may be used for diagnostic marker development. These sequence variations are being analyzed by testing golden nematode populations from other geographic regions to verify their utility for diagnostic marker development further.
Sub-Objective 2.1: Characterize candidate effector protein-encoding genes and their associated host proteins contributing to nematode parasitism and virulence. Plant-parasitic nematodes, including the two potato cyst nematode species (PCN; Globodera rostochiensis and G. pallida), are among the most devastating plant pathogens that cause significant crop yield losses annually. These nematode pests secrete a wide range of secretory proteins (called effectors) into host root cells to induce disease. It is now known that PCNs encode a large group of effector proteins; however, the exact function of these effectors and the nature of the host cellular program that they may target to promote successful infection are largely unknown. Understanding how nematode effectors function in host root cells is necessary for developing effective and sustainable methods for PCN control. Studies this year focused on the confirmation of host targets of a group of effectors (e.g., Gr29D09 and GrSPRYSEC effectors) that were previously demonstrated to have a role in host defense suppression. Several candidate proteins in the host plant potato were confirmed as targets of these effectors.
Most importantly, we identified and characterized a novel effector named PIMP4 (plant immunity manipulating protein 4). Our studies revealed that PIMP4 effectors from cyst nematodes play a key role in interfering with plant immunity (or plant defenses). Interestingly, PIMP4 effectors contain two conserved motifs, one of which was shown to be responsible for targeting autophagy-related proteins in the host cell. Autophagy is a major protein degradation process in eukaryotes that has been recently recognized as a battlefield in plant-pathogen interactions. For the first time, our studies have discovered that plant-parasitic cyst nematodes have evolved effectors to manipulate the host autophagy system to enable successful infection. By screening existing databases using bioinformatics tools, we further identified a large group of effectors from PCN that contain one or more conserved motifs that may be capable of mediating the interaction with autophagy-related proteins in the host cell. Understanding how this novel group of effectors affect host autophagy and other cellular processes will uncover the molecular mechanisms of PCN infection of potato but may also suggest vulnerable points that can be manipulated to generate novel and durable resistance in potato against PCN.
Researchers in Ithaca, New York, continued to make progress on understanding the signaling pathways mediated by nematode secreted CLE effectors, mimics of plant CLE (CLAVATA3/Embryo Surrounding Region) peptide hormones that regulate many aspects of plant growth and development. New host receptors and transcription factors downstream of CLE signaling were identified and are under further characterization. We also identified CLE genes in the potato host that are regulated during nematode infection. Our studies have added new knowledge on CLE peptide signaling in plant-nematode interactions and suggest components in the CLE signaling pathways that may be targeted to develop novel nematode resistance in potato plants.
Sub-Objective 3.1: Determine the resistance of potato clones and wild potato species to PCN pathotypes. Researchers in Ithaca, New York, continued the collaboration with potato breeders at Cornell University and other major U.S. potato breeding programs to screen potato breeding clones for resistance against the golden nematode. A total of 117 breeding clones were screened using marker test and pot bioassays and 91 of them showed resistance to Ro1 or Ro2 of the golden nematode. These clones will be retested for nematode resistance multiple times and further evaluated in fields by University collaborators until they may be released as a golden nematode-resistant variety. We also screened 14 existing European varieties and identified 9 varieties to have Ro2 resistance. These resistant varieties may serve as an alternative tool for Ro2 control in New York. We continued to screen wild potato clones obtained from the U.S. Potato Genebank for nematode resistance. A total of 30 clones were confirmed to have extreme resistance to Ro2. These clones are being evaluated for resistance to G. pallida in collaboration with researchers at the University of Idaho. Most importantly, we identified one wild potato clone (named Y1-5) that exhibits strong and broad-spectrum resistance to both PCN species. A mapping population was further obtained from crosses between Y1-5 and a susceptible parent clone. This mapping population will be used for the cloning of the novel resistance gene(s) present in Y1-5. The identification of new resistant potato germplasm and novel resistance genes will accelerate the development of potato varieties with novel and/or durable PCN resistance, which are critically needed to support PCN control and eradication programs, and to help sustain the profitability of the U.S. potato industry.
Accomplishments
1. Identification of a novel group of nematode-secreted effector proteins. Plant-parasitic nematodes utilize secreted effector proteins to induce disease. Identification of nematode effectors and understanding how they function in the host root cell is necessary to develop effective and sustainable means for nematode control. ARS scientists in Ithaca, New York, discovered, for the first time, that cyst nematodes have evolved effectors that target the host plant autophagy system, a major intracellular degradation system that has been recently linked to plant defenses against pathogen infection. Our studies have added new knowledge on the molecular basis of plant-nematode interactions and suggested methods for generating engineered resistance in crop plants including soybean and potato.
2. Two previously released GN resistant potato varieties now rank as the most popular chipping varieties grown in the U.S. Potato cyst nematodes, including the golden nematode and the pale cyst nematode, are quarantine pests important to agriculture. Deploying resistant varieties is the most effective and sustainable means for combating potato cyst nematodes. ‘Lamoka’ and ‘Waneta’ are two golden nematode resistant varieties previously developed and released through collaboration between ARS scientists in Ithaca, New York, and potato breeders at Cornell University. These two varieties are now ranked as the 1st and 4th most widely grown chipping varieties in the U.S. The commercial success of these two varieties has helped ensure the continued effectiveness of golden nematode quarantine in the U.S.
Review Publications
Dandurand, L.M., Zasada, I.A., Wang, X., Mimee, B., Dejong, W., Novy, R.G., Whitworth, J.L., Kuhl, J. 2019. Current status of potato cyst nematodes in the United States and Canada. Annual Review of Phytopathology. 57:117-133. https://doi.org/10.1146/annurev-phyto-082718-100254.
Mondal, S., Wintermantel, W.M., Gray, S.M. 2021. Virus and helper component interactions favour the transmission of recombinant potato virus Y strains. Journal of General Virology. 102(6). https://doi.org/10.1099/jgv.0.001620.
Wang, X., Yang, H., Veronneau, P., Thurston, D., Mimee, B. 2021. Genome resources of two pathotypes of the potato cyst nematode Globodera rostochiensis from New York. Phytopathology. https://doi.org/10.1094/PHYTO-09-20-0403-A.
Dejong, W., Halseth, D.E., Plaisted, R.L., Wang, X., Perry, K.L., Qu, X., Paddock, K.M., Falise, M., Christ, B.J., Porter, G.A. 2020. Waneta, a variety with excellent chip color out of cold storage, long tuber dormancy, and resistance to the Golden Cyst Nematode. American Journal of Potato Research. 97:580-585. https://doi.org/10.1007/s12230-020-09806-z.
Da Silva, W., Kutnjak, D., Xu, Y., Xu, Y., Giovannoni, J.J., Santiago, E., Gray, S.M. 2020. Transmission modes affect the population structure of potato virus Y in potato. PLoS Pathogens. 16(6). https://doi.org/10.1371/journal.ppat.1008608.
