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.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	3130	1120	50%
206	1310	1160	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 206 - Basic Plant Biology;

Subject Of Investigation
1310 - Potato; 3130 - Nematodes;

Field Of Science
1120 - Nematology; 1160 - Pathology;

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.