Source: UNIV OF IDAHO submitted to NRP
A NOVEL STRATEGY TOWARDS RESISTANCE TO POTATO CYST NEMATODE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
Reporting Frequency
Annual
Accession No.
1032109
Grant No.
2024-67013-42425
Cumulative Award Amt.
$298,928.00
Proposal No.
2023-10076
Multistate No.
(N/A)
Project Start Date
Jun 1, 2024
Project End Date
May 31, 2026
Grant Year
2024
Program Code
[A1112]- Pests and Beneficial Species in Agricultural Production Systems
Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
(N/A)
Non Technical Summary
The potato cyst nematode (PCN) Globodera pallida was first detected in the United States in 2006 and has been one of the major production constraints for potato and can cause up to 100% yield loss. To successfully infest and colonize potato roots, PCN produces proteins, called effectors, and uses the stylet, a needle-like apparatus, to deliver them into potato root cells. One of the major functions of these effectors is to destroy plant defense system, including the one called pattern-triggered immunity (PTI) which is conferred by pattern recognition receptor (PRR). We have found PCN used a specific effector RHA1B that can degrade the potato PRR termed StNILR1 thereby overcoming potato defense. Thus, we will use the combination of biochemical and genetic approaches to engineer StNILR1 protein - make it not degradable by RHA1B effector, and transfer this "new engineered" StNILR1 gene into potato to possibly make it resistant to PCN. Toward this end, we propose two research objectives: 1.) Engineer RHA1B degradation resistant StNILR1 mutants; 2.) Manipulate the StNILR1 gene in potato towards resistance to PCN.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2123130112050%
2061310116050%
Goals / Objectives
Our long-term goal is to develop novel, innovative and environmentally-sound methods for improved resistance to the agriculturally important cyst nematode (Globodera pallida) in potato. In general, as the first level of resistance to invading pathogens and pests, plants use pattern recognition receptors (PRRs) to detect invading pathogens thereby triggering immune signaling and resistance, presumably, in the case of plant-nematode interactions, through perceiving nematode-associated molecular patterns (NAMPs). We have recently discovered that the potato (Solanum tuberosum) NEMATODE-INDUCED leucine-rich repeat receptor-like kinase 1 (StNILR1) is the immune receptor of the nematode NAMP Ascaroside #18 (Ascr18), mediating Ascr18-triggered immune signaling and resistance to plant-parasitic nematodes. Unfortunately, the potato cyst nematode G. pallida has evolved a specific effector RHA1B, which is a functional ubiquitin ligase, to target and degrade StNILR1, thereby destroying StNILR1-mediated plant resistance and facilitating nematode parasitism. Thus, we propose two objectives to manipulate the StNILR1 gene towards resistance to G. pallida in potato: 1.) Engineer RHA1B degradation resistant StNILR1 mutants; 2.) Manipulate the StNILR1 gene in potato towards resistance to G. pallida.
Project Methods
The following approaches will be adopted to achieve our objectives:Objective 1: Engineer RHA1B degradation resistant StNILR1 mutants.In our preliminary experiments we have found that the G. pallida RHA1B effector targets the immune receptor StNILR1 for degradation. In this objective, we seek to engineer StNILR1 mutants that are resistant to RHA1B degradation but retain the function of recognition of the nematode NAMP Ascr18. Towards this end, we will use two approaches: 1.) We will generate a StNILR1 deletion mutant without the PEST motif by conventional sub-subcloning method and use site-directed mutagenesis to generate RHA1B degradation-resistant SlNILR1 mutants with point-mutations. The resulting mutants will be verified by co-expression with RHA1B in N. benthamiana leaves. 2.) Once RHA1B degradation resistant StNILR1 mutants are generated, verification of the functionality of these mutants will be conducted by transiently expressing them in Arabidopsis nilr1 mutant protoplasts. Transfected nilr1 protoplasts will be treated with Ascr18 and the immune signaling will be determined by qPCR analysis on the induction of the immune marker gene PR4.Objective 2. Manipulate the StNILR1 gene in potato towards resistance to G. pallida. In this objective, the functional StNILR1 mutants (containing epitope Flag tag) will be fused with its native nematode-inducible promoter or CaMV 35 promoter and introduced into the susceptible potato cultivar Desiree by well-established Agrobacterium-mediated transformation. Multiple transgenic lines will be generated and verified by PCR analysis using the StNILR1 promoter- or CaMV 35S promoter-specific primer together with the Flag tag-specific primer. All transgenic lines will be further verified for the stability of StNILR1 mutant proteins upon G. pallida infection by Western blotting using the anti-Flag antibody. Possibly gained resistance rendered by the engineered StNILR1 immune receptor in those transgenic potato lines will be determined by G. pallida infection, with the non-transgenic potato plants serving as a control.

Progress 06/01/24 to 05/31/25

Outputs
Target Audience:Through Potato Expo and potato conference we presented our research to potato growers and industry colleagues. Through professional conferences, we presented research results and interacted with peers in the field of plant-nematode interactions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student and oneundergraduate studenthave been trained during the report period time. How have the results been disseminated to communities of interest?The relevant research results have been presented to potato growers at the Idaho Potato Conference. What do you plan to do during the next reporting period to accomplish the goals?We will continue to generate the ProStNILR1::StNILR1?PEST construct and introduce it into potato via conventional tissue culture-base transgenic approach.

Impacts
What was accomplished under these goals? Objective 1. Engineer RHA1B degradation resistant StNILR1 mutants. We also conducted site-directed mutagenesis to generate alanine substitution at the PEST motif in the full length StNILR1 and obtained five alanine substation mutants (termed StNILR1Mut). When transiently co-expressed these StNILR1Mut mutants with RHA1B in N. benthamiana leaves, all of them were degraded by RHA1B, suggesting alanine substitution at the PEST motif is not sufficient to stabilize StNILR1 in response to RHA1B action. We have also generated a StNILR1 deletion mutant without the PEST motif (termed StNILRΔPEST mutant) and subcloned into the plant expression vector pTEX and pBIN. The resulting pTEX::StNILRΔPEST construct will be transiently expressed in Arabidopsis nilr1 mutant protoplasts, and their expression will be verified by Western blotting. Transfected nilr1 protoplasts will be treated with Ascr18 and the immune signaling will be determined by qPCR analysis on the induction of the immune marker gene PR4. StNILRΔ245-278 partially restored, as manifested by partial induction of the immune marker gene PR4 by Ascr18. Objective 2. Manipulate the StNILR1 gene in potato towards resistance to G. pallida. In this objective, the functional StNILR?PEST gene will be fused with its native nematode-inducible promoter and introduced into potato to confer resistance to G. pallida. To this end, a 2.5kb promoter fragment has been PCR-amplified from potato and subcloned into the plant expression vector to replace the original CaMV 35S promoter.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Huang L., Yuan Y, Ramiez C, Xia C., Zhang C., Kud J, Kuhl JC, Caplan A, Dandurand L-M, Xiao F* (2024) The potato RNA metabolism machinery is targeted by the cyst nematode effector RHA1B for successful parasitism. The Plant Cell. 2024 Sep 26:koae264. doi: 10.1093/plcell/koae264. Online ahead of print. PMID: 39325717