CHARACTERIZATION OF THE CYST NEMATODE EFFECTOR AND HOST SUSCEPTIBILITY GENES TO DEVELOP NEMATODE RESISTANCE IN POTATO

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1027802
• Grant No.: 2022-67013-36138
• Cumulative Award Amt.: $681,158.00
• Proposal No.: 2021-08446
• Project Start Date: Jan 1, 2022
• Project End Date: Dec 31, 2025
• Grant Year: 2022
• 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 infect and colonize potato roots, PCN produces proteins, called effectors, and uses the stylet, a needle-like apparatus, to deliver them into potato root cells. All these effectors have different biochemical activities in potato cells and function together to help develop a feeding site (called syncytium) for nematode development and growth. Thus, in order to eventually generate potato varieties resistant to PCN, it is important to understand how effectors interact with each other and manipulate host genes to overcome plant defense and establish the feed site. Although several plant-parasitic nematode effectors have been shown to suppress plant defense signaling, the molecular basis by which effectors manipulate host plants is largely unknown, mainly because we do not know what kind of biochemical activities effectors have and how they deal with the plant defense response inside host root cells.We have recently found a PCN effector RHA1B, which is an enzyme termed ubiquitin ligase responsible for degradation of proteins in all eukaryotic cells. Significantly, RHA1B can suppress plant defense signaling, suggesting RHA1B could arm G. pallida with a unique advantage in nematode parasitism. Particularly, RHA1B could target plant proteins encoded by genes termed susceptibility genes which are important for successful colonization of nematode. In other words, we will be able to make potato plants resistant to nematode if we can genetically make these genes not functional or remove them. Thus, to further investigate the molecular basis by which G. pallida manipulates host potato plants for successful colonization and to identify the host susceptibility genes that are essential for G. pallida infection, with an ultimate goal of generation of G. pallida-resistant potatoes via loss of susceptibility, we propose three research objectives:Determine RHA1B can function as a master effector that can affect other effectors' during infection of host potato roots;Determine what genes in host potato are affected by RHA1B effector and how these susceptibility genes are manipulated to help produce feeding site for nematode;Generate G. pallida-resistant potato by genetically manipulating these susceptibility genes, which will be achieved by making thesegenes not functional through gene-editing technique.In brief, the proposed project will help us understand the molecular basis of how the potato cyst nematode (G. pallida) infects the host plant (potato) and eventually use the evolved knowledge to develop novel, innovative and environmentally-sound ways for improved resistance to nematode.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	3130	1040	70%
212	1310	1040	30%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1310 - Potato; 3130 - Nematodes;

Field Of Science
1040 - Molecular biology;

Keywords

globodera pallida

metaeffector

potato cyst nematode

susceptibility gene

ubiquitin ligase

Goals / Objectives
Our long-term goal is to understand the molecular basis by which the potato cyst nematode (PCN), Globodera pallida, manipulates the physiological processes of host potato plants for successful colonization, and to eventually use the evolved knowledge to develop cultivars with improved resistance to G. pallida. Particularly, our research focuses on elucidating the biological significance of G. pallida effectors during establishment of the feeding site. Although several plant-parasitic nematode effectors have been shown to suppress plant defense signaling, the mechanistic basis by which effectors manipulate host defense is largely unknown, mainly due to lack of understanding of the biochemical characteristics of the effectors. We have recently identified the novel G. pallida effector RHA1B, which is the first E3 ubiquitin ligase effector found in eukaryotic pathogens. Significantly, RHA1B can suppress defense signaling and function in concert with three host ubiquitin E2s when ubiquitinating in vitro. These results suggest that the ability of RHA1B to exploit multiple host E2s could arm G. pallida with a unique advantage in parasitism, in which various RHA1B-E2 combinations provide the parasite with the ability to affect a wider range of host plant physiological processes via the manipulation of both proteolytic and non-proteolytic protein processes in infected hosts. Thus, to further investigate the mechanistic basis by which G. pallida manipulates host physiological processes for successful colonization and to identify the host susceptibility genes that are essential for G. pallida infection, with an ultimate goal of generation of G. pallida-resistant potatoes via loss of susceptibility, we propose three research objectives: 1) Determine the role of RHA1B as a potential metaeffector; 2) Determine the host targets of the RHA1B ubiquitin ligase; 3) Generate G. pallida-resistant potato via loss of susceptibility.

Project Methods
The following approaches will be adopted to achieve our objectives:Objective 1: Determine the role of RHA1B as a potential metaeffector.In this objective, we seek to determine whether, besides targeting host factors, RHA1B also acts as a metaeffector to post-translationally modify other effectors, and what may be the biological consequence of this higher-order regulation.To identify RHA1B-interacting effectors (RIEs), we will use two approaches: 1.) Bioinformatic analysis of the genomic region surrounding the RHA1B sequence to identify neighboring genes whose encoded proteins are good candidates for potential RHA1B interactors; 2.) comprehensive yeast two-hybrid (Y2H) screening against an esophageal gland-specific cDNA library using RHA1B as a bait.To determine the biological significance of RHA1B-effector interplay, we will first verify whether RIEs identified above are substrates of RHA1B. RIEs will be expressed in E. coli to generate recombinant protein and used for RHA1B-mediated in vitro ubiquitination assays. Once verified as substrates, the biological consequence of ubiquitination of such effectors by RHA1B will be tested. Potential outcomes to be tested include: 1.) degradation of effectors mediated by RHA1B (tested by Western blotting analysis following co-expression of RHA1B and RIEs in N. benthamiana); 2) change of in planta localization of effectors by RHA1B (determined by fluorescence microscopy of GFP-fused RIEs); 3) change of activity of effectors promoted by RHA1B (applicable if any specific enzymatic activity is detected for RIEs).To determine the role of RHA1B in parasitism, we will use the in-planta RNA interference (RNAi) approach to eventually silence the RHA1B gene in PCN attacking transgenic potatoes. Upon generation of transgenic potato plants expressing dsRNA of RHA1B, the possible gained resistance to G. pallida in those transgenic potato plants will be determined.Objective 2. Detemine the host targets of RHA1B.To identify RHA1B-interacting proteins (RIPs), the 12 putative RIPs identified by Y2H and AP-MS, the full-length cDNAs of these three chaperone genes will be cloned from potato and constructed into the relevant expression vectors for co-IP, in vitro ubiquitination, and in vivo degradation assays.To determine the putative targets of RHA1B, first, we will verify interactions between RIPs and RHA1B in planta by co-IP assay; Second, the RIPs and chaperones confirmed by co-IP will be cloned into the pMAL-c2 vector to obtain the recombinant proteins from E. coli, followed by in vitro ubiquitination assays to determine whether they can be ubiquitinated by RHA1B; Third, the RIPs and chaperones confirmed by in vitro ubiquitination will be further examined to determine whether RHA1B promotes their degradation by the proteasome.Objective 3. Generate G. pallida-resistant potato via loss of susceptibility.Loss of susceptibility could be a novel strategy to develop G. pallida resistance in potato. Towards this end, we will further characterize the putative susceptibility genes identified in Objective 2 and genetically manipulate them using CRISPR and RNAi techniques. We speculate that RHA1B indirectly manipulates these genes by ubiquitin-mediated modifications of intermediate regulators, of which CCR4-NOT and transcription factors are key players. Thus, once verified in Objective 2, putative susceptibility genes identified by both Y2H and AP-MS (particularly CCR4-NOT10, Caf1, KIN12B, WRKY and bZIP transcription factors) will be genetically manipulated towards obtaining nematode resistance.To generate transgenic potato plants with knockout/knockdown of susceptibility genes, We will generate and characterize individual transgenic potato plants with CRISPR/CRISPR-associated9 (Cas9) endonuclease-based knockouts of up to 15 identified susceptibility genes in potato. CRISPR constructs will then be used to generate transgenic potato via Agrobacterium-mediated transformation. Knockout of the target susceptibility genes will be verified by DNA sequencing of target genes. However, it is theoretically possible that the CRISPR/Cas9 system might not render efficient editing on some susceptibility genes in tetraploid potato. If this is the case, we will use an RNAi-based hairpin RNA construct to knock down recalcitrant susceptibility genes in potato.To determine the role of susceptibility genes in the formation of syncytium in the potato host plant, we will inoculate susceptibility gene-knockout/knockdown and WT potato plants with G. pallida to compare the development of a syncytium. We will infect 4-week-old potato grown in microscopy rhizosphere chambers with hatched G. pallida J2s and harvest infected roots for microscopic studies at 2, 4, 7 and 14 days post inoculation. Root cross-sections will be carefully evaluated using light and transmission electron microscopy to monitor the formation of syncytia.To assess the resistance of transgenic potato plants with knockout/knockdown of susceptibility genes, multiple independent transgenic lines and non-transgenic control plants will be inoculated with G. pallida cysts (10 cysts per plant) in the root zone. To evaluate infection rate, half of tested plants will be subjected for an acid fuchsin assay to score all nematode life stages six weeks post-inoculation. To evaluate reproductive success, fully formed cysts will be recovered from the dry soil of remaining plants using an elutriator ten weeks post-inoculation. We will determine the number of cysts formed per plant, number of eggs per cyst, egg viability (Meldola blue staining), and calculate the nematode reproduction factor (Rf=Pf-newly produced eggs/Pi-initial egg population).

Progress 01/01/24 to 12/31/24

Outputs
Target Audience:We have reached a very broad audience. Through Potato Expo and potato conference we presented our research to potato growers and industry colleagues. Through professional conferences and publications, 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?Two postdocs, one graduate student and four undergraduate students have been trained in the PI's and co-PI's labs during the report period time. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue to determine the biological significance of RHA1B-RIEs interplay and the role of RHA1B in parasitism, focusing on determining whether RHA1B ubiquitinates RIEs and generating/characterizing transgenic potato plants expressing RHAB1-Ri construct was created (Objective 1); we will also determine the role of StNILR1 in basal level resistance to nematode and whether it also plays a role in resistance to other pathogens, including oomycete Phytophthora infestans (objective 2); and we will keep working on the cellular basis of the role of StCAF1-NOT10 deadenylase in potato-G. pallida interactions, focusing on investigating syncytium development in G. pallida-infected StCAF1-KD and StNOT10-KD transgenic potato roots (objective 3).

Impacts
What was accomplished under these goals? ?Objective 1. Determine the role of RHA1B as a potential metaeffector. We have identified five RHA1B neighboring genes designated as RIE1-5 and transiently co-expressed them in N. benthamiana leaves with a wild-type RHA1B or an E3 ligase deficient form of RHA1B (RHA1BC135S). Significantly, the stability of two of them, RIE1 and RIE4, was negatively impacted by co-expression with RHA1B in an E3-dependent manner. In planta interaction between HA-RIE1 (ME1) or HA-RIE4 (ME4) with GFP-RHA1B was tested using co-immunoprecipitation assay (IP anti-HA) after the transient co-expression in N. benthamiana leaves. We also determined the role of these RIEs in interference of plant defense signaling. We co-expressed RIEs with the potato Gpa2 R protein and its corresponding avirulent G. pallida effector RBP1 in Nicotiana benthamiana leaves and found RIE4 can suppress HR cell death caused by Gpa2-RBP1. The HR suppression activity of RIE4 was further verified by its activity on suppression of HR caused by Rx1 (resistance to the virus), Prf (resistance to the bacterium) and Rpi-blb1 (resistance to the oomycete). In addition, we found ROS production in response to flg22 in N. benthamiana leaves is inhibited by expression of all tested effectors (RIE1, RIE4, and RHA1B), suggesting these effectors play a role in suppression of generic PTI defense triggered by PAMPs. Objective 2. Determine the host targets of the RHA1B ubiquitin ligase. We characterized the 12 putative RIPs identified by Y2H and AP-MS, of which RHA1B interacts with two subunits (StNOT10 and StCAF1) of a highly conserved RNA deadenylase complex CcR4-NOT. When transiently expressed in N. benthamiana leaves, RHA1B triggered degradation of StNOT10 and StCAF1 in a ubiquitin ligase activity-dependent manner. Moreover, the in vitro ubiquitination assay indicates that RHA1B can specifically ubiquitinate StNOT10 protein. We next determined the deadenylase activity of StCAF1 in vitro and further explored the genes that are regulated by StCAF1 deadenylase in the context of potato-G. pallida interaction. Since CAF1 has been reported to recruit the RNA binding protein, we determined the potato PUMILIO5 (StPUM5) protein, which binds to a specific motif (UGUACAUG) within the 3' untranslated region (3'-UTR) of target gene mRNAs (Ref), to target mRNA for deadenylation. We conducted Co-IP assays and found the StPUM5 interacts with StCAF1 in vivo and in vitro. These findings suggest that StPUM5 serves as an RNA-binding protein. In Drosophila, the CAF1-based deadenylase deadenylates cyclinA mRNA to disrupt mitosis and cyclin genes are reported to be essential for early development of syncytia. We hypothesized that StPUM5 might directly target potato CyclinA mRNA, presumably by binding to specific motifs within their 3'-UTRs. Among six potato CycA (StCycA) genes, only StCycA2 (ID: PGSC0003DMP400004562) contains a putative PUMILIO binding motif (UGUACAUG) in its 3'-UTR, marking it as a prime candidate for StPUM5-mediated regulation. To verify this possibility, RNA immunoprecipitation (RIP)-Chip assay was carried out to determine that StPUM5 specifically binds to StCycA2 mRNA. Further G/I tailing assay was conducted to verify the poly-A tail length of StCycA2 mRNA in the WT and transgenic StCAF1-KD and StPUM5-KD plants. The results indicate that when StCAF1 or StPUM5 was knocked down, the poly-A tail length of StCycA2 mRNA was markedly enriched. Significantly, the subsequent qRT-PCR assay indicated elevated StCycA2 mRNA levels in StCAF1-KD and StPUM5-KD transgenic plants compared to WT plants, suggesting that StCAF1 deadenylates StCycA2 mRNA to target it for decay, thereby down-regulating the StCycA2 gene at the post-transcriptional level. Thus, our results suggest a novel parasitic mechanism by which G. pallida effector manipulates host RNA metabolism machinery to positively regulate syncytium development and facilitate nematode parasitism. In addition, we have found that the potato homolog of NILR1, termed StNILR1, also recognizes nematode-associated molecular pattern (NAMP) ascaroside #18 (Ascr18) to activate immune signaling and resistance against G. pallida. Isothermal titration calorimetry (ITC) assays revealed a direct binding of the StNILR1 ectodomain (Ecto-StNILR1) to Ascr18. The role of StNILR1 in basal level resistance against G. pallida was determined using both gain-of-function and loss- in basal level resistance against G. pallida was determined using both gain-of-function and loss-of-function approaches. Overexpression of StNILR1 (StNILR1-OX) imparted resistance to G. pallida, whereas knockdown of StNILR1 (StNILR1-KD) by RNA interference (RNAi) resulted in increased susceptibility. Significantly, our co-IP assay indicated RHA1B interacts with StNILR1 in plant cells leaves via Agrobacterium-mediated transient expression. It is notable that the proteasome inhibitor MG115 was included in the Agrobacterium inocula to prevent possible degradation of StNILR1 triggered by RHA1B. We further examined the effect of RHA1B on StNILR1 protein levels without the presence of MG115 and found that StNILR1 protein accumulated well in plant cells when co-expressed with the empty vector but was not detected when co-expressed with RHA1B, suggesting RHA1B promotes StNILR1 degradation. Significantly, when StNILR1 was co-expressed with the RHA1B ligase-deficient mutant RHA1BC63S, in which the conserved Cys was substituted with a Ser in the RING domain, the RHA1B-triggered degradation of StNILR1 was abolished and the accumulation of StNILR1 protein resumed, suggesting RHA1B triggers proteasome degradation of StNILR1, dependent on its ubiquitin ligase activity. Moreover, our in vitro ubiquitination assay indicated that the recombinant RHA1B protein is able to ubiquitinate the recombinant StNILR1 protein (particularly the extracellular domain of StNILR1) in vitro. Thus, we conclude that the potato cyst nematode G. pallida has evolved the RHA1B effector, which is a functional ubiquitin ligase, to target StNILR1 for ubiquitination-mediated proteasome-dependent degradation, thereby promoting parasitism. Objective 3. Generate G. pallida-resistant potato via loss of susceptibility. To establish genetic evidence supporting the role of the CCR4-NOT deadenylase complex in nematode parasitism, we have generated multiple transgenic potato lines overexpressing either StCAF1 (StCAF1-OX) or StNOT10 (StNOT10-OX) and observed any altered responses to G. pallida. Based on quantification of StCAF1 or StNOT10 mRNA levels in the transgenic potato lines by qRT-PCR, three transgenic lines for each transgene were selected for nematode infection assay. The results indicated that both StCAF1-OX and StNOT10-OX transgenic plants were less susceptible to G. pallida. Additionally, we employed RNA-interference (RNAi) to generate loss-of-function transgenic potato lines with knockdown of StCAF1 (StCAF1-KD) or StNOT10 (StNOT10-KD) gene. Three transgenic lines with significant repression of either gene were selected for nematode infection assay. Significantly, knockdown of either StCAF1 or StNOT10 results in increased susceptibility to nematode infection, suggesting the CCR4-NOT deadenylase complex's contribution to resistance against G. pallida. In addition, to genetically determine the role of StCycA2, which is a regulatory target of CCR4-NOT-StPUM5 module, during G. pallida parasitism, we generated transgenic potato plants with StCycA2 being overexpressed (StCycA2-OX) or knocked down (StCycA2-KD) and determined their altered susceptibility to G. pallida. We found, in comparison to WT potato plants, StCycA2-OX plants were more susceptible to G. pallida infection, whereas StCycA2-KD plants exhibited enhanced resistance to G. pallida.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Huang L., Yuan Y, Ramiez C, Zhao Z., Chen T., Griebel T., Kud J, Kuhl JC, Caplan A, Dandurand L-M, Xiao F* (2024) A receptor for dual ligands governs plant immunity and hormone response and is targeted by a nematode effector. Proceedings of the National Academy of Sciences. 2024 Oct 15;121(42): e2412016121. doi: 0.1073/pnas.2412016121. Epub 2024 Oct 10.
• 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

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:We have reached a very broad audience. Through Potato Expo and potato conference we presented our research to potato growers and industry colleagues. Through professional conferences and publications, we presented research results and interacted with peers in the field of plant-nematode interactions Changes/Problems:Since October 2022, the co-PI, Dr. Joanna Kud, has moved to University of Arkansas located at Fayetteville Arkansas as an Assistant Professor. Dr. Kud is continuing working on the sponsored project as proposed and has made significant progress. What opportunities for training and professional development has the project provided?Two postdocs, one graduate student and four undergraduate students have been trained in the PI's and co-PI's labs during the report period time. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue to determine the biological significance of RHA1B-RIEs interplay and the role of RHA1B in parasitism, focusing on determining with RHA1B interacts with and ubiquitinates RIEs (Objective 1); we will also verify the ubiquitination of another putative target (StNILR1) by RHA1B, and determine whether StNILR1 plays an important role in resistance to nematode (objective 2); and we will begin to determine the cellular basis of the role of StCAF1 and StCycA2 in potato-G. pallida interactions, focusing on whether StNOT10 and StCAF1 affect the syncytium development in potato roots upon infection by G. pallida (objective 3).

Impacts
What was accomplished under these goals? Objective 1. Determine the role of RHA1B as a potential metaeffector. All five RHA1B neighboring genes designated as RIE1-5 were cloned into a binary vector and transiently co-expressed in N. benthamiana leaves with a wild-type RHA1B or an E3 ligase deficient form of RHA1B (RHA1BC135S). Significantly, Stability of RIE1 and RIE4 was dramatically reduced in the presence of RHA1B, but not the RHA1BC135S mutant, suggesting RHA1B may regulate RIE1 and RIE4. We also determined the role of these RIEs in interference of plant defense signaling. We co-expressed RIEs with the potato Gpa2 R protein and its corresponding avirulent G. pallida effector RBP1 in Nicotiana benthamiana leaves and found RIE4 can suppress HR cell death caused by Gpa2-RBP1. The HR suppression activity of RIE4 was further verified by its activity on suppression of HR caused by Rx1 (resistance to the virus), Prf (resistance to the bacterium) and Rpi-blb1 (resistance to the oomycete). Objective 2. Determine the host targets of the RHA1B ubiquitin ligase. We characterized the 12 putative RIPs identified by Y2H and AP-MS. Full-length cDNAs of these RIPs were cloned from potato and constructed into the relevant expression vectors for co-IP, in vitro ubiquitination, and in vivo degradation assays. We found RHA1B interacts with two subunits (StNOT10 and StCAF1) of a highly conserved RNA deadenylase complex CcR4-NOT. When transiently expressed in Nicotiana benthamiana leaves, RHA1B triggered degradation of StNOT10 and StCAF1 in a ubiquitin ligase activity-dependent manner. Moreover, the in vitro ubiquitination assay indicates that RHA1B can specifically ubiquitinate StNOT10 protein. We next determined the deadenylase activity of StCAF1 in vitro and further explored the genes that are regulated by StCAF1 deadenylase in the context of potato-G. pallida interaction. Since CAF1 has been reported to recruit the RNA binding protein, we determined the potato PUMILIO5 (StPUM5) protein, which binds to a specific motif (UGUACAUG) within the 3' untranslated region (3'-UTR) of target gene mRNAs (Ref), to target mRNA for deadenylation. We conducted Co-IP assays and found the StPUM5 interacts with StCAF1 in vivo and in vitro. These findings suggest that StPUM5 serves as an RNA-binding protein. In Drosophila, the CAF1-based deadenylase deadenylates cyclinA mRNA to disrupt mitosis and cyclin genes are reported to be essential for early development of syncytia. We hypothesized that StPUM5 might directly target potato CyclinA mRNA, presumably by binding to specific motifs within their 3'-UTRs. Among six potato CycA (StCycA) genes, only StCycA2 (ID: PGSC0003DMP400004562) contains a putative PUMILIO binding motif (UGUACAUG) in its 3'-UTR, marking it as a prime candidate for StPUM5-mediated regulation. To verify this possibility, RNA immunoprecipitation (RIP)-Chip assay was carried out to determine that StPUM5 specifically binds to StCycA2 mRNA. Further G/I tailing assay was conducted to verify the poly-A tail length of StCycA2 mRNA in the WT and transgenic StCAF1-KD and StPUM5-KD plants. The results indicate that when StCAF1 or StPUM5 was knocked down, the poly-A tail length of StCycA2 mRNA was markedly enriched. Significantly, the subsequent qRT-PCR assay indicated elevated StCycA2 mRNA levels in StCAF1-KD and StPUM5-KD transgenic plants compared to WT plants, suggesting that StCAF1 deadenylates StCycA2 mRNA to target it for decay, thereby down-regulating the StCycA2 gene at the post-transcriptional level. Thus, our results suggest a novel parasitic mechanism by which G. pallida effector manipulates host RNA metabolism machinery to positively regulate syncytium development and facilitate nematode parasitism. Objective 3. Generate G. pallida-resistant potato via loss of susceptibility. To establish genetic evidence supporting the role of the CCR4-NOT deadenylase complex in nematode parasitism, we have generated multiple transgenic potato lines overexpressing either StCAF1 (StCAF1-OX) or StNOT10 (StNOT10-OX) and observed any altered responses to G. pallida. Based on quantification of StCAF1 or StNOT10 mRNA levels in the transgenic potato lines by qRT-PCR, three transgenic lines for each transgene were selected for nematode infection assay. The results indicated that both StCAF1-OX and StNOT10-OX transgenic plants were less susceptible to G. pallida. Additionally, we employed RNA-interference (RNAi) to generate loss-of-function transgenic potato lines with knockdown of StCAF1 (StCAF1-KD) or StNOT10 (StNOT10-KD) gene. Three transgenic lines with significant repression of either gene were selected for nematode infection assay. Significantly, knockdown of either StCAF1 or StNOT10 results in increased susceptibility to nematode infection, suggesting the CCR4-NOT deadenylase complex's contribution to resistance against G. pallida. In addition, to genetically determine the role of StCycA2, which is a regulatory target of CCR4-NOT-StPUM5 module, during G. pallida parasitism, we generated transgenic potato plants with StCycA2 being overexpressed (StCycA2-OX) or knocked down (StCycA2-KD) and determined their altered susceptibility to G. pallida. We found, in comparison to WT potato plants, StCycA2-OX plants were more susceptible to G. pallida infection, whereas StCycA2-KD plants exhibited enhanced resistance to G. pallida.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Huang L, Yuan Y, Lewis C, Kud J, Kuhl JC, Caplan A, Dandurand LM, Zasada I, Xiao F (2023). NILR1 perceives a nematode ascaroside triggering immune signaling and resistance. Curr Biol. 2023 Sep 25;33(18):3992-3997.e3. doi: 10.1016/j.cub.2023.08.017. Epub 2023 Aug 28. PMID: 37643618

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:We have reached a very broad audience. Through Potato Expo and potato conference we presented our research to potato growers and industry colleagues. Through professional conferences and publications, we presented research results and interacted with peers in the field of plant-nematode interactions. Changes/Problems:Since October 2022, the co-PI, Dr. Joanna Kud, has moved to University of Arkansas located at Fayetteville Arkansas as an Assistant Professor. Dr. Kud is continuing working on the sponsored project as proposed and has made significant progress. What opportunities for training and professional development has the project provided?Two graduate students and four undergraduate students have been trained in the PI's and co-PI's labs during the report period time. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue to determine the biological significance of RHA1B-RIEs interplay and the role of RHA1B in parasitism using the in planta RNA interference (RNAi) approach (Objective 1); we will also verify the in vivo ubiquitination of CcR4-NOT10, CAF1 and NILR1 by RHA1B, and determine whether CAF1 is a functional deadenylase that may regulate mRNA encoded by genes involved in resistance or susceptibility to nematode (objective 2); and we will begin to generate in planta expression constructs overexpressing CRISPR and RNAi constructs harboring guide RNA or gene fragment targeting CcR4-NOT10, CAF1 or NILR1, respectively. The resulting constructs will be introduced into potato via Agrobacterium-mediated transformation (objective 3).

Impacts
What was accomplished under these goals? Objective 1. Determine the role of RHA1B as a potential metaeffector. We have cloned all five RHA1B neighboring genes designated as RIE1-5 into a binary vector and transiently co-expressed in N. benthamiana leaves with a wild-type RHA1B or an E3 ligase deficient form of RHA1B (RHA1BC135S). Significantly, Stability of two of them, RIE1 and RIE4, was negatively impacted by the co-expression with RHA1B in an E3-dependent manner as indicated by lack of accumulation of both proteins in presence of the wild type RHA1B but not its mutant version. Furthermore, a weak interaction was detected between RIE4 and RHA1BC135S, but not wild type RHA1B using Y2H method. In addition, all cloned RIEs were tested for their ability to suppress a hypersensitive response cell death triggered by either co-expression of potato Gpa2 R protein and its corresponding avirulent G. pallida effector RBP1 or auto-active resistance proteins conferring resistance to other pathogens (Rx1 - resistance to the virus, Prf resistance to the bacterium, Rpi-blb1 resistance to the oomycete). Out of five RIEs, only RIE4 was able to suppress HR triggered by all the tested resistance proteins. Objective 2. Determine the host targets of the RHA1B ubiquitin ligase. We have conducted both Y2H and co-IP assay to determine whether SGT1, HSP90, or RAR1 interact with RHA1B and no detectable interactions was observed, suggesting none of these three common chaperons is the target of RHA1B. We then characterized the 12 putative RIPs identified by Y2H and AP-MS. Full-length cDNAs of these RIPs wer cloned from potato and constructed into the relevant expression vectors for co-IP, in vitro ubiquitination, and in vivo degradation assays. We found RHA1B interacts with two subunits (CcR4-NOT10 and CAF1) of a highly conserved RNA processing/regulating complex CcR4-NOT and the pattern recognition receptor NILR1 that has been shown to play an important role in nematode resistance. When transiently expressed in Nicotiana benthamiana leaves, RHA1B triggered degradation of CcR4-NOT10, CAF1 and NILR1 in a ubiquitin ligase activity-dependent manner. Moreover, the in vitro ubiquitination assay indicates that RHA1B can specifically ubiquitinate CcR4-NOT10 protein. Thus, our results strongly suggest RHA1B targets at least three host proteins, CcR4-NOT10, CAF1 and NILR1 for ubiquitination promoting their degradation. Objective 3. Generate G. pallida-resistant potato via loss of susceptibility. We plan to begin conducting research of Objective 3 in year 2023.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Kud J, Pillai SS, Raber G, Caplan A, Kuhl JC, Xiao F, Dandurand LM. (2022) Belowground Chemical Interactions: An Insight Into Host-Specific Behavior of Globodera spp. Hatched in Root Exudates From Potato and Its Wild Relative, Solanum sisymbriifolium. Front Plant Sci. 2022 Jan 12;12:802622. doi: 10.3389/fpls.