Conferring resistance to soybean cyst nematode using decoy engineering

Abstract

The present disclosure provides compositions for conferring resistance to soybean cyst nematode in soybean plants by expression of soybean PBS1 derivatives that can be cleaved by soybean cyst nematode proteases. The present disclosure also provides a method of conferring resistance to soybean cyst nematode in soybean plants, as well as soybean plants, plant parts and cells with resistance to soybean cyst nematode syncytium formation.

Description

IU-2025-033-02-WCCONFERRING RESISTANCE TO SOYBEAN CYST NEMATODE USING DECOY ENGINEERINGSTATEMENT OF GOVERNMENTAL RIGHTS

[0001] This invention was made with government support under20216701334260, and 20226701136552 awarded by the National Institute of Food and Agriculture, and 201731 awarded by the National Science Foundation. The government has certain rights in the invention.CROSS-REFERENCE

[0002] This application claims the benefit of U.S. Provisional Application No.63 / 763,574, filed on February 26, 2025, which is hereby incorporated by reference.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] The sequence listing contained in the file named IU-2025-033-02-WO SL.xml, created on February 25, 2026, and having a size of 10,743 bytes is incorporated herein by reference.BACKGROUND

[0004] The Soybean Cyst Nematode (SCN; Heterodera glycines) is an obligate endoparasite that is responsible for yield losses exceeding $1 billion annually in North America (Arjoune et al. Plant Methods 18:110-148, 2022). Current methods to mitigate economic losses include crop rotations with non-host plants and the use of resistant soybean varieties. However, the overuse of resistant soybean varieties, which are primarily derived from a single source of resistance (PI 88788), have caused shifts in SCN populations (Niblack et al., Plant Health Prog.9:29, 2008; Allen et al., Plant Health Prog., 18:19-27, 2017), diminishing the efficacy of these varieties (Mitchum et al., Plant Dis. 91:1473-1476, 2007; McCarville et al., Plant Health Prog. 18:146-155, 2017). Development of durable SCN preventionIU-2025-033-02-WCstrategies remains a challenge, requiring a deeper understanding of SCN virulence mechanisms.SUMMARY OF THE INVENTION

[0005] The Soybean Cyst Nematode (SCN) presents a unique challenge to disease resistance strategies because its life cycle depends entirely on the formation of a permanent feeding site known as a syncytium. This process involves extensive host cell remodeling, including cell wall digestion and protoplast fusion, to create a nutrient sink essential for nematode development. The syncytium serves as a nutrient source providing sugars, starches, fatty acids, amino acids, and B vitamins necessary for nematode development and reproduction. Without a viable syncytium, cyst nematodes are unable to complete their life cycle.

[0006] While protease-activated decoy systems (such as those utilizing PBS1 and RPS5) have been successfully deployed to detect and counter pathogens in other crop species, their application to SCN has been prevented by a lack of identified targets. Specifically, no effector proteases secreted by SCN during the critical window of syncytium formation have been characterized for use in such decoy systems. Consequently, there remains a need for compositions and methods that can specifically recognize syncytium-associated proteolytic activity to trigger a resistance response and interfere with SCN survival.

[0007] Accordingly, provided herein are compositions for conferring resistance to soybean cyst nematodes in soybean plants. The compositions generally comprise a soybean PBS1 modified to include a recognition sequence cleaved by the soybean cyst nematode effector protease CPR1.

[0008] In some embodiments, provided herein are soybean PBS1 proteins comprising a soybean cyst nematode effector protease CPR1 cleavage site sequence, the soybean cyst nematode effector protease CPR1 cleavage site sequence being located within the activation loop of the PBS1 proteinIU-2025-033-02-WQ

[0009] In certain embodiments, the soybean cyst nematode effector protease CPR1 cleavage site sequence is capable of being cleaved by a soybean cyst nematode effector protease CPR1 protease comprising the amino acid sequence of SEQ ID NO: 1 .

[0010] In certain embodiments, the soybean cyst nematode effector protease CPR1 cleavage site sequence comprises the amino acid sequence Leu-Gly-Thr. In certain embodiments, the soybean cyst nematode effector protease CPR1 cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the activation loop comprises amino acid positions 227-250 of SEQ ID NO: 6. In certain embodiments, the soybean cyst nematode effector protease CPR1 cleavage site sequence is located between amino acid positions 232-247 of SEQ ID NO: 6.

[0011] In certain embodiments, the soybean PBS1 protein comprises a structure represented by the formula:N-A-Cwherein:N is a N-terminal region of the soybean PBS1 protein, the N-terminal region comprising at least 90 % sequence identity to the amino acid sequence of SEQ ID NO: 9;A is the activation loop, the activation loop comprising the soybean cyst nematode effector protease CPR1 cleavage site sequence; andC is a C-terminal region of the soybean PBS1 protein, the C-terminal region comprising at least 90 % sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the activation loop comprises the soybean cyst nematode effector protease CPR1 cleavage site sequence and at least 90 % sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0012] In some embodiments, provided herein are methods for conferring resistance to soybean cyst nematodes in soybean plants. In certain embodiments, the methods comprises the steps of transforming soybean plant cells with a nucleic acid sequence comprising a soybean PBS1 gene modified to include a novel recognition sequence cleaved by the soybean cyst nematode effector protease CPR1.IU-2025-033-02-WG

[0013] In certain embodiments, the recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a soybean PBS1 protein, wherein the endogenous AvrPphB cleavage site of the PBS1 protein is replaced with a heterologous recognition sequence for cleavage by the soybean cyst nematode effector protease CPR1.

[0014] In certain embodiments, the recombinant nucleic acid molecule wherein the heterologous cleavage site for CPR1 is LLGTGP.

[0015] In certain embodiments, the recombinant nucleic acid molecule encodes GmPBSI , wherein the heterologous protease cleavage site is located from about amino acid position 230 to about amino acid position 245 of SEQ ID NO: 6.

[0016] In certain embodiments, the modified soybean PBS1 protein has a heterologous sequence cleavable by the CPR1 protease of soybean cyst nematode, wherein the modified substrate protein is encoded by the recombinant nucleic acid comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a soybean PBS1 protein, wherein the endogenous AvrPphB cleavage site of the PBS1 protein is replaced with a heterologous recognition sequence for cleavage by the soybean cyst nematode effector protease CPR1.

[0017] In certain embodiments, the modified soybean PBS1 protein consists of SEQ ID NO:7.

[0018] In certain embodiments, the provided is a vector comprising the recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a soybean PBS1 protein, wherein the endogenous AvrPphB cleavage site of the PBS1 protein is replaced with a heterologous recognition sequence for cleavage by the soybean cyst nematode effector protease CPR1.

[0019] In certain embodiments, included herein is a transformed soybean plant cell comprising the recombinant nucleic acid comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a soybean PBS1 protein, wherein the endogenous AvrPphB cleavage site of the PBS1 protein is replaced with a heterologous recognition sequence for cleavage by the soybean cyst nematode effector protease CPR1.

[0020] In certain embodiments, the transformed soybean plant comprises the recombinant nucleic acid comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a soybean PBS1 protein, wherein the endogenous AvrPphB cleavage site of the PBS1 protein is replaced with a heterologous recognition sequence for cleavage by the soybean cyst nematode effector protease CPR1.

[0021] In certain embodiments, the provided herein is a transgenic seed of the transformed soybean plant.

[0022] In some embodiments, provided herein are methods of protecting soybean plants from infection by soybean cyst nematodes, the method comprising the steps of transforming the cells of a soybean plant with a recombinant nucleic acid comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a soybean PBS1 gene, wherein the endogenous AvrPphB cleavage site encoded by the PBS1 gene is replaced with a heterologous cleavage sequence for the soybean cyst nematode effector protease CPR1 , growing the cells into mature soybean plants expressing the PBS1 protein having the CPR1 cleavage sequence.

[0023] In some embodiments, provided herein are plants, plant parts, seeds, and cells from the soybean plant arising from the transformed soybean cells.

[0024] In some embodiments, provided herein are soybean plants expressing a PBS1 protein having a heterologous amino acid sequence that is cleaved by the CPR1 protease of soybean cyst nematode.

[0025] In some embodiments, provided herein are methods of protecting a soybean plant from infection by soybean cyst nematode, the method comprisingthe steps of editing a nucleotide sequence that encodes a soybean PBS1 protein within a soybean plant cell, wherein the endogenous AvrPphB cleavage site of the PBS1 protein is edited to encode a heterologous amino acid sequence that can be cleaved by the soybean cyst nematode effector protease CPR1 and growing the cell into a mature soybean plant expressing the modified PBS1 protein havingthe heterologous amino acid sequence that can be cleaved by CPR1.IU-2025-033-02-WG

[0026] In some embodiments, provided herein are plant, plant parts, seeds, and cells from the soybean plant arising from the transformed soybean cells of claim 13.

[0027] In some embodiments, provided herein are soybean plants expressing the soybean PBS1 protein having a heterologous amino acid sequence that is cleaved by the CPR1 protease of soybean cyst nematode.BRIEF DESCRIPTION OF THE FIGURES

[0028] The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the description, serve to explain the principles of the disclosure.

[0029] FIGs. 1 A and 1 B illustrate the structural domains and a catalytic residue of the CPR1 protein, and show that CPR1 expression is substantially higher in PA3 than MM10 gland populations at the parasitic J2 life stage. FIG. 1A: Schematic of the CPR1 protein. SP=signal peptide, 129= I29 inhibitor domain, C1 Peptidase= protease domain. Numbers represent amino acid position. Amino acid 323 (bold) is the catalytic cysteine residue. FIG.1 B: Bar graph depicting normalized read counts of CPR1 in glands of PA3 and MM10 populations at the parasitic J2 (pJ2) life stage. The data represent mean values ±SEM of three biological replicates for each population. Statistical analysis employed a two-tailed unpaired t-test, P<0.05 (*).

[0030] FIGs. 2A, 2B, 2C, and 2D show data demonstrating that wild-type CPR1 , but not its protease-inactive variant (C323S), suppresses RPS5D266E-induced cell death in N. benthamiana leaves, as confirmed by protein expression and reduced electrolyte leakage. FIG. 2A: Images showing cell death induced by auto active RPS5 (RPS5D266E:5xMYC) is suppressed when co-expressed with CPR1 :mCherry. The indicated constructs were transiently expressed in N. benthamiana using dexamethasone-inducible promoters. Leaf images are representative of 10 leaves. 10 / 10 leaves co-expressing RPS5D266E and empty vector (ev), and 0 / 10 leaves co-expressing RPS5D266E and CPR1 displayed tissue collapse. Images were taken under white light and ultraviolet (UV) light. Images taken 24h post geneIU-2025-033-02-WGexpression induction. FIG. 2B: Image showing RPS5D266E mediated cell death is not suppressed by the protease inactive variant (CPR1C323S). 8 / 8 leaves co-expressing RPS5D266E and CPR1 C323S and 2 / 8 leaves co-expressing RPS5D266E and CPR1 displayed tissue collapse. Images taken 24h post gene expression induction. FIG. 2C: Immunoblot showing expression of the RPS5D266E and CPR1 C323S or CPR1. RUBISCO was used as a loading control. FIG. 2D: Graphical depiction of data from electrolyte leakage assay of N. benthamiana leaves co-infiltrated with RPS5D266E and ev, CPR1, CPR1C323S, or CPR1(mat). MgCl2 treated leaves were used as a buffer control. Readings were taken from 4 individual leaf samples, containing 3 leaf discs each from different areas of the leaf avoiding any mechanical damage near injection sites. Error bars represent ±SEM. Electrolyte leakage assay was repeated twice with consistent results.

[0031] FIGs 3A and 3B demonstrate that CPR1(mat) is expressed in N. benthamiana leaf cells and localizes to mitochondria, where it co-localizes with GmPBS1-1. FIG. 3A: Confocal images showing the expression and subcellular co-localization of GmPBSI-1 :mCherry and CPR1 (mat):sYFP in N. benthamiana leaves. Images were collected 5 to 8 h post induction of gene expression by dexamethasone application and 1 h post injection of MitoTracker Red. Scale bars = 20 pm. FIG. 3B: Confocal images showing the expression of CPR1(mat):sYFP and mitochondria stained with MitoTracker Red. Images were collected 5 to 8 h post induction of gene expression by dexamethasone application and 1 h post injection of MitoTracker Red. Scale bars = 20 pm.

[0032] FIGs 4A, 4B, and 4C demonstrate that CPR1 interacts with and promotes the degradation of a truncated soybean enzyme (GmBCATI A1-74), indicatinga role for CPR1 in regulating the stability of GmBCATI through proteolytic cleavage in plant cells. FIG. 4A: Immunoblots showing GmBCATI A1-74:4xMYC co-immunoprecipitates (co-IPs) with the protease-inactive CPR1(mat)C323S:GFP. The indicated proteins were transiently coexpressed in N. benthamiana and leaves harvested 21 h post dexamethasone application. GFP-tagged bait proteases were immunoprecipitated using GFP-Trap agarose beads. AvrPphBC98S was used as a negative control. Co-IPs were repeated three times with consistent results. FIG. 4B: Immunoblots showing GmBCAT1A1-74 accumulation isreduced when co-expressed with active CPR1 variants. GmBCATI A1 -74 was co-expressed with empty vector (ev), active CPR1 , protease inactive CPR1 C323S, or CPR1 (mat). Relative expression values of GmBCATI were normalized to the ev control and quantified using ImageJ. RUBISCO was used as a loading control. Cleavage tests were performed at least twice with consistent results. All constructs used were under control of dexamethasone inducible promoters. FIG. 4C: Immunoblots showing GmBCATI (full-length minus signal peptide) accumulation is reduced when co-expressed with CPR1(mat) but not ev or CPR1 C323S. Relative expression values of GmBCATI were normalized to the ev control and quantified using ImageJ. RUBISCO was used as a loading control. Cleavage tests were performed at least twice with consistent results. All constructs used were under control of dexamethasone inducible promoters.

[0033] FIG. 5A shows Confocal images showing the subcellular colocalization of GmBCATI :sYFP and mitochondria stained with Mitotracker Red approximately 5-6 hours post dexamethasone application and 1 hour post addition of Mitotracker Red. Scale bars = 20 pm.

[0034] FIGs 5Aand 5B show that GmBCATI localizes to mitochondria in plant cells and depict the evolutionary relationships among BCAT proteins from soybean and Arabidopsis based on phylogenetic analysis. FIG. 5B: Illustration of Maximum Likelihood phylogenetic analysis of soybean and Arabidopsis BCAT proteins. Numbers at branch points represent percent total of 1000 bootstraps that support the indicated tree topology.

[0035] FIGs 6A, 6B, and 6C demonstrate that silencing CPR1 expression in H. glycines J2 nematodes using dsRNA significantly reduces CPR1 transcript levels and results in decreased nematode penetration of soybean roots compared to controls. FIG. 6A: Graph depicting qRT-PCR analysis of CPR1 transcript abundance in H. glycines J2s after soaking in CPR1-dsRNA (2 mg / ml and 3 mg / ml concentration) and dsRNA-GFP (negative control) at 24 h. Three biological and three technical replicates were included. This experiment was repeated three times with similar results. The H. glycines GAPDH gene (CA939315.1) was used for data normalization across samples. The data represent mean values ±SEM. Statistical analyses employed One-way ANOVA tests, with significance set as P<0.05. AIU-2025-033-02-WGDunnett’s multiple comparison test showed P was significant at <0.0001 (****). FIG. 6B: Images showing nematode penetration assay on soybean roots. J2 worms that had been soaked in dsRNA were inoculated onto root tips of 5-day old soybean seedlings placed in a pluronic gel (23% Pluronic F-127) and then incubated for 24 h. Roots were then stained with acid fuchsin to reveal the nematodes. FIG 6C: Graphical depiction of CPR1 -silenced nematodes display reduced penetration of soybean roots in comparison to controls (J2 worms treated with dsRNA targeting GFP). Statistical analysis employed an unpaired t-test. P<0.01 (**).

[0036] FIGs. 7A and 7B show that CPR1 -expressing soybean roots have significantly fewer nematode cysts per gram compared to controls. FIG. 7A: Representative images of cysts harvested and counted from CPR1 -expressing roots using Nemacounter, which was designed in house at Iowa State University. Images show a mixed population of root debris and cysts (left) and the Nemacounter program detecting and counting the cysts specifically (right). FIG. 7B: Graph depicting cysts per gram of soybean root weight were measured from roots expressing CPR1 :mCherry+RUBY, CPR1C323S:mCherry+RUBY, and mCherry+RUBY. Roots were collected from 10, 10, and 8 individual composite soybean plants, respectively. Statistical analysis employed a Welch’s unpaired, t-test comparing roots expressing CPR1 or CPR1 C323S to control roots expressing mCherry. P<0.01 (**).

[0037] FIGs. 8A and 8B show that CPR1 does not cleave or reduce accumulation of wild-type GmPBS1-1, but specifically cleaves and reduces accumulation of the GmPBSI LLGTGP variant in N. benthamiana leaves. FIG.8A: Immunoblot of N. benthamiana leaf samples co-expressing wild-type GmPBS1-1:mCherry with empty vector (ev), AvrPphB:GFP, AvrPphBC98S:GFP (protease inactive), CPR1(mat)C323S:GFP (protease inactive), or CPR1 (mat):YFP. Numbers at bottom of lanes indicate relative amounts of GmPBSI protein in each lane. FIG 8B: Immunoblot of N. benthamiana leaf samples coexpressing GmPBS1LLGTGPwith empty vector (ev), AvrPphB:GFP, CPR1(mat), or CPR1(mat)C323S, shows reduction in GmPBS1LLGTGPaccumulation in CPR1(mat) but not controls, which indicates it is being cleaved by CPR1 . Numbers at bottom of lanes indicate relative amounts of GmPBSI protein in each lane.IU-2025-033-02-WG

[0038] FIGs. 9A, 9B, and 9C show that the GmPBS1LLGTGPdecoy protein is not toxic to soybean roots. FIG: 9A: Diagram of GmPBSI decoy construct used to generate transgenic soybean roots. RUBY indicates a marker that causes transformed cells to produce the red pigment betalin (from beets), which enables identification of transgenic roots based on their red color. FIG 9B: Transient expression of the construct shown in panel A in a leaf from Nicotiana benthamiana. Note the red coloration in the leaf and absence of any necrosis (top panel). Bottom panelshows an immunoblotto detect expression of the GmPBSILLGTGPdecoy protein in N. benthamiana leaves, confirming that this construct expresses the decoy protein. FIG. 9C: Soybean roots from composite soybean plants expressing the GmPBSILLGTGPdecoy protein in roots. The red roots indicate roots that are successfully transformed.

[0039] FIG. 10 shows data demonstrating that soybean roots expressing the GmPBSILLGTGPdecoy protein display enhanced resistance to infection by soybean cyst nematodes. Composite soybean plants were generated with roots expressing either wildtype GmPBSI (a negative control) or the GmPBSILLGTGPdecoy protein. Roots were then inoculated with soybean cyst nematode J2-stage worms (2,000 worms per plant), which were allowed to infect for 33 days, and then cysts per gram root rate were determined. In two independent experiments, we observed an approximately 50% reduction in cyst formation per gram root tissue.DETAILED DESCRIPTION

[0040] To reprogram host cells and evade the plant immune system (Jones and Dangl 2006; Sato et al. 2019), cyst nematodes secrete a diverse repertoire of effector proteins. Effectors are broadly defined as proteins secreted by a pathogen during infection to alter the host cell’s structure and / or function (Hogenhout et al. 2009). In cyst nematodes, effectors are synthesized primarily in the esophageal gland cells (two sub-ventral and one dorsal) of the nematode and then secreted through a hollow mouth spear called the stylet. The sub-ventral gland cells are more prominent during early stages of infection and involved in the secretion of cell-wall degrading enzymes during root penetration (Pellegrin et al. 2024),IU-2025-033-02-WGwhereas the dorsal gland cells play a pivotal role during sedentary stages and are thought to facilitate suppression of plant immune responses and initiating and maintaining the syncytium (Davis et aL 2004).

[0041] Nematologists have employed various approaches to identify effectors, which are reviewed in (Davis et aL 2004; Vieira and Gleason 2019). The availability of an annotated SCN genome (Masonbrink et al. 2019b) and the ability to isolate and purify nematode gland cells for transcriptomic analysis (Maier et al. 2013) have both facilitated identification of novel SCN effectors. Using these resources, we specifically aimed to identify SCN proteases that are secreted during infection and function inside plant cells, as our overarching goal is to engineer a plant-produced decoy substrate that, when cleaved by an SCN protease effector, elicits a plant immune response.

[0042] SCN relies on living cells to form a syncytium. We expected the PBS1 decoy system, which induces cell death upon activation, would be effective in conferring resistance to SCN. As a first step toward development of a PBS1 decoy effective against SCN, we needed to identify SCN effector proteases that contribute to virulence. Such proteases have been described in bacterial (Figaj et al. 2019), fungal (Chandrasekaran et al.2016), viral (Rodamilans et al. 2018), and nematode (Antonino de Souza Junior et al. 2014) plant pathogens. However, little is known about effector proteases in cyst nematodes specifically.

[0043] In addition to identifying candidate SCN effector proteases, we also aimed to identify the targets of these proteases inside soybean cells. Identification of such targets will provide insight into the plant processes that SCN modifies to promote virulence and will also aid in the identification of preferred cleavage sequences for each protease, which will be required for future decoy engineering.

[0044] Described herein is the identification of an SCN effector protease - Cysteine Protease 1 (CPR1; Hg_chrom8_TN10mRNA_16195). Gland transcriptome analysis showed that CPR1 is expressed in parasitic-stage gland cells. Transient expression in Nicotiana benthamiana showed that CPR1 localizes, in part, to mitochondria and can suppress cell death triggered by RPS5. Proximity-based labeling experiments in transgenic soybean rootsidentified a soybean branched-chain amino acid aminotransferase (GmBCATI; Glyma.06G050100) as a putative target, which was further confirmed through coimmunoprecipitation. Most significantly, co-expression of CPR1 variants with GmBCATI showed that GmBCATI accumulation is reduced in a protease-dependent manner, which suggests it is a substrate of CPR1. Silencing of CPR1 in SCN reduced nematode penetration frequency, while expression of CPR1 in transgenic soybean roots enhanced susceptibility. These results show that CPR1 contributes to SCN virulence and represents an ideal candidate for development of a decoy substrate.

[0045] Described herein are recognition sequences cleaved by the soybean cyst nematode effector protease CPR1. Exemplary recognition sequences include, but are not limited to, the CPR1 cleavage sequence within GmBCATI (SEQ ID NO:5) and related cleavage sequences.

[0046] Described herein is a soybean PBS1 gene (SEQ ID NO: 6) modified to include a recognition sequence cleaved by the soybean cyst nematode effector protease CPR1. One of skill in the art would understand that soybean has three different copies of GmPBSI and that the methods described herein are applicable to any GmPBSI . A GmPBSI gene having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence homology to SEQ ID NO: 6 may be utilized herein as long has it contains an AvrPphB cleavage site. In one embodiment, the PBS1 gene is modified to replace, through insertion of an exogenous sequence orgenetic editing, the endogenous AvrPphB cleavage site (GDKSHVS) of the PBS1 protein with a heterologous recognition sequence for cleavage by the soybean cyst nematode effector protease CPR1. Preferably, the heterologous protease cleavage site of the PBS1 protein is located between about amino acid position 230 to about amino acid position 245 in reference to SEQ ID NO:6. In some embodiments, such as SEQ ID NO: 7, the endogenous AvrPphB cleavage cite of the PBS1 protein is replaced with SEQ ID NO: 5.

[0047] Non limiting examples of soybean cyst nematode-cleavable soybean PBS1 proteins include soybean PBS1 proteins comprising a soybean cyst nematode effector protease CPR1 cleavage site sequence, the soybean cyst nematode effector protease CPR1 cleavage site sequence being located within the activation loop of the PBS1 protein. AIU-2025-033-02-WQsoybean cyst nematode effector protease CPR1 cleavage site sequence generally refers to and includes an amino acid sequence that can be cleaved by a soybean cyst nematode effector protease CPR1 protease comprising the amino acid sequence of SEQ ID NO: 1. Assays for identification of cleavage of an amino acid sequence include, but are not limited to, those described throughout the Examples. Exemplary cyst nematode effector protease CPR1 cleavage site sequences, include but are not limited to, the amino acid sequence Leu-Gly-ThrorSEQ ID NO 5.

[0048] The activation loop generally refers to and includes amino acid positions 227-250 of SEQ ID NO: 6. For example, non-limiting positions of the cleavage site sequence positions within the activation loop include instances where the soybean cyst nematode effector protease CPR1 cleavage site sequence is located between amino acid positions 232-247 of SEQ ID NO: 6. In certain instances, the soybean cyst nematode effector protease CPR1 cleavage site sequence replaces the native AvrPphB cleavage site sequence. In certain instances, the soybean cyst nematode effector protease CPR1 cleavage site sequence is inserted into the native activation loop sequence or comprises a substitution of residues within the activation loop (e.g., SEQ ID NO: 10). Additional non-limiting examples of the position of the soybean cyst nematode effector protease CPR1 cleavage site sequence include instances where it is located between amino acid positions 232-247 of SEQ ID NO: 6 and / or as positioned in SEQ ID NO: 10.

[0049] Examples of soybean PBS1 proteins further include, but are not limited to, soybean PBS1 proteins comprising a structure represented by the formula:N-A-C(Formula 1)wherein:N is a N-terminal region of the soybean PBS1 protein, the N-terminal region comprising at least 85 %, at least 90 %, at least 95 %, at least 98 %, at least 99 %, or 100 % sequence identity to the amino acid sequence of SEQ ID NO: 9;A is the activation loop, the activation loop comprising the soybean cyst nematode effector protease CPR1 cleavage site sequence; andIU-2025-033-02-WQC is a C-terminal region of the soybean PBS1 protein, the C-terminal region comprising at least 85 %, at least 90 %, at least 95 %, at least 98 %, at least 99 %, or 100 % sequence identity to the amino acid sequence of SEQ ID NO: 11. Additional non-limiting examples of the activation loop of Formula 1 include instances wherein the activation loop comprises the soybean cyst nematode effector protease CPR1 cleavage site sequence and has at least 85 %, at least 90 %, at least 95 %, at least 98 %, at least 99%, or 100 % sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0050] As described herein also contemplated are nucleic acids and plants (e.g., soybean plants, seeds, and / or plant cells) comprising the soybean cyst nematode cleavable soybean PBS1 proteins described herein.

[0051] Nucleic Acid Constructs

[0052] As used herein, "recombinant," when used in connection with a nucleic acid molecule, means a molecule that has been created or modified through deliberate human intervention such as by genetic engineering. For example, a recombinant nucleic acid molecule is one having a nucleotide sequence that has been modified to include an artificial nucleotide sequence or to include some other nucleotide sequence that is not present within its native (non-recombinant) form.

[0053] Further, a recombinant nucleic acid molecule has a structure that is not identical to that of any naturally occurring nucleic acid molecule or to that of any fragment of a naturally occurring genomic nucleic acid molecule spanning more than one gene. A recombinant nucleic acid molecule also includes, without limitation, (a) a genetically edited sequence combining a portion of the native coding sequence is replaced with an artificial sequence or the sequence from a non-native source, (b) a nucleic acid molecule having a sequence of a naturally occurring genomic orextrachromosomal nucleic acid molecule, but which is not flanked by the coding sequences that flank the sequence in its natural position; (c) a nucleic acid molecule incorporated into a construct, expression cassette or vector, or into a host cell's genome such that the resulting polynucleotide is not identical to any naturally occurring vector or genomic DNA; (d) a separate nucleic acid molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR) or arestriction fragment; and (e) a recombinant nucleic acid molecule having a nucleotide sequence that is part of a hybrid gene (i.e., a gene encoding a fusion protein). As such, a recombinant nucleic acid molecule can be modified (chemically or enzymatically) or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded. A nucleic acid molecule (or its complement) that can hybridize to any of the uninterrupted nucleotide sequences described herein, under either highly stringent or moderately stringent hybridization conditions, also is within the scope of the present disclosure.

[0054] Compositions of the present disclosure also include nucleic acid constructs, such as expression cassettes or vectors, having plant promoters operably linked with a nucleic acid molecule that encodes a substrate protein of a pathogen-specific protease with a heterologous pathogen-specific protease recognition sequence for use in transforming plant cells, plant parts and plants. In addition, the constructs can include a nucleic acid molecule that encodes a modified PBS1 gene that is not native / not endogenous to the plant cell, plant part or plant to be transformed. As used herein, "nucleic acid construct" means an oligonucleotide or polynucleotide composed of deoxyribonucleotides, ribonucleotides or combinations thereof having incorporated therein the nucleotide sequences described herein. The nucleotide construct can be used for transforming organisms such as plants. In this manner, plant promoters operably linked to a nucleotide sequence for a modified substrate protein of a pathogen-specific protease as described herein are provided in nucleic acid constructs for expression in a plant cell, plant part or plant.

[0055] As used herein, "expression cassette" means a nucleic acid molecule having at least a control sequence operably linked to a coding sequence.

[0056] As used herein, "operably linked" means that the elements of the expression cassette are configured so as to perform their usualfunction. Thus, control sequences (i.e., promoters) operably linked to a coding sequence are capable of effecting expression of the coding sequence. The control sequences need not be contiguous with the coding sequence so long as they function to direct the expression thereof. Thus, for example, interveninguntranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promoter sequence still can be considered "operably linked" to the coding sequence.

[0057] As used herein, a "coding sequence" or "coding sequences" means a sequence that encodes a particular polypeptide and is a nucleotide sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at a 5' (amino) terminus and a translation stop codon at a 3' (carboxy) terminus. A transcription termination sequence will usually be located 3’ to the coding sequence.

[0058] As used herein, "control sequence" or "control sequences" means promoters, polyadenylation signals, transcription and translation termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for replication, transcription and translation of a coding sequence in a recipient host cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

[0059] As used herein, a "promoter" means a nucleotide region comprising a nucleic acid (i.e., DNA) regulatory sequence, wherein the regulatory sequence is derived from a gene or synthetically created that is capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. A number of promoters can be used in the expression cassette, including the native soybean promoter.

[0060] Alternatively, promoters can be selected based upon a desired outcome. Such promoters include, but are not limited to, "constitutive promoters" (where expression of a polynucleotide sequence operably linked to the promoter is unregulated and therefore continuous), tissue-specific promoters (where expression occurs only in specific tissues, such as roots), "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.),and "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.).

[0061] As used herein, "plant promoter" means a promoter that drives expression in a plant such as a constitutive, inducible (e.g., chemical-, environmental-, pathogen- or wound-inducible), repressible, tissue-preferred or other promoter for use in plants. Soybean root specific promoters useful in the constructs, vectors, and methods of the invention are known to those of skill in the art. (sysbio.unl.edu / RGPDB / soybean.php).

[0062] The control sequence(s) and / or the coding sequence therefore can be native / analogous to the host cell or to each other. Alternatively, the control sequence(s) and / or coding sequence can be heterologous to the host cell or to each other. As used herein, "heterologous" means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same / analogous species, one or both are substantially modified from their original form and / or genomic locus, orthe promoter is not the native promoter for the operably linked polynucleotide.

[0063] The expression cassette can include other control sequences 5' to the coding sequence. For example, the expression cassette can include a 5' leader sequence, which can act to enhance translation. Other expression enhancing sequences can be used, for example, in trans and the like.

[0064] The expression cassette also can include a transcriptional and / or translational termination region that is functional in plants. The termination region can be native with the transcriptional initiation region (i.e., promoter), can be native with the operably linked coding sequence, can be native with the plant of interest, or can be derived from another source (i.e., foreign or heterologous to the promoter, the coding sequence, the plant host cell, or any combination thereof). Termination regions are typically located downstream (3'direction) from the coding sequence.

[0065] The expression cassette also can include one or more linkers. As used herein, "linker" means a nucleotide sequence that functions to link one element of the expression cassette with another without otherwise contributing to the transcription or translation of a nucleotide sequence of interest when present in the expression cassette. The linker can include plasmid sequences, restriction sequences and / or sequences of a 5'-untranslated region (5'-UTR). Alternatively, the linker further can include nucleotide sequences encoding the additional amino acid residues that naturally flank the heterologous protease recognition sequence in the substrate protein from which it was isolated. The length and sequence of the linker can vary and can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250 nucleotides or greater in length.

[0066] Gene Editing

[0067] In addition to traditional methods of plant transformation described above, it is understood by those of skill in the art that targeted genetic modification of the nucleotide sequence that encodes a PBS1 protein in the soybean plant may be performed to create a recombinant nucleic acid without inserting exogenous genetic material into the soybean plant genome. In such embodiments of the invention, a soybean plant cell that includes the endogenous PBS1 protein is modified by using a gene editing system, such as sequencespecific nucleases including ZFNs, homing endonucleases, TAL-effector nucleases, CRISPR associated Cas9 and Cas12 systems, and a guide RNA to modify the endogenous AvrPphB cleavage site of the PBS1 with a heterologous recognition sequence for cleavage by the soybean cyst nematode effector protease CPR1. As an example approach for how the endogenous AvrPphB cleavage sequence in a GmPBSI gene can be replaced with a CPR1 cleavage sequence, those skilled in the art can use the CR / SPR-mediated insertion of exon (CRISPIE) strategy as described by Zhong etal., eLife 2021 Jun 8;10:364911.

[0068] Methods of Transforming Soybean Plants

[0069] While methods of plant transformation are described in the examples herein, it is understood by those of skill in the art that numerous methods for plant transformation have been developed and are available, including biological and physical plantIU-2025-033-02-WGtransformation protocols. See, e.g., Miki etal., “Procedures for Introducing Foreign DNAinto Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, Eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88. In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, e.g., Gruber eta / ., “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, Eds. (CRC Press, Inc., Boca Raton, 1993), pp.89-119. Plant transformation methods include the natural transformation system of Agrobacterium, or direct gene transfer through microprojectile-mediated transformation or sonication.

[0070] BreedingwithTransformed Soybean Plants

[0071] In some embodiments, the soybean cyst nematode resistant plants described herein provide a source of breeding material that may be used to develop new soybean varieties. Plant breeding techniques known in the art and used in a soybean plant breeding program include, but are not limited to, recurrent selection, mass selection, bulk selection, backcrossing, pedigree breeding, restriction fragment length polymorphism (RFLP) enhanced selection, genetic marker enhanced selection, making double haploids and transformation. Often combinations of these techniques are used. There are many analytical methods available to evaluate the result of a cross. The oldest and most traditional method of analysis is the observation of phenotypic traits, but genotypic analysis may also be used.

[0072] The present disclosure provides methods for producing a soybean seed by crossing a first parent soybean plant with a second parent soybean plant, where the first or the second parent soybean plant is the soybean cyst nematode resistant plant described herein, with the other parent soybean plant being of a different soybean line. Also provided are methods for producing a soybean seed where both the first and the second parent soybean plants are soybean plants of the soybean cyst nematode resistant plants described herein. The present disclosure thus provides a multitude of breeding methods involving the soybean cyst nematode resistant plants described herein, including, for example, selfing, backcrosses, hybrid production, crosses to populations, and the like.

[0073] The present disclosure also provides methods for producing a soybean plant by crossing the soybean cyst nematode resistant plants described herein with a second, different soybean plant and growingthe resultant progeny seed, and repeatingthe crossing and growing steps with progeny plants from 0 to 7 times. All plants produced using the soybean cyst nematode resistant plants described herein are within the scope of the present disclosure, includingthose developed from varieties derived from the soybean cyst nematode resistant plants described herein.

[0074] The soybean cyst nematode resistant plants described herein can be used in the development of further soybean plants. In one embodiment, a method for developing a derived progeny soybean plant in a soybean breeding program includes utilizing soybean cyst nematode resistant plants described herein or a plant part thereof as a source of breeding material and selecting a progeny plant increased resistance to soybean cyst nematodes, and / or selecting a progeny plant with molecular markers in common with the soybean cyst nematode resistant plants described herein.

[0075] The soybean cyst nematode resistant plants described herein provide a new locus, loci and / or trait that may be introgressed into an inbred soybean line. Direct transformation and backcrossing are two important methods that can be used to accomplish such an introgression.

[0076] Reproduction of the soybean cyst nematode resistant plants described herein can occur by tissue culture and regeneration. Tissue culture of various tissues of soybean and regeneration of plants therefrom is well known in the art, with methods having been widely published. The present disclosure thus provides regenerative cells which, upon growth and differentiation, produce soybean plants having resistance to soybean cyst nematodes.

[0077] The term “tissue culture” refers to a composition including isolated cells of the same type or cells of different types, or a collection of such cells organized into parts of a plant. Examples of tissue cultures include protoplasts, calli, plant clumps, and plant cells that can generate tissue culturesthatform intact plants or parts of plants, such as embryos,pollen, flowers, seeds, glumes, panicles, leaves, stems, roots, root tips, anthers, and the like. Means for preparing and maintaining plant tissue cultures are well known in the art.

[0078] As used herein phrases such as “growing the seed” or “grown from the seed” include embryo rescue, isolation of cells from seed for another use in tissue culture, as well as traditional growing methods.EXAMPLESExample 1: Identification of SCN Effector Protease CPR1

[0079] To identify candidate SCN effector proteases, we searched SCN esophageal gland cell RNA-seq data (Maier et al., Mol. Plant-Microbe Interact. 34:1084-1087, 2021) for transcripts annotated as proteases, using the TN10 genome as a guide (Masonbrink et al., BMC Genomics 20:1-14, 2019). We confirmed the presence of the protease domains using MEROPS peptidase and NCBI domain prediction databases. We selected those predicted to harbor signal peptides (SignalP5.0) and lack transmembrane domains (TMHMM 2.0). To further prioritize our list of proteases, we compared esophageal gland cell expression to whole worm samples (Maier et aL, Plant-Microbe Interact. 34:1084-1087, 2021). Since SCN effectors are synthesized in gland cells (Gao et al., Mol. Plant-Microbe Interact. 14:1247-1254,2001; Noon etaL, Phytopathology, 105:1362-1372, 2015), we selected proteases that were upregulated in glands compared to the whole worm. In this pursuit, we were especially interested in identifying and characterizing cysteine proteases, due to their previous success in PBS1 decoy engineering.

[0080] Of the proteases identified in the SCN gland cell RNA-seq data, CPR1 was the top priority candidate as it was annotated as a cysteine protease and is highly conserved in the SCN genomes available (early release data available on SCNBase) (Masonbrink et aL, Database 2019:baz111). CPR1 (SEQ ID NO: 1) is 565 amino acids long and predicted to belong to the C1 Peptidase superfamily (FIG. 1A). The protease harbors a cathepsin propeptide inhibitor domain (I29), which is known to form an alpha helical structure that blocks its own substrate-binding site in cis to prevent it from binding to substrates. The catalytic cysteine residue was identified as cysteine 323 based on alignment to similar proteases that were identified in the MEROPS peptidase database (Rawlings et al., NucleicIU-2025-033-02-WGAcids Res., 46:D624-D632, 2017). SignalP5.0 identified a signal peptide spanning from amino acids 1 -16. TMTHH predicted that the protease lacked transmembrane domains (FIG.1A).

[0081] Analysis of gland cell RNA-seq data from two different SON populations, PA3 and MM10, prepared from parasitic J2 (pJ2) nematodes, as detailed in (Maier et al. 2021), revealed that CPR1 transcripts are more abundant in glands of MM10 pJ2 worms (FIG. 1 B), which was determined using normalized read counts from three biological replicates. This is noteworthy as SCN population MM10 is defined as an HG type 1-7, meaning this population is capable of reproducing on all seven resistant soybean indicator lines (Niblack et al., J. Nematol. 34:279-288, 2003). SCN population PA3 is defined as an HG type 0, meaningthis population is incapable of reproducin on all seven resistant soybean indicator lines. The elevated expression of CPR1 in the MM10 population suggests that CPR1 may aid in overcoming resistance.Example 2: CPR1 Suppresses RPS5-Mediated Cell Death

[0082] A common role of effectors is to suppress plant immune responses, including both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). To determine whether CPR1 can suppress ETI-dependent cell death known as the hypersensitive response (HR), we used an auto-active RPS5 mutant (RPS5D266E) that elicits an HR when overexpressed in N. benthamiana without an effector present (Qi et al., Plant Physiol.158:1819-1832, 2012). To perform these assays, we first co-expressed RPS5D266E with either CPR1 (lacking its signal peptide) or an empty vector (ev) control. At 24 h post gene expression induction, the area of the leaf infiltrated with RPS5D266E and CPR1 lacked cell death while the area infiltrated with RPS5D266E and the ev control exhibited complete tissue collapse. We confirmed that the area infiltrated with the protease contained living cells using ultraviolet light as the chlorophyll auto-fluoresces red in living cells (FIG.2A). This result showed that in the presence of CPR1, ETI-dependent HR was suppressed and that CPR1 can function inside of N. benthamiana cells. While screening CPR1 for ETI suppression activity, a second SCN protease was screened in parallel (20453) that wasIU-2025-033-02-WGunable to suppress RPS5-mediated HR. 20453 served as a second negative control for this assay. To test whether CPRI’s HR suppression activity required protease function, we mutagenized the catalytic cysteine residue of CPR1 to a serine at amino acid position 323. Co-expression of RPS5D266E with the inactive protease (CPR1 C323S) resulted in complete tissue collapse (FIG. 2B), indicating that the protease activity of CPR1 is required for HR suppression.

[0083] Suppression of the RPS5-induced HR by CPR1 could potentially be caused by interference with RPS5 accumulation, either via destabilization or inhibition of transient transformation. To rule out these explanations, we assessed RPS5D266E accumulation using an immunoblot. Neither wild-type CPR1 nor CPR1C323S inhibited RPS5D266E accumulation (FIG. 2C). Since the expression level of full-length RPS5D266E was similar in both the inactive and active protease samples, and because RPS5 is not conserved in soybean, RPS5 is unlikely to be a direct target of CPR1.

[0084] Immunoblot analyses of the above samples also revealed a putative selfcleavage product in the wild type CPR1 protein at approximately 70 kDa that was missing from the CPR1C323S samples (FIG. 2C). Based on its size, such self-processing would remove the N-terminal inhibitor domain of CPR1 , and thus would be expected to activate it. We therefore generated a “mature” variant (CPR1(mat)) lacking the inhibitor domain to mimic the processed version of the protease. (SEQ ID. NO: 2)

[0085] To further assess whether protease activity and proteolytic processing influenced the cell death suppression observed, we transiently co-expressed RPS5D266E with ev, CPR1 , CPR1 C323S, or CPR1 (mat) in N. benthamiana leaves. Three leaf discs from four different leaves per condition were harvested approximately 3.5 h post gene expression induction and an electrolyte leakage assay was performed to measure cell death via conductivity. Readings were taken at 4, 6, 8, 12.5, 15, and 26 h post gene expression induction. As expected, the samples containing RPS5D266E co-expressed with either ev or CPR1 C323S resulted in the highest levels of cell death. When RPS5D266E was co-expressed with CPR1 or CPR1(mat), reduced cell death was observed. Notably, the samples expressing CPR1(mat) resulted in the lowest levels of cell death, except for the MgCl2IU-2025-033-02-WQcontrol (FIG. 2D). To further support the conclusion that the lack of cell death was not due to lack of expression of RPS5D266E, representative leaf tissue was harvested from the electrolyte leakage assay and subjected to immunoblot analysis. All samples were found to be expressing the protease variants and RPS5D266E at similar levels. Together, these results show that CPR1 is capable of suppressing RPS5-mediated HR in a proteasedependent manner. Furthermore, removal of the inhibitor domain of CPR1 enhances its cell death suppression activity.Example 3: CPR1 Localization.

[0086] To generate a PBS1 decoy protein that can be cleaved by an SCN protease, we assessed whether CPR1 co-localizes with the soybean PBS1 protein (GmPBSI a), as partial co-localization is needed to facilitate cleavage of PBS1 (Qi et al., Plant Physiol. 164:340-351 , 2014; Pottinger et al., Mol. Plant-Microbe Interact. 33:932-944, 2020). When GmPBSI a:mCherry was transiently co-expressed with CPR1 (mat):sYFP in N. benthamiana leaves, the CPR1 :sYFP fluorescence overlapped with the GmPBSI a:mCherry fluorescence at the cell periphery, but numerous puncta-like structures (FIG. 3A) reminiscent of mitochondria were also observed. To assess whether CPR1 might be partially localizing to mitochondria, we stained N. benthamiana leaves expressing CPR1(mat):sYFP with MitoTracker Red. The CPR1 puncta co-localized with the mitochondrial marker (FIG. 3B), confirming that CPR1 partially localizes to mitochondria. Consistent with this observation, analysis of the CPR1 (mat) sequence using iPSORT (Bannai etal., Bioinformatics 18:298-305, 2002) revealed a predicted mitochondrial localization signal. Although CPR1 appears to be partially localized to mitochondria, its presence at the cell periphery suggests that it should also have access to PBS1 , which is targeted to the plasma membrane by acylation on its N-terminus.Example 4: Identification of Candidate Substrates of CPR1

[0087] Identification of CPR1 targets enables determination of CPRI’s preferred cleavage sequence, which can then be inserted into a PBS1 decoy. To identify potentialsubstrates of CPR1 in planta, we used a proximity-based biotin labeling system (miniTurbo; mT) (Zhang et al., J. Vis. Exp.: e60728, 2020) that we optimized for use in transgenic soybean roots. Specifically, we generated composite soybean plants consisting of wild-type shoots and transgenic roots expressing CPR1 variants fused to a miniTurbo biotin ligase (CPR1 :miniTurbo). To facilitate identification of transgenic roots, we included the RUBY reporter gene in ourT-DNA construct (He et al., Hortic. Res.7:152-157, 2020). By 21-32 days post inoculation (dpi) of soybean hypocotyls with Agrobacterium rhizogenes K599 strains, RUBY-colored hairy roots expressing CPR1 (with and without protease activity and with and without the inhibitor domain), were readily observed with no significant growth defects observed based upon fresh weight of transgenic tissue. These composite plants were then treated with exogenous biotin, and the roots were harvested 4 h later. As a negative control, we generated composite soybean plants expressing YFP:miniTurbo. Biotinylated proteins were purified using streptavidin beads and prepared for mass spectrometry. A total of 277 soybean proteins were identified by mass-spectrometry analysis across all four of the CPR1:miniTurbo variant samples. Of these, we focused on proteins that were present in CPR1 datasets and absent in datasets of other SON effectors. Based upon our analysis, we selected 12 soybean proteins to test for direct interaction and cleavage.Table 1 : Top Priority Targets Identified Using miniTurbo-based Proximity LabelingIU-2025-033-02-WG(* genes that were successfully cloned from soybean root cDNA)

[0088] Ten out of twelve of these proteins were enriched in CPR1 datasets compared to the YFP:miniTurbo datasets. While we included different CPR1 variants with and without protease activity and / or the inhibitor domain, there were no significant enrichments between the CPR1 samples that influenced our selection of top priority candidates. Notably, several of the selected proteins have links to SCN infection and / or plant immunity from prior studies. Using soybean cDNA, we successfully cloned seven of the twelve proteins for further analysis.

[0089] Proximity labelling enabled us to identify GmBCATI as a putative target of CPR1 . The mature-inactive protease variant, CPR1(mat)C323S, was shown to interact with GmBCATA1-74(FIG. 4A). When co-expressed with CPR1 or CPR1 (mat), GmBCATA1-74exhibited a significant reduction in accumulation in comparison to the empty vector control or a sample expressing the inactive protease, CPR1C323S(FIG. 4B), indicating that GmBCATI is a substrate of CPR1 protease activity. BCATs are enzymes that catalyze the last step of leucine, isoleucine, and valine (branched-chain amino acids; BCAA) biosynthesis orthe first step of theircatabolism. In Arabidopsis, five of the six functional BCATs identified have been shown to localize to either the chloroplast, cytosol, or mitochondria (Diebold et al., Plant Physiol. 129:540-550, 2002). GmBCATI has previously been shown to be upregulated upon drought stress and has been proposed to regulate induction of autophagy during stress responses (Do et aL, Cryobiology and Cryotechnology 68:17-22, 2022). Similarly, BCATs from soybean have been shown to be induced upon drought stress (Shim et al., Plant Physiol. 191:1435-1447, 2023), while a mitochondrial BCAT from wheat (TaBCATI) was found to be a positive regulator of susceptibility to the wheat rust fungus (Corredor-Moreno et aL, Plant Cell 33:1728-1747, 2021 ).IU-2025-033-02-WGExample 5: CPR1 interacts with GmBCATI

[0090] To confirm interactions between CPR1 and the soybean proteins identified from the miniTurbo experiments, we first performed co-immunoprecipitation (co-IP) assays in N. benthamiana leaves. To stabilize the putative interactions, we used the mature variant of CPR1 lacking protease activity (CPR1 (mat)C323S) with a C-terminal GFP tag as a bait protein. As a negative control, inactive AvrPphB (AvrPpBC98S), was used as the bait protein. Of the proteins identified in the miniTurbo datasets, we prioritized soybean branched-chain amino acid aminotransferase 1 (GmBCATI) (Glyma.06G050100), as it was absent in the YFP:miniTurbo negative control and all other SCN effector datasets. (SEQ ID NO: 3) We cloned GmBCATI from soybean root cDNA but left off the N-terminal 74 amino acids (GmBCATIA1-74) (SEQ ID NO: 4), which was predicted to contain a transmembrane domain that could potentially complicate co-IP analysis. GmBCATIA1 74was found to co-IP with CPR1(mat)C323Sbut not with AvrPphB0983(FIG. 4A). In parallel to testing the interaction between GmBCATI and CPR1, we also tested a second putative soybean target that was annotated as a CMP / dCMP-type deaminase domain-containing protein (Glyma.09G080100), using the same bait proteins. Glyma.09G080100 did not co-IP with CPR1, further supporting the specificity of CPRTs interaction with GmBCATI.

[0091] To further characterize the interaction between CPR1 and GmBCATI, we assessed whether GmBCATI protein could be proteolytically cleaved by CPR1. When GmBCATIA1-74was transiently co-expressed with CPR1 or CPR1(mat) in N. benthamiana leaves, a consistent reduction in GmBCATIA1-74protein accumulation was detected in comparison to the samples expressing the empty vector (ev) control or CPR1C323S(FIG. 4B). We also transiently co-expressed the full-length version of GmBCATI with ev, CPR1C323S, or CPR1(mat) in N. benthamiana leaves. As expected, accumulation of full-length GmBCATI was also reduced in the presence of CPR1 (mat) (FIG. 4C). Notably, there was no detectable reduction in GmBCATIA1-74accumulation when expressed with the CPR1(mat)C323Sinactive protease (FIG. 4A), further indicating that GmBCATI is likely a substrate of CPR1.

[0092] In addition to GmBCATI, we also investigated whether SNAP11 (GLYMA_11G234500) was a target of CPR1 as it appeared in the mass spec datasets fromIU-2025-033-02-WGminiTurbo and is known to play a role in additive SCN resistance (Lakhssassi et al., Sci Rep 7:45226, 2017; Shaibu et aL, Front Plant Sci 13:939763, 2022). However, we were unable to detect a direct interaction between CPR1 and SNAP11 using co-IP or evidence that CPR1 cleaves SNAP11.

[0093] We used LOCALIZER (Sperschneider et aL, Sci. Rep. 7:44598, 2017) to predict the subcellular localization of GmBCATI. LOCALIZER returned no predicted targeting sequences for GmBCATI . However, in a study that showed GmBCATI is upregulated upon drought stress (Do et al., Cryobiology and Cryotechnology 68:17-22, 2022), iPSORT was used to identify a putative mitochondrial localization signal. We thus assessed whether GmBCATI localizes to mitochondria using MitoTracker Red and observed co-localization in puncta (FIG. 5A). The localization of CPR1 in mitochondria (FIG. 3B) strongly suggest this is the site in which targeting of GmBCATI occurs.

[0094] Since there is less known about soybean BCATs in comparison to those identified in Arabidopsis, we performed phylogenetic analysis to identify GmBCATI orthologs (FIG. 5B). A total of 9 soybean BCATs were identified, along with the 7 previously described Arabidopsis BCATs (Diebold et al., Plant Physiol. 129:540-550, 2002). GmBCATI was determined to be most closely related to Arabidopsis BCAT1. Notably, Glyma.04G049200.1 p is 95% identical to GmBCATI at the amino acid level, and thus likely represents a homoeolog derived from the most recent genome duplication event in soybean. Arabidopsis BCAT1 localizes to mitochondria and contributes to BCAA degradation (Schuster and Binder, Plant Mol. Biol. 57:241-254, 2005; Binder, Arabidopsis Book 8:e0137, 2010). If GmBCATI also participates in BCAA degradation, CPR1 may be targeting GmBCATI to manipulate BCAA levels during infection. Notably, BCAA levels in syncytia induced by Heterodera schachtii on Arabidopsis roots are elevated compared to uninfected roots (Anwar et aL, Russ. J. Nematol. 24:49-59, 2016). In the same study, enzymes involved in BCAA biosynthesis, including Arabidopsis BCAT3 (Knill et al., Plant Physiol. 155:1960-1975, 2008), were found to be upregulated in syncytia compared to uninfected roots. This increase in biosynthetic BCATs combined with proteolytic removal ofIU-2025-033-02-WGcatabolic BCAT1 by CPR1 could account for the elevated levels of BCAAs observed in syncytia.Example 6: CPR1 Contributes to SCN virulence

[0095] To assess whether CPR1 contributes to the virulence of SCN, we used RNA interference (RNAi) technology to silence CPR1. Designing a gene-specific probe is crucial for RNAi to avoid off-target effects, so we first conducted a BLASTn analysis of CPR1 to identify similarities in the SCN genome (https: / / blast.scnbase.org / blastn). A gene-specific probe was then designed to specifically target CPR1. This probe template was then amplified using PCR resultingin a CPR1- specific amplicon. In brief, a dsRNA probe targeting a region common to all CPR1 isoforms was synthesized using in vitro transcription. In parallel, a GFP-specific probe was prepared and used to control for toxic effects of soaking or dsRNA ingestion by the nematode. Since the optimal dsRNA concentration for efficient gene silencing was uncertain, we tested two concentrations: 2 mg / ml or 3 mg / ml of the CPR1 dsRNA. After soaking, the nematodes were harvested, washed, and subjected to RT-qPCR to assess the effect of dsRNA soaking on the CPR1 transcript. Soaking in dsRNA resulted in an approximately 85% reduction in CPR1 transcripts for both concentrations of dsRNA tested (FIG. 6A).

[0096] We next investigated the effect of CPR1 gene silencing on nematode parasitism. We used CPR1 -silenced nematodes to perform penetration assays by infecting soybean seedlings with approximately 120 CPR1 -silenced or control J2 nematodes. After allowing the nematodes to infect soybean seedlings for 24 h, the nematodes inside the roots were stained with acid fuchsin (Bybd et aL, J. NematoL 14:142-143, 1983) and counted (FIG. 6B). Seedlings inoculated with CPR1 -silenced J2 nematodes showed a significant decrease (33%, P=0.0011) in penetration ability compared to those treated with GFP dsRNA as a control (FIG. 6C). Together these results suggest that CPR1 expression promotes early stages of infection.

[0097] To further assess CPRI’s contribution to virulence, we generated soybean composite plants (Fan et al. 2020) with transgenic roots that were expressingIU-2025-033-02-WQCPR1 :mCherry+RUBY, CPR1 C323S:mCherry+RUBY, or mCherry+RUBY. These composite soybean plants were generated using Williams 82, which is susceptible to the TN10 SCN population. Approximately 2000 SCN TN10 eggs were inoculated on 23-day old plants. Nematodes were allowed to infect and develop for 28 days prior to harvesting. Cysts per gram of root tissue were counted for each biological replicate (FIG. 7A). Roots expressing CPR1 or CPR1C323S resulted in a significant increase in susceptibility compared to roots expressing mCherry+RUBY alone (FIG. 7B), further indicating that expression of the protease, even without protease activity, enhances virulence.Example 7: Testing GmPBS1LLGTGPdecoy for cleavage by CPR1

[0098] As proof of principle, we transformed soybean with a modified version of one of its own PBS1 genes (soybean has three PBS1 genes) expressed under a moderately expressed constitutive promoter. GmPBSI was engineered to contain the LLGTGP cleavage site for CPR1 from GmBCATI . SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7. Thus, the soybean PBSfgene was modified to encode a protein that could be cleaved by SCN protease CPR1.

[0099] As shown in FIG. 8A, CPR1 does not cleave or prevent the accumulation of wildtype GmPBS1-1 in N. benthamiana leaf samples co-expressing wild-type GmPBSI-1 :mCherry with empty vector (ev), AvrPphB:GFP, AvrPphBC98S:GFP (protease inactive), CPR1(mat)C323S:GFP (protease inactive), or CPR1 (mat):YFP. In contrast, in samples coexpressing GmPBSLLGTGPand CPR1(mat), but not in samples co-expressing the inactive variant (C323S(mat)C323S), GmPBS1LLGTGPaccumulation was reduced, which indicates it is being cleaved by CPR1. (FIG. 8B).Example 8: Expression of the modified GmPBSI decoy protein in soybean roots is nontoxic and confers enhanced resistance to soybean cyst nematode infection.

[0100] As shown in FIGs. 9A-C, expression of the modified GmPBSI protein (SEQ ID NO:7) in soybean roots did not affect the growth of soybean roots, thus is not toxic by itself. We next tested whether soybean roots expressing this modified GmPBSI protein had enhanced resistance to infection by soybean cyst nematodes. Roots of composite soybeanplants expressingthe modified GmPBSI protein were inoculated with J2-stage worms (2,000 worms per plant) and allowed to infect for 33 days. At the end of this time, roots were removed from pots and the number of cysts on each plant were counted and numbers divided by the weight of each root system. FIG. 10 shows that roots expressing the modified GmPBSI protein had 50% fewer cysts compared to plants transformed with an unmodified (wild-type) GmPBSI protein. This finding confirms that the decoy GmPBSI protein (SEQ ID NO:7) confers enhanced resistance to infection by soybean cyst nematodes.Example 9: Materials and Methods

[0101] Identification of candidate effector proteases expressed in SCN gland cells:Methods for isolation of SCN gland cells and purification of gland cell RNA have been described previously (Maier et al., Mol. Plant-Microbe Interact, 2013), as has the generation of RNA-seq data from SCN gland cell RNA. To identify candidate effector proteases expressed in gland cells, we searched these RNA-seq data (available in SCNbase; Masonbrinket al., Database: baz111, 2019) forgenes annotated as proteases or peptidases, using the TN10 genome annotation as a guide (Masonbrink et al, BMC Genomics 20:1-14, 2019). We confirmed the presence of the protease domains using MEROPS peptidase and NCBI domain prediction databases. We selected those predicted to harbor signal peptides (SignalP5.0) and lacktransmembrane domains (TMHMM 2.0) and prioritized those that were more highly expressed in gland cells than in whole worms. The accession number for the protease CPR1 has recently been updated as is now designated as Hg_chrom8_TN10mRNA_16195.

[0102] Plant growth and maintenance: N. benthamiana seeds were planted directly on Sun Gro propagation mix (Sun Gro Horticulture, Agawam, MA) supplemented with Osmocote® 14-14-14 slow-release fertilizer. Plants were maintained in a growth room with a temperature of 21 to 23°C, a relative humidity ranging from (30-70%), and a 16-h light and 8-h dark photoperiod. For the composite soybean plants, soybean seeds [Glycine max (L.) Merr.] of cultivar Williams 82 were planted directly on Pro-mix soil in 6-inch pots supplemented with Osmocote 14-14-14 slow-release fertilizer. Plants were maintained in aIU-2025-033-02-WQgrowth chamber set to a temperature of 22°C to 24°C, a relative humidity of 60%, and a 16-h light and 8-h dark photoperiod with an average light intensity at pot level of 300 p Einsteins m-2 s-1. For penetration assays, Glycine max seeds (cultivar Williams 82) were surface sterilized with 70% ethanol for 2 min and then with 50% bleach for 10 min, followed by three rinses in sterile water. Sterilized seeds were placed on wet filter paper with 10 mM morpholinoethanesulfonic acid (MES) buffer [pH 6.5] inside a Petri plate and incubated in a growth chamber at 26°C for 5 days.

[0103] Growth of bacterial strains: Agrobacterium tumefaciens GV3101 and Agrobacterium rhizogenes K599 strains were grown on Luria-Bertani (LB) plates for 2 days at 28°C in an incubator. Liquid LB cultures were grown at 28°C overnight on a shaker. Escherichia coli Top10 cells were grown overnight at 37°C either on LB plates (incubator) or LB liquid (shaker). Antibiotics were used at the following concentrations kanamycin 50 pg / ml, carbenicillin 100 pg / ml, gentamycin 10 pg / ml, and spectinomycin 50 pg / ml for selection during growth. All overnight incubation periods were for approximately 12 to 16 h.

[0104] Generation of plant expression constructs: The coding sequence of CPR1 (lacking the signal peptide) was synthesized and cloned into a pUC57-Amp plasmid (GeneWiz). For cloning CPR1 constructs used in cleavage assays and localization, the coding sequence was amplified from the pUC57-Amp plasmid with primers adding attB1 and attB4 sites for Gateway-cloning into pBSDONR(P1-P4) (Qi et al., Plant Physiol.158:1819-1832, 2012). The pBSDONR-CPR1 construct was sequence verified (Eurofins) and used as a template for site-directed mutagenesis to mutagenize the catalytic cysteine residue (CPR1C323S) or amplify without the N-terminal I29 inhibitor domain. Site-directed mutagenesis was performed as described in (Edelheit et al., BMC Biotechol.9:61 -68, 2009). pBSDONR-CPR1 and pBSDONR-CPR1 C323S served as templates to amplify the “mature” CPR1 variants with or without protease activity respectively. In brief, the mature variant (CPR1(mat)) was generated by PCR amplifying the coding sequence beginning at Ala 71, which is the first amino acid after the predicted I29 inhibitor domain. The PCR product was amplified with primers that incorporated a start codon and attB1-attB4 sites for Gateway cloning. Using multisite Gateway LR cloning, pBSDONR constructs carrying the differentCPR1 variants (CPR1, CPR1C323S, CPR1(mat), or CPR1 (mat)C323S) were cloned into dexamethasone (DEX) inducible vectors pBAV154 with a C-terminal miniTurbo fusion or pTA7001 with a Cterminal mCherry or sYFP2 fusion. All C-terminal protein fusions were sourced from pBSDONR (P4r-P2) vectors that harbored the coding sequences for the fluorescent proteins. These constructs were then transformed into A. tumefaciens GV3101. Gateway LR cloning was also used to assemble pTA7001-AvrPphBC98S:eGFP, pTA7001-GmBCATI :sYFP, and pTA7001 - PBS1 :mCherry.

[0105] For the constructs used in the composite soybean plants, a combination of Gateway and Golden Gate assembly was utilized to insert multiple transcription units into a single T-DNA. Any coding sequence containing Bbsl or Bsal sites was first adapted for use in the Golden Gate system by using site directed mutagenesis to remove endogenous Bbsl and Bsal sites. The coding sequence of the three different variants of CPR1 were first fused with the miniTurbo tag and cloned into pBAV154 via Gateway cloning for consistency with previous constructs, then amplified from pBAV154 with primers to add overhangs for Golden Gate assembly , and finally cloned into the Levell plCH47732 plasmid (Addgene catalog # 48000) with a CaMV 35S short promoter (plCH51277; Addgene catalog # 50268), and a CaMV 35S terminator (plCH41414; Addgene catalog# 50337). The screenable marker RUBY (He et al. 2020) was amplified from pCAMBIA2300 (previously generated and gifted by Sebastian S. Cocioba, Binomica Labs) with primers for Golden Gate assembly into the Level 1 plCH47742 plasmid (Addgene catalog # 48001 ) with a double 35S promoter (plCH51277; Addgene catalog # 50268) and the same 35S terminator. Level 1 constructs were assembled using Bsal-HFv2 restriction enzyme and T4 DNA Ligase. Level 2 constructs were assembled into plCH89921 with the plCH47732- protease:miniTurbo, plCH47742-RUBY, and end linker (plCH41744; addgene catalog # 48017) using a Bbsl-HF restriction enzyme and T4 DNA Ligase. The MoClo Plant Parts Kit was a gift from Nicola Patron (Addgene kit # 1000000047) (Engler et al., ACS Synth. Biol. 3:839-843, 2014). The MoClo Toolkit was a gift from Sylvestre Marillonnet (Addgene kit # 1000000044) (Weber et al., PLoS One 6:e16765, 2011; Werner et al., Bioeng. Bugs 3:38-43, 2012).

[0106] To generate the constructs used in the co-immunoprecipitation and cleavage assays (pTA7002-BCAT1 A1-74:4xMYC, pTA7002-GmBCAT1 :4xMYC, and pTA7002-CPR1(mat)C323S:eGFP), we used NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). pTA7002 was linearized using Xhol and Spel restriction enzymes (New England Biolabs). GmBCATI was PGR amplified with terminal extensions complementary to the resulting pTA7002 ends. For the full-length GmBCATI , the N-terminus was synthesized by TwistBio, PCR amplified, and assembled. Each tagwas amplified from the MoClo Plant Parts Kit plasmids with extensions complementary to the gene of interest (GmBCATI or CPR1 (mat)C323S) at the N-terminus or the vector at the C-terminus.

[0107] RNA extractions and cDNA synthesis: Soybean root and leaf tissue was harvested from 7 to 14-day old soybean plants. Hairy root tissue expressing YFP:miniTurbo+RUBY was harvested 4 to 5 weeks post infection. All tissue was flash frozen in liquid nitrogen at the time of harvesting. Tissue was ground to a fine powder in a chilled mortar with a pestle under liquid nitrogen. Approximately 0.5 grams of tissue was transferred to a 2 ml microcentrifuge tube for RNA extraction. 1.0 ml of TRIzol reagent (Thermo Scientific) was added to the plant tissue powder and vortexed for 30 s. Samples were incubated on a rocker for 10 min at room temperature. 200 pl chloroform was added to the sample, vortexed for 30 s, and incubated on a rocker for 5 min at room temperature. Samples were then centrifuged at 12,000xg, 4°C, for 15 min. Approximately 400 pl of the supernatant was transferred to a new tube and mixed with 400 pl of isopropanol. Samples were incubated at a minimum of 2 h, with a maximum of overnight, at -20°C to precipitate the RNA. Samples were then centrifuged at 12,000xg, 4°C, for 10 min to pellet the RNA. The supernatant was carefully removed. The RNA pellet was washed 3 times with 1 ml 75% ethanol, vortexed to mix and centrifuged at 7,500xg, 4°C, for 5 min. The pellet was dried for approximately 15 min under a sterile hood before resuspending in 50 pl of water. The RNA concentration was measured using a Nanodrop One instrument (Thermo Scientific). A DNase treatment was performed with 2 pg of the RNA prior to cDNA synthesis. 1 pg of RNA, the Thermo Verso cDNA Synthesis Kit (Thermo Scientific), and an Anchored Oligo dT primerIU-2025-033-02-WGwas used to generate soybean cDNA. The resulting cDNA concentration was measured usingthe Nandrop One and used as a template for cloning of soybean genes.

[0108] Transient expression in N. benthamiana: A. tumefaciens (GV3101) cells were scraped from Luria-Bertani (LB) plates and resuspended in 10 mM MgCl2. Final OD600 were adjusted based upon experiment as specified in figure legends. Resuspensions were supplemented with 100 pM acetosyringone, gently mixed by inverting, and incubated at room temperature for 2 to 3 h. After incubation, cultures were infiltrated into leaves of 4 to 6-week-old N. benthamiana plants with a needleless syringe. Plants were sprayed with 50 pM dexamethasone (Thermo Scientific), 0.02% Tween-20, 40 to 45 h post infiltration and tissue were harvested for analysis 4 to 21 h post induction, as specified in figure legends.

[0109] Cell death suppression and electrolyte leakage assay: Mixtures of A. tumefaciens carrying pTA7001-RPS5D266E:5xMYC (OD600 of 0.150) and pTA7001-CPR1 variants with a C-terminal mCherry tag (OD600 of 0.400) were prepared for transient expression. Gene expression was induced 40 to 45 h post infiltration via dexamethasone. For visual analysis of cell death, images were taken under white and ultraviolet light 24 h post gene expression induction. For each replicate, ten to twelve leaves were injected from at least six different plants. Plants exhibiting tissue collapse prior to gene expression induction were not scored or imaged for cell death. At leasttwo independent replicates were performed for all cell death assays. For electrolyte leakage, 3 leaf discs per treatment from 4 different leaves were rinsed three times in deionized water approximately 3.5 h post gene expression induction. Leaf discs were floated in sterile deionized water with 0.001 % Tween-20. Measurements were taken with a conductivity meter (HORIBA Scientific) at 4, 6, 8, 12.5, 15, and 26 h post gene expression induction. Measurements for each condition and timepoint were imported into GraphPad to calculate the standard error of the mean (SEM) and generate a graph.

[0110] Protein isolation and immunoblots: For protein isolation from N. benthamiana, leaf tissue was harvested 4 to 21 h post gene expression induction via dexamethasone, weighed, and flash frozen in liquid nitrogen. Tissue was ground in ice-cold protein extraction buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.1% Nonidet P-40 [Sigma-Aldrich], 1%protease inhibitor cocktail [Sigma-Aldrich], 2 mM 2,2’-dipyridal disulfide) with a chilled mortar and pestle. Cell debris was removed by centrifuging twice at 10,000xg, 4°C, for 10 min. N. benthamiana proteins were prepared in sodium dodecyl sulfate (SDS) loading buffer supplemented with 5% beta-mercaptoethanol and heat denatured in a heating block at 95°C for 10 min. The proteins were separated on a 4 to 20% Trist-glycine stain free polyacrylamide 10- well gel (Bio-Rad) at 170 V for approximately 1 h in 1x Tris / glycine / SDS running buffer. Loading controls were obtained by imagingthe stain-free polyacrylamide gel using the stain-free gel setting on a ChemiDoc™ Imaging System (Bio-Rad). Proteins were transferred to nitrocellulose membrane (Cytiva) at 300 milliamps for 1 h. Membranes were stained in Ponceau for approximately 1 min, washed in deionized water, and imaged. Membranes were blocked in 5% nonfat dry milk (w / v) in 1x TBS-T overnight at 4°C on a shaker. Membranes were then incubated for 1 h at room temperature on a rocker with horseradish peroxidase (HRP)-conjugated c-MYC antibody (Invitrogen; Cat. No. MA1-81357), (HRP)-conjugated HA antibody (Roche; Cat. No. 12013819001), GFP monoclonal antibody (Proteintech; Cat. No. 66002-1 -Ig), or RFP monoclonal antibody (Chromotek; Cat. No. 6G6) to detect proteins. Membranes were washed three times for 15, 5, and 5 min in 1x Tris-buffered saline (TBS) with 0.1% Tween-20 (TBS-T). The anti-GFP blots and anti-RFP blots were then incubated with HRP-conjugated goat anti-mouse antibody (Abeam) for 1 h at room temperature on a rocker. The anti-MYC, anti-RFP, and anti-mouse antibodies were diluted to a concentration of 1:5000 in 5% milk. The anti-GFP antibody was diluted to a concentration of 1 :1000 in 5% milk. Membranes were washed an additional three times in 1x TBS-T after which they were incubated with ProtoGlow chemiluminescent substrate (National Diagnostics) for 5 min and exposed on ChemiDoc™ Imaging System (Bio-Rad) using custom settings for exposure time.

[0111] Fluorescence microscopy: Proteins tagged with sYFP or mCherry were transiently expressed in N. benthamiana using a dexamethasone inducible promoter. 40 to 45 h post A. tumefaciens infiltration, attached leaves were sprayed with 50 pM dexamethasone and 0.01% Tween-20. At 5 to 8 h post transgene induction, leaves were imaged using a Leica Stellaris 8 FALCON Confocal microscope with a 63x water objective.IU-2025-033-02-WGFor sYFP (when co-expressed with mCherry), a 514 nm excitation wavelength was used, and the images were viewed between 526 to 569 nm wavelength of emission. For mCherry, a 587 nm excitation wavelength was used, and the images were viewed between 600 to 640 nm wavelength of emission. For MitoTracker Red (Molecular Probes), lyophilized powder was resuspended in dimethyl sulfoxide to a concentration of 1 mM. A working solution of 100 pM was prepared in 10mM MgCl2and infiltrated into N. benthamiana leaves 4 to 6 h post transgene induction and at least 30 min priorto imaging. Leaves were incubated and kept in the dark. For MitoTracker Red, a 579 nm excitation wavelength was used, and the images were viewed between 590 to 620 nm wavelength of emission. When sYFP was co-expressed with MitoTracker Red a 513 nm excitation wavelength was used and the images were viewed between 523 to 540 nm wavelength of emission. A minimum of three leaves were imaged between at least two independent replicates.

[0112] Generation of composite soybean plants: The protocol for generation of composite plants was optimized from (Fan et al., BMC Biotechnol. 9:61-68, 2020), with minor modifications. In brief, A. rhizogenes K599 strains carrying T-DNA constructs harboring protease variantsand the RUBY screenable marker were prepared on LB solid and in LB liquid media. Prior to infection, liquid cultures were centrifuged at 4,000xg for 5 min, the pellet was washed once with %-strength Gamborg’s liquid [pH 5.75] (plantMedia), and re-centrifuged. Bacterial pellets were resuspended in 14 strength Gamborg’s [pH 5.75] (plantMedia) and adjusted to an OD600 of 1.000 to 1.200. Slant cuts 0.5-1 inch long were made on hypocotyls of 7-day old soybean seedlings, right below the cotyledons, with a sterile razor. A. rhizogenes K599 strains were scraped from plates and lathered on the cut site of the scion. The scions were then placed in 1020 plastic flats filled with coarse vermiculite soaked in 14-strength Gamborg’s media [pH 5.75]. The infected plants were maintained under clear plastic humidity bags to maintain high humidity inside a growth chamber set to 23 to 24°C, 60% humidity, and a 16-h light / 8-h dark photoperiod for the first 7 to 9 days. Plants were carefully removed from the vermiculite at approximately 10 days post inoculation with A. rhizogenes K599 strains to remove non-transgenic (non-red) tissue.Plants were maintained under standard soybean growth conditions (described above) in vermiculite or soil from 3 weeks post infection.

[0113] Proximity-based labeling in composite soybean plants: The protocol for miniTurbo-mediated proximity labelingwas optimized from (Zhang et al., J. Vis. Exp.:360728, 2020) for use in soybean roots. In brief, transgenic soybean roots expressingthe visual RUBY marker were submerged in 250 pM biotin (prepared in dimethyl sulfoxide) in 14-strength Gamborg’s.The roots were vacuum infiltrated for 1 min and maintained in a growth chamber for 4 h. Beakers were covered in foil to prevent light exposure to roots during the incubation. Approximately 1.5-2.0 gram of tissue was harvested from each sample set and flash frozen in liquid nitrogen. Proteins from root tissue were extracted in two volumes of RIPA buffer (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 1 mM EDTA, 1% NP40 [v / v], 0.1% SDS [w / v], 0.5% sodium deoxycholate [w / v], 1 mM DTT, 1 % protease inhibitor cocktail [Sigma Aldrich]). Free biotin was removed from the samples using 10 ml ZebaTM Spin Desalting Columns 7K MWCO (Thermo Scientific). Bradford analysis was used to quantify protein concentration post desalting. Samples were adjusted to 4 mg / ml and then incubated with 100 pl of streptavidin Dynabeads overnight at 4°C. The beads were collected on a magnetic rack for 5 min and the protein supernatant was removed. A series of washes were performed as described in (Zhang et al., J. Vis. Exp.:360728, 2020). The beads were captured on a magnetic rack for 5 min between each wash. All washes were performed at room temperature. The last wash buffer (50 mM ammonium bicarbonate) was removed, and the beads were frozen at -80°C until proteins were digested and analyzed via mass spectrometry.

[0114] Protein digestion and mass spectrometry: Individual samples containing streptavidin beads and associated proteins were denatured in 8 M urea in 100 mM ammonium bicarbonate. Samples were incubated for 45 min at 57°C with 10 mM Tris(2-carboxyethyljphosphine hydrochloride to reduce cysteine residue side chains. These side chains were then alkylated with 20 mM iodoacetamide for 1 h the dark at 21 °C. The urea was diluted to 1 M using 100 mM ammonium bicarbonate. A total of 0.4 pg trypsin (Promega) was added, and the samples were digested for 14 h at 37°C. The resulting peptide solution wasIU-2025-033-02-WGcentrifuged at 2000 ref for 1 min to pellet the beads. The supernatant was transferred to fresh tubes and desalted using ZipTip pipette tips (EMD Millipore), dried down and resuspended in 0.1% formic acid. Peptides were analyzed by LC-MS on an Orbitrap Fusion Lumos equipped with an Easy NanoLCI 200. Buffer A was 0.1 % formic acid in water. Buffer B was 0.1 % formic acid in 80% acetonitrile. Peptides were separated on a 90-min gradient from 0% B to 35% B. Peptides were fragmented by HCD at a relative collision energy of 32%. Precursor ions were measured in the Orbitrap with a resolution of 60,000. Fragment ions were measured in the Orbitrap with a resolution of 15,000. Data were analyzed using Proteome Discoverer (2.5) to interpret and quantify the relative amounts in a label free quantification manner. Data was searched against the Glycine max proteome downloaded on 10 / 26 / 2022. Trypsin was set as the protease with up to two missed cleavages allowed. Carbamidomethylation of cysteine residues was set as a fixed modification. Oxidation of methionine and protein N-terminal acetylation were set as variable modifications. A precursor mass tolerance of 10 ppm and a fragment ion quantification tolerance of 0.05 Da were used. Data was quantified using the Minora feature detector node within Proteome Discoverer.

[0115] Co-immunoprecipitation and cleavage assays: For co-immunoprecipitations (co-IPs), N. benthamiana leaves co-expressing the bait (CPR1(mat)C323S:GFP or AvrPphBC98S:GFP) and the putative soybean target protein, with Cterminal 4xMYC protein fusions, were harvested 21 h post gene expression induction for protein isolation. Bait proteins were infiltrated at an OD600 of 0.400 and 0.300 respectively. GmBCAT1-74 was infiltrated at an GD600 of 0.500. Protein was extracted in ice-cold co-IP extraction buffer (10% glycerol, 25 mM Tris-HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl, 10 mM dithiothreitol, 1x protease inhibitor cocktail [Sigma-Aldrich], 1 mM phenylmethylsulfonyl fluoride), 1 ml of protein lysate was incubated with 20 pl GFP-trap agarose (Chromotek) beads that were previously equilibrated with co-IP extraction buffer according to manufacturer’s directions at 4°C, on a rotator, for 2 h. Beads were washed 5 times with co-IP extraction buffer supplemented with 0.1% NP-40. After the third wash, beads were transferred to a new 1.5 ml microcentrifuge tube for the final two washes. For analysis of proteins captured on GFP-IU-2025-033-02-WGtrap beads for co-IP assays, beads were boiled in SDS loading buffer + 10% betamercaptoethanol at 95°C for 10 min. Samples were centrifuged at 2,500xg for 3 min to separate beads and protein samples and then analyzed by immunoblot analysis as described above. The co-IP assays were repeated three times. For cleavage assays, N. benthamiana leaves co-expressing the CPR1 variants with a Cterminal mCherry fusion protein (OD= 0.400) and GmBCATI (or GmBCATI*1'74) with a C-terminal sYFP or 4xMYC protein fusion (OD= 0.500), were harvested between 4 and 21 h post gene induction for immunoblot analysis. Cleavage tests were performed at least two times.

[0116] Phylogenetic analysis of Arabidopsis and soybean BCAT proteins: The Arabidopsis BCAT1 amino acid sequence (At1g10060) was downloaded from the NCBI Protein database (Genbank accession number BAH19488). The proteomes of soybean (Glycine max W82.a6.v1 , (Valliyodan et al. 2019) and Arabidopsis (Araportl 1 , (Cheng et al., Plant J 89:789-804, 2017) were obtained from the Phytozome 13 online database (Goodstein et al., Nucleic Acids Res. 40:D1178-1186, 2012). These were then queried with the Arabidopsis BCAT1 sequence using BLASTp (2.13.0+) (Altschul et al., J. Mol. Biol. 215:403-410, 1990; Altschul et al., Nucleic Acids Res.25:3389-3402, 1997). We retained all proteins with alignment scores above 300. Secondary protein isoforms were removed, retaining the best scoring isoform from each gene. Amino acid alignments were created using Clustal Omega 1.2.4 (Sievers and Higgins, Mehtods Mol. Biol. 2231:3-16, 2021). A Maximum Likelihood tree was created using RaxML 8.2.12 in PROTGAMMAAUTO mode with 1000 bootstraps (Kozlov et al., Bioinformatics 35:4453-4455, 2019). Trees were displayed usig iTOL V6.9.1 (Letunic and Bork, Nucleic Acids Res.:1-5, 2024), with BCAT-like genes pruned from the tree due to more distant evolutionary relationships. This software pipeline is available at: github.com / ISUgenomics / 2024_Baum_SCN_Protease_Collaboration.

[0117] dsRNA synthesis and purification: A 289 bp fragment of the CPR1 gene and a 255 bp dsRNA targeting a synthetic GFP (Green Fluorescent Protein) gene were amplified using primers with T7 promoter sequences added to their 5’ ends. These PCR products were then used for in vitro transcription using T7 RNA polymerase. The synthetic GFP gene, not naturally present in the nematode, served as a negative control in the experiments. ToIU-2025-033-02-WGfacilitate octopamine-induced ingestion by nematodes, the size of dsRNA for both genes was kept below 300 bp. Sense and antisense RNA were synthesized in a single in vitro reaction using the MEGAscript® RNAi Kit (Thermo Scientific) according to the manufacturer’s instructions, with an incubation period of 8 h to enhance RNA yield. The resulting dsRNA product was purified using an ethanol precipitation protocol (Green and Sambrook, Cold Spring Harb. Protoc. 2020:101717) and its integrity assessed using agarose gel electrophoresis. Its concentration and quality were assessed using a Nanodrop spectrophotometer (Thermo Scientific).

[0118] RNA interference: RNAi soaking was conducted following the protocols outlined by (Sukno et al., J. Nematol. 39:145-152, 2007) with slight modifications. Approximately 10000 nematodes were used per biological replicate of freshly hatched H. glycines J2 worms, soaked in a mixed buffer containing 2 mg / ml and 3 mg / ml dsRNA in 1 / 4 M9 buffer (43.6 mM Na2HPO4, 22 mM KH2PO4, 2.1 mM NaCl, 4.7 mM NH4CL), 1 mM spermidine, and 50 mM octopamine at 26°C on a rotator covered with aluminum foil to maintain a dark environment. J2 worms incubated in dsRNA of the GFP gene were utilized as the control. After 24 h of incubation, J2 worms were washed three times with Nemawash (10 mM MES buffered water [pH 6.5], 0.01% Tween-20) through brief centrifugation to remove external dsRNA. Three biological replicates were used for each gene.

[0119] Reverse transcription quantitative PCR (RT-qPCR): RT-qPCR was employed to investigate the impact of RNAi on the expression of CPR1. Total RNA was isolated from H. glycines J2 worms following treatment with CPR1 -dsRNA or GFPdsRNA control using the Nucleospin microRNA kit (Macherey-Nagel, Hoerdt, France). First-strand cDNA was synthesized and served as a template for PCR. RT-qPCR was conducted usingiTaq universal SYBR Green super mix (Bio Rad) on a Bio Rad CFX96 Real-Time PCR Machine. Each reaction included 2 pl of each primer [10 pM / pl], 5 pl of cDNA, 10 pl SYBR Green super mix, and 1 pl of nuclease-free water, making a final volume of 20 pl. Thermocycler conditions comprised an initial denaturation cycle at 94°C for 30 s, followed by 40 cycles at 94°C for 5 s and 58°C for 34 s. The reaction concluded with the determination of the dissociation curve for all amplicons. The experimental design incorporated three biological replicates and threeIU-2025-033-02-WGtechnical replicates. GAPDH and Actin served as the internal control to normalize the reaction.

[0120] Nematode penetration assays: Five-day-old surface sterilized soybean seedlings were used for the experiments. A 23% Pluronic F-127 (PF-127) (Sigma-Aldrich) gel was prepared. SCN infection was assessed in a 6- well tissue culture plate. Four milliliters of Pluronic gel were poured into each well, and seedlings were placed in each well at 15-20°C. After the gel solidified, approximately 120 J2s / 50 pl of H. glycines (TN10) were inoculated at the root tip of each seedling using a pipette tip, and plates were stored at28°C for 24 h. Three biological replicates were utilized for each treatment, totaling 47 plants for GFP and 47 plants for CPR1 in the analysis. Eight plates were employed for each treatment in the experiment. After soaking, nematodes were washed twice with Nemawash solution (10 mM MES buffer [pH 6.5], 0.01 % Tween-20). Subsequently, the nematodes were counted four times, and the final average of the four replicates were calculated. The concentration of the nematodes was adjusted to 120 J2 suspended in 50 pl of Pluronic gel (120 J2s / 50 pl). After 24 h, plants were harvested from the gel by briefly positioning the plates over an ice bath. This slight decrease in temperature caused the gel to liquify, facilitating the extraction of plantlets without causing damage to the root system. For staining, roots underwent a 2-min treatment with 1% bleach followed by immersion in boiling acid fuchsin solution (Sigma-Aldrich) for 2 to 3 min. Subsequently, the roots were immersed in acidified glycerol (10 drops) and leftfor destaining. Nematodes were counted under a dissecting microscope.

[0121] Nematode infection assays of composite soybean plants: Transgenic composite soybean plants were removed from vermiculite again at approximately 21 days post inoculation with A. rhizogenes K599 strains to remove non-transgenic tissue as before, then were placed in 8-inch containers filled with a mixture of two-parts sand to one-part field soil that had previously been steam sterilized. Plants were allowed to recover for two additional days and then inoculated with 2000 eggs of SCN population TN10. Nematodes were allowed to infect and develop to adulthood (approximately 28 days), at which time the root systems were removed from conetainers and the cysts from each root system were collected in a manner similar to Kandoth et al., Plant Physiol. 155:1960-1975, 2011. Wetweight measurements were recorded for each root system. Cysts from replications of each transgenic construct were transferred to individual wells of 12 well plates. Each well was then photographed using a Zeiss Stemi SV11 microscope with AxioCam HRc and Axiovision SE64 V4.9.1 software at 1040x1040 pixel resolution and each photograph (see representative photograph) was used as input for an Al software-based cyst counting program (Nemacounter) (github.com / DjampaKozlowski / NemaCounter) (Mejias et al., bioRxiv:2024.2007.2007.602381). Cysts per gram wet weight was determined for 8-10 plants for each construct.EQUIVALENTSAND SCOPE

[0122] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present invention is not intended to be limited to the above, but rather is as set forth in the appended claims.

[0123] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0124] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses and descriptive terms, from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0125] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps.

[0126] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranged can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0127] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of the ordinary skill in the art, which will depend in parton how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5% or up to 1% of a given value. Alternatively, the term can mean within an order of magnitude, for example within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaningwithin an acceptable error range forthe particular value should be assumed.

[0128] The term sequence identity or percent (%) sequence identity refers to the percentage of identical nucleotides or amino acids in a sequence compared to a reference sequence, when the sequences are aligned using a sequence alignment program such as BLAST or ClustalW, with default parameters. Sequence identity is calculated by comparingthe aligned positions in the two sequences and dividin the number of identical residues by the total number of positions compared, multiplied by 100 %.

[0129] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the method of the invention can be excluded from anyone or more claims, for any reason, whether or not related to the existence of prior art.

[0130] Each of the foregoing patents, patent applications and references is hereby incorporated by reference, particularly for the teaching referenced herein.SEQUENCES

Claims

CLAIMS1. A soybean PBS1 protein comprising a soybean cyst nematode effector protease CPR1 cleavage site sequence, the soybean cyst nematode effector protease CPR1 cleavage site sequence being located within the activation loop of the PBS1 protein.

2. The soybean PBS1 protein of claim 1, wherein the soybean cyst nematode effector protease CPR1 cleavage site sequence is capable of beingcleaved by a soybean cyst nematode effector protease CPR1 protease comprising the amino acid sequence of SEQ ID NO: 1 .

3. The soybean PBS1 protein of claim 1 or claim 2, wherein the soybean cyst nematode effector protease CPR1 cleavage site sequence comprises the amino acid sequence Leu-Gly-Thr.

4. The soybean PBS1 protein of cany one of claims 1-3, wherein the soybean cyst nematode effector protease CPR1 cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 5.

5. The soybean PBS1 protein of any one of claims 1-4, wherein the activation loop comprises amino acid positions 227-250 of SEQ ID NO: 6.

6. The soybean PBS1 protein of any one of claims 1-5, wherein the soybean cyst nematode effector protease CPR1 cleavage site sequence is located between amino acid positions 232-247 of SEQ ID NO: 6.

7. The soybean PBS1 protein of any one of claims 1-6, wherein the soybean PBS1 protein comprises a structure represented by the formula:N-A-Cwherein:N is a N-terminal region of the soybean PBS1 protein, the N-terminal region comprising at least 90 % sequence identity to the amino acid sequence of SEQ ID NO: 9;A is the activation loop, the activation loop comprisingthe soybean cyst nematode effector protease CPR1 cleavage site sequence; andIU-2025-033-02-WQC is a C-terminai region of the soybean PBS1 protein, the C-terminai region comprising at least 90 % sequence identity to the amino acid sequence of SEQ ID NO: 11.

8. The soybean PBS1 protein of claim 7, wherein the activation loop comprises the soybean cyst nematode effector protease CPR1 cleavage site sequence and at least 90 % sequence identity to the amino acid sequence of SEQ ID NO: 10.

9. A nucleic acid molecule encoding the soybean PBS1 protein of any one of claims 1- 8.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A plant cell, plant, or plant seed comprising the nucleic acid molecule of claim 9 or vector of claim 10.

12. A recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a soybean PBS1 protein, wherein the endogenous AvrPphB cleavage site of the PBS1 protein is replaced with a heterologous cleavage site for the soybean cyst nematode effector protease CPR1.

13. The recombinant nucleic acid molecule of claim 12, wherein the heterologous cleavage site for CPR1 is LLGTGP.

14. The recombinant nucleic acid molecule of claim 12, wherein the heterologous protease cleavage site is located between about amino acid position 230 to about amino acid position 245 in reference to SEQ ID NO:6.

15. A modified soybean PBS1 protein having a heterologous sequence that can be cleaved by the CPR1 protease of soybean cyst nematode, wherein the modified substrate protein is encoded by the recombinant nucleic acid molecule accordingto claim 12.

16. The modified soybean PBS1 protein of claim 15 consisting of SEQ ID NO:7.

17. A vector comprising the recombinant nucleic acid molecule accordingto claim 12.

18. Atransformed soybean plant cell comprising the recombinant nucleic acid molecule accordingto claim 12.

19. Atransformed plant comprisingthe recombinant nucleic acid molecule accordingto claim 12.

20. A transgenic seed of the transformed plant of claim 19.

21. A method of protecting a soybean plant from infection by soybean cyst nematode, the method comprisingthe steps of:transforming the cells of a soybean plant with a recombinant nucleic acid comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a soybean PBS1 protein, wherein the endogenous AvrPphB cleavage site of the PBS1 protein is replaced with a heterologous amino acid sequence that can be cleaved by the soybean cyst nematode effector protease CPR1; and growing the cells into mature soybean plants expressing the modified PBS1 protein having the heterologous amino acid sequence that can be cleaved by CPR1.

22. Plants, plant parts, seeds, and cells from the soybean plant arising from the transformed soybean cells of claim 21.

23. Plants of claim 22 expressing the soybean PBS1 protein having a heterologous amino acid sequence that is cleaved by the CPR1 protease of soybean cyst nematode.

24. A method of protecting a soybean plant from infection by soybean cyst nematode, the method comprisingthe steps of:editing a nucleotide sequence that encodes a soybean PBS1 protein within a soybean plant cell, wherein the endogenous AvrPphB cleavage site of the PBS1 protein is edited to encode a heterologous amino acid sequence that can be cleaved by the soybean cyst nematode effector protease CPR1; and growing the cell into a mature soybean plant expressing the modified PBS1 protein having the heterologous amino acid sequence that can be cleaved by CPR1.

25. Plants, plant parts, seeds, and cells from the soybean plant arising from the transformed soybean cells of claim 24.

26. Plants of claim 25 expressingthe soybean PBS1 protein having a heterologous amino acid sequence that is cleaved by the CPR1 protease of soybean cyst nematode.

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