Fusarium virguliforme is one of the most serious fungal pathogens. It causes sudden death syndrome (SDS) in soybean. In the U.S., the estimated soybean yield suppression from SDS is valued at over $0.6 billion. More than 80 quantitative trait loci (QTL), each providing small SDS resistance effect, are reported. Therefore, the SDS resistance is partial resistance. The major genes conferring robust SDS resistance, comparable to major genes such as Rps1-k that confers complete race-specific Phytophthora resistance, are most likely absent in soybean. The soybean Rps1-k locus contains two genes encoding coiled coil (CC) - nucleotide binding site (NBS) – leucine-rich repeat region (LRR) intracellular receptor proteins and this class of resistance proteins are abbreviated as NLR. This project is undertaken to create a novel NLR gene conferring complete SDS resistance. If we are successful, such a gene will complement the currently exploited SDS resistance QTL for SDS resistance and protect annual soybean yield losses valued millions of dollars in the soybean growing areas, where SDS is prevalent.

In rice, an NLR receptor protein Pikm-1 functions with its helper NLR, Pikm-2, to confer resistance against the rice blast fungus, Magnaporthe oryzae. Pikm-1 recognizes the M. oryzae Avr-Pik effector protein through its unique integrated domain (ID) located in between the CC and NBS domains. The mutant forms of the ID can recognize additional M. oryzae effectors. Recently, it was shown that through modification or replacement of the ID one should be able to create genes that will provide resistance against other pathogens in other crop species. In the modified Pikm-1/Pikm-2 system, we can replace the ID with small antibody (pikobodies) molecules raised against the very important effectors or pathogenicity factors of the pathogen.

In the last two decades, our molecular studies conducted to understand how F. virguliforme causes SDS allowed us to gather the necessary components to engineer this rice gene for conferring robust and complete SDS resistance in transgenic soybean lines. Briefly, in 2011 we described the isolation of the F. virguliforme FvTox1 gene that encodes the FvToxin 1 involved in foliar SDS development in soybean. Subsequently, we have shown that mutation of the FvTox1 gene eliminates about 80% of the foliar SDS symptoms suggesting that it is a major pathogenicity determinant of the pathogen for SDS foliar development.

We have shown that overexpression of the plant antibodies generated against FvTox1 toxin reduces foliar SDS development. Subsequently, we generated nine synthetic genes that encode small FvTox1-interacting peptides. The FvTox1-interacting peptide genes are being tested in transgenic soybean lines to determine if any of them enhances SDS resistance. In a recent unpublished study, Dong and Bhattacharyya groups showed that a sensor developed using one of the FvTox1-interacting peptides can detect FvTox1 in femtomole concentration suggesting that the sensor is extremely sensitive to detect a very tiny or trace amount of the toxin. This sensor study and our two additional published reports established that the FvTox1-interacting peptides can be used to modify the Pik1m-1 for activating Pikm-2 for inducing possible robust SDS resistance.

We are the only group who have reported the FvTox1 cloning and subsequently production of anti-FvTox1 plant antibodies and then FvTox1-interacting peptides. These resources, created over the years, provide us with the opportunity to engineer the Pikm-1/Pikm-2 system to generate a novel artificial SDS resistance gene that will provide soybean with complete protection against F. virgulifiorme.

The rationale of this project is that, once we establish that any one of the 11 peptides including two plant anti-FvTox1 antibodies and nine FvTox1-interacting peptides able to detect FvTox1 to activates the Pikm-2 and generates cell death known as hypersensitive response (HR) in leaves of a model wild tobacco plant, Nicotiana benthamiana, bioengineering of soybean for robust and complete SDS resistance will be feasible. Considering the FvTox1 is the major toxin for foliar SDS development, this is an ideal target for recognition by the engineered Pikm-1 receptor protein, in which ID will be replaced by one of the 11 FvTox1 interacting peptides. Such as modified Pikm-1 receptor is expected activate the Pikm-2 in soybean roots as soon as the root infected SDS pathogen releases the FvTox1 to the vascular root tissues. Rapid HR will restrict any further growth of the SDS pathogen. Thus, robust complete SDS resistance will be generated.

The natural NLR genes have limited life span because the pathogens start to accumulate mutations in the effector proteins recognized by the NLR receptor proteins. Usually, the most complex NLR genes such as Rps1-k containing two functional NLR genes was first introduced in the early 1980s and was providing resistance against most of the P. sojae isolates available at the time. Now, a large number of P. sojae isolates can overcome the resistance encoded by Rps1-k. In this project, selection of the crucial toxin FvTox1, which is involved in foliar SDS development, will provide durability of the to be created SDS resistance gene. FvTox1 is not an effector protein for recognition by the modified Pikm-1/Pikm-2 system. The study of fvtox1 mutants suggested that FvTox1 toxin, produced by the pathogen, is a major toxin for foliar SDS development. The fvtox1 mutants failed to produce severe foliar SDS symptoms. In a study, we observed that the levels of symptoms developed by the mutants were reduced to approximately over 2.5-fold of that produced by the wild-type isolate. These results suggest that the accumulation mutation in FvTox1 could also weaken the pathogen for causing foliar SDS. To make the system durable, in our approach, we will use 11 DNA molecules to modify the Pikm-1 protein. The 11 sequences encoding FvTox1-interacting peptide molecules are distinct from each other; and expected that each of these 11 molecules bind to different regions of the FvTox1 protein. We expect that more than one of these 11 molecules, engineered individually into Pikm-1, will bind to FvTox1 in planta and activate the Pikm-2 receptor. If we can identify more than one of these engineered Pikm-1 proteins that activate the Pikm-2 receptor protein, we will stack these functional engineered Pikm-1 genes along with Pikm-2 in transgenic soybean lines under the support of a renewal grant proposal.

The rationale behind our novel single gene system is that when the recognition of a FvTox1 mutant protein by a modified Pikm-1 protein is lost, the additional effective modified Pikm-1 protein(s) stacked in the cultivar is expected to continue recognition of the mutant FvTox1 protein and provide the robust/potent SDS resistance. Therefore, if we are successful in getting the Pikm-1/Pikm-2 system to work in soybean, our novel single gene for SDS resistance is expected to confer complete and durable SDS resistance. The long-term effectiveness of the modified Pikm-1 gene(s) and Pikm-2 incorporated in an SDS susceptible cultivar will be evaluated for several years under the field conditions to determine the durability of this single gene for robust SDS resistance, under the support of a renewal grant proposal.

Our approach of stacking multiple modified Pikm-1 genes, each carrying a distinct FvTox1-interacting peptide molecule is expected to generate a durable single-gene mediated SDS resistance. It’s however unknow if that is the case, until we evaluate the gene for a long-term. Therefore, under the support of a renewal grant proposal we will incorporate the selected gene(s) into both SDS-resistant and SDS-susceptible cultivars through marker-assisted backcrossing. Our field study of the lines developed through back-crossing for at least three seasons should establish the utility of the gene(s) for providing soybean with robust and complete SDS resistance. We will consider utilizing the novel SDS resistance single gene in the currently available SDS resistant cultivars for commercial cultivation. Placing the novel gene(s) in the SDS resistant cultivars through marker assisted backcrossing will mitigate the possible negative impact of any possible failure of the novel gene(s) to continue providing soybean with the robust SDS resistance.

Novelty of the Proposed Research: We will be the first one to explore the rice Pikm-1 and Pikm-2 gene system in engineering an NLR gene to provide soybean with complete SDS resistance. Soybean already has the necessary downstream signal pathway genes for expression of single gene-mediated resistance. For example, the signal pathways for NLR proteins such as Rps genes for Phytophthora resistance could be used by the modified rice Pikm-1 and Pikm-2 gene system for mediating SDS resistance. It’s already shown that the Pikm-2 gene functions in eudicot Nicotiana benthamiana. Therefore, the rice Pikm-1 and Pikm-2 gene system is expected to work in eudicot soybean as well.

The goal of this project is to generate and test the effectiveness of 11 modified Pikm-1 genes in providing soybean with complete SDS resistance. The following two objectives will be conducted to accomplish the goal of this project.

Objective 1: Determine if any one of the 11 modified Pikm-1 genes activates the Pikm-2 gene in absence FvTox1.

Objective 2: Determine if any of the modified Pikm-1 genes that do not activate the Pikm-2 gene in absence of FvTox1 can confer robust SDS resistance following F. virguliforme infection.

The research activities to be conducted are described below under each of the two objectives.

Objective 1: Determine if any one of the 11 modified Pikm-1 genes activates the Pikm-2 gene in absence FvTox1.
We have generated 11 DNA molecules that encode FvTox1-interacting peptides or anti-FvTox1 plant antibodies. The two anti-FvTox1 plant antibodies were tested in transgenic lines and can neutralize the FvTox1 toxin (Gao and Bhattacharyya 2012). The nine FvTox1-interacting peptides have been tested in transgenic soybean lines. Some of these peptides can also reduce foliar SDS symptom development in transgenic soybean plants (unpublished). We will generate 11 modified Pikm-1 genes to determine if any of them can self-activate the Pikm-2 gene to result in hypersensitive cell death response (HR) in absence of FvTox1. Those modified Pikm-1 genes that failed to cause HR in absence of FvTox1 will be selected to determine if any of these modified Pikm-1 genes can generate complete foliar SDS resistance in stable transgenic soybean plants under the Obj. 2.
We will modify the Pikm-1 gene as described by Kourelis et al. (2023). The ID will be replaced with any of the selected nine FvTox1-interacting peptides or two anti-FvTox1 plant antibodies, and 11 fusion Pikm-1 genes will be generated (Fig. 2B). Each of the 11 fusion Pikm-1 genes will then be tested for possible activation HR in absence of FvTox1. This aspect of the study will be rapidly conducted using the transient assay system in N. benthamiana described by Kourelis et al. (2023) (Fig. 1). The fusion Pikm-1 genes that activate Pikm-2 and causes HR in absence of FvTox1 will be eliminated for further study.
The Pikm-1 genes that do not cause HR in absence of FvTox1 will be selected to determine if they can activate HR in presence of FvTox1. To test this, we will co-express both modified Pikm-1 genes and Pikm-2 along with the FvTox1 gene in N. benthamiana as in Fig. 1. The modified Pikm-1 genes that activate Pikm-2 in presence of FvTox1 will be selected for generating stable transgenic lines under Obj. 2.

Objective 2: Determine if any of the modified Pikm-1 genes that do not activate the Pikm-2 gene in absence of FvTox1 can confer robust SDS resistance following F. virguliforme infection.
We will generate stable transgenic soybean lines for each of the modified Pick-1 gene that do not activate Pickm-2 in absence of FvTox1 in N. benthamiana system in Obj. 1. We will generate at least 5 independent transgenic lines for each of the selected modified Pickm-2. If more than five modified Pickm-2 genes to be tested, we will conduct three independent transgenic events for each gene and the gene showing HR following F. virguliforme infection will be selected for developing additional independent transgenic lines. In Year 3 of this project, the selected lines with HR following F. virguliforme infection and exhibiting SDS resistance in growth chambers will be selected for evaluation of responses to F. virguliforme in the field.

Project milestones & deliveries: We expect to deliver the following by years.

Year 1: The 11 modified Pikm-1 genes generated and co-expressed with Pikm-2 in N. benthamiana. The modified Pikm-1 genes that do not activate Pikm-2 in N. benthamiana will be identified and used in co-expression with FvTox1 in N. benthamiana.

Year 2: The modified Pikm-1 genes that do not activate Pikm-2 in N. benthamiana in absence of FvTox1 but activate in presence of FvTox1 will be expressed in stable transgenic soybean lines.

Year 3: The transgenic lines carrying the modified Pikm-1 genes and Pikm-2 will be tested for their responses to F. virguliforme infection under growth chamber and field conditions.

Update:
Research Needs: Fusarium virguliforme is one of the most damaging fungal pathogens. It causes sudden death syndrome (SDS) in soybean. In the U.S., the estimated soybean yield suppression from F. virguliforme is valued at up to $0.6 billion. More than 80 quantitative trait loci (QTL), each providing small SDS resistance effect, are reported. The SDS resistance governed by natural SDS resistance QTL provide soybean with only partial resistance. The major genes conferring complete SDS resistance unlikely present in the nature. The major genes such as Rps1-k that confers race-specific Phytophthora resistance provides complete resistance against certain Phytophthora sojae isolates or races. The soybean Rps1-k locus contains two genes encoding coiled coil (CC) - nucleotide binding site (NBS) – leucine-rich repeat region (LRR) intracellular receptor proteins and this class of resistance proteins are abbreviated as NLR (Gao et al. 2005).
The creation of a novel NLR gene conferring complete SDS resistance is an important research need. If we are successful, such a gene will complement the currently exploited SDS resistance QTL for SDS resistance and protect annual soybean yield losses valued over $300 millions across the soybean growing areas, where F. virguliforme is prevalent. The goal of this project is to generate a synthetic NLR gene that confers complete SDS resistance.
It has been demonstrated that the NLR receptor proteins Pikm-1 and Pikm-2 conferring resistance against the rice blast fungus, Magnaporthe oryzae can be modified to provide immunity of a wild tobacco species Nicotiana benthamiana against the Potato Virus X (PVX) (Kourelis et al. 2023).
We have applied the same system to generate an NLR receptor conferring complete resistance against F. virguliforme.
To determine if the proposed system can generate single NLR genes for providing complete SDS resistance in transgenic soybean plants, we are developing a transient system in wild-type tobacco N. benthamiana. In this approach, we will transiently express each of the modified 11 Pikm-1 receptors with each of the two FvTox1 proteins encoded by the FvTox1 gene.
To accomplish our goal, we proposed to modify two vectors received from Sophien Kamoun, Sainsbury Laboratory, England. If we are successful in showing that one or more of the 11 modified Pikm-1 genes generate HR following co-expression with one or both FvTox1 proteins, we will express that modified Pickm-1 gene in stable transgenic soybean lines. Our lab has recently established the soybean transformation protocol and we will generate transgenic soybean plants in the Year 2 of this project as proposed in our funded proposal.
In Year 1, the proposed deliverables are:
o The 11 modified Pikm-1 genes generated and co-expressed with Pikm-2 in N. benthamiana.
o The modified Pikm-1 genes that do not activate Pikm-2 in N. benthamiana in absence of FvTox1 will be identified.
o The modified Pikm-1 genes that initiate HR in N. benthamiana in presence of FvTox1 will be identified.

Our progress in the last six months is summarized under each of the above three deliverables:

1. The 11 modified Pikm-1 genes generated and co-expressed with Pikm-2 in N. benthamiana: The pJK-B2-0529 vector provided by Dr. Kamoun carrying the modified Pikm-1 gene that contains the anti-GFP pico-antibody (very small antibody raised against GFP) for binding to the GFP protein expressed from the pPVX-001. The pJK-B2-0529 vector is a large plasmid (16 kilo bases). Therefore, more than one site is found for most of the restriction endonuclease enzymes and therefore engineering this plasmid is complex. The strategy to be followed for developing 11 modified pJK-B2-0529 vectors is described below.

The DNA sequence encoding the anti-GFP pico-antibody will be replaced by each of the 11 synthetic genes generated for expressing two anti-FvTox1 plant antibodies and nine FvTox1-interacting peptides.

Towards delivering this deliverable, we have accomplished the following:
I. We have synthesized the 689-base pair NarI-NsiI fragment containing the AscI-PacI-AvrII cloning sites to replace the 1 kb NarI-NsiI fragment containing the anti-GFP piko-antibody in the pJK-B2-0529 vector.
II. The 689-base pair NarI-NsiI fragment, to be cloned into the 8 kb SacII-MluI fragment, is being cloned in the pBlueScript vector.
III. The 8 kb SacII-MluI fragment has been being cloned into the modified pBlueScript vector which we developed by placing an MluI site in between BamHI and EcoRI sites as shown below.
IV. Once the 689-base pair NarI-NsiI fragment containing the AscI-PacI-AvrII restriction sites is cloned into the 8 kb SacII-MluI fragment in pBlueScript, the modified SacII-MluI fragment will then be used to replace the 8 kb SacII-MluI fragment of the pJK-B2-0529 vector.
V. We will then clone each of the 11 synthetic DNA fragments encoding each of the nine FvTox1-interacting peptide and two anti-FvToxI plant antibodies in the AscI -AvrII sites of the modified pJK-B2-0529 vector.
VI. The resultant 11 modified pJK-B2-0529 vectors will then be transformed in Agrobacterium tumefaciens for transient expression in N. benthamiana along with each of the two modified pJK-PVX-001 constructs that have been engineered to carry each of two FvTox1 DNA fragments described under the Deliverable # 2.

2. The modified Pikm-1 genes that do not activate Pikm-2 in N. benthamiana in absence of FvTox1 will be identified. To accomplish this deliverable, we have replaced the green fluorescence protein gene (GFP) from the pJK-PVX-001 construct, obtained from Dr. Kamoun, with either FvTox1-1 or FvTox1-2. FvTox1-1 and FvTox1-2 encode the entire FvTox1 protein or processed matured FvTox1 toxin, respectively (Brar et al. 2011).

We have already cloned both FvTox1-1 and FvTox1-2 genes and used in replacing the GFP gene of the pPVX-001 construct developed in the vector developed from Potato Virus X (PVX). The steps involved in cloning the FvTox1-1 and FvTox1-2 are described below.

I. For cloning the FvTox1 genes, we digested the pJK-PVX-001 construct with NheI and SacI and the large vector fragment was purified and stored. The small fragment is the GFP fragment and was used in PCR cloning of the two FvTox1 gene fragments.

II. Two-step PCR was applied to fuse a section of the small NheI and SacI fragment with the two FvTox1 fragments. The two NheI and SacI fragments containing FvTox1 sequences were sequenced to confirm that there was no mutation added during the PCR, and then cloned into the large NheI and SacI fragment, i.e., the rest of the pJK-PVX-001 construct. The two modified pJK-PVX-001 constructs containing the FvTox1-1 and FvTox1-2 DNA fragments were named pPVX-FvTox1-1 and pPVX-FvTox1-2.

III. The two modified pJK-PVX-001 constructs, pPVX-FvTox1-1 and pPVX-FvTox1-2 were transformed into A. tumefaciens. The A. tumefaciens isolates carrying either pPVX-FvTox1-1 or pPVX-FvTox1-2 were identified by conducting PCR and were used to infect N. benthamiana for transient expression of the pPVX-FvTox1-1 and pPVX-FvTox1-2 toxin. The very preliminary results suggest that neither the FvTox1-1 nor FvTox1-2 induced any hypersensitive response (HR) in N. benthamiana following transient expression. N. benthamiana is a nonhost for F. virguliforme and FvTox1 is expected not to induce in HR in this nonhost plant. If the observed phenotype “absence of HR” is reproduced in two subsequent experiments, this model system will be ideal for investigating the interactions of FvTox1-1 and FvTox1-2 with the modified 11 Pikm-1 genes carrying either of the nine FvTox1-interacting peptides or two anti-FvTox1 plant antibody genes.

3. The modified Pikm-1 genes that initiate HR in N. benthamiana in presence of FvTox1 will be identified. The activities for this deliverable will be started as soon as we have a few of the 11 modified pJK-B2-0529 vectors are available and we expect to complete this task before the end of Year 1.

Self-evaluation:

Project milestones & deliveries:
By the end of Year 1, it will be known:
1. If any of the 11 modified Pikm-1 genes that do not activate the Pickm-2 NLR protein in absence FvTox1.
2. If any of the modified Pikm-1 genes that do not activate Pikm-2 do activate HR in presence of FvTox1.
Self-evaluation: Our progress is in the right track. We already cloned the two forms of the FvTox1 gene and generated two modified vectors pPVX-FvTox1-1 and pPVX-FvTox1-2. We have started to study if any of these two vectors causes any hypersensitive response (HR) in N. benthamiana. Considering N. benthamiana is a nonhost to F. virguliforme, therefore we do not expect observe any HR responses following transient expression of the two genes in N. benthamiana leaves. The results of the first assay support this expectation.
We are also close to developing the 11 modified Pikm-1 genes in the pJK-B2-0529 vector. Once the modified 11 Pikm-1 genes are developed, we will determine if any of these genes can activate the Pikm-2 receptor protein in presence of FvTox1 and produce HR. We expect to complete all research activities and deliver all deliverables proposed for Year 1 by September 30, 2024.
Year 2: The modified Pikm-1 genes that do not activate Pikm-2 in N. benthamiana in absence of FvTox1 but activate in presence of FvTox1 will be expressed in stable transgenic soybean lines.
Year 3: The transgenic lines carrying the modified Pikm-1 genes and Pikm-2 will be tested for their responses to F. virguliforme infection under growth chamber and field conditions.

References
Brar H.K., and Bhattacharyya, M.K. (2012) Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants. Mol. Plant-Microbe Interact. 25:817-824.
Brar H.K., Swaminathan, S., and Bhattacharyya, M.K. (2011) The Fusarium virguliforme toxin FvTox1 causes foliar sudden death syndrome-like symptoms in soybean. Mol. Plant-Microbe Interact. 24:1179-1188.
Gao, H., Narayanan, N., Ellison, L., and Bhattacharyya, M.K. (2005) Two classes of highly similar coiled coil-nucleotide binding-leucine rich repeat genes isolated from the Rps1-k locus encode Phytophthora resistance in soybean. Mol. Plant-Microbe Interact. 18:1035-1045.
Kourelis J., Marchal C., Posbeyikian A., Harant A., Kamoun S. (2023) NLR immune receptor-nanobody fusions confer plant disease resistance. Science. 379:934-939.
Wang, B., Swaminathan, S., and Bhattacharyya, M.K. (2015) Identification of Fusarium virguliforme FvTox1-interacting synthetic peptides for enhancing foliar sudden death syndrome resistance in soybean. PLoS ONE 10: e0145156.

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Updated November 15, 2024:
Research Needs: Fusarium virguliforme is one of the most damaging fungal pathogens. It causes sudden death syndrome (SDS) in soybean. In the U.S., the estimated soybean yield suppression from F. virguliforme is valued at up to $0.6 billion. More than 80 quantitative trait loci (QTL), each providing small SDS resistance effect, are reported. The SDS resistance governed by natural SDS resistance QTL provide soybean with only partial resistance. The major genes conferring complete SDS resistance unlikely present in the nature. The major genes such as Rps1-k that confers race-specific Phytophthora resistance provides complete resistance against certain Phytophthora sojae isolates or races. The soybean Rps1-k locus contains two genes encoding coiled coil (CC) - nucleotide binding site (NBS) – leucine-rich repeat region (LRR) intracellular receptor proteins and this class of resistance proteins are abbreviated as NLR (Gao et al. 2005).

The creation of a novel NLR gene conferring complete SDS resistance is an important research need. If we are successful, such a gene will complement the currently exploited SDS resistance QTL for SDS resistance and protect annual soybean yield losses valued over $300 millions across the soybean growing areas, where F. virguliforme is prevalent. The goal of this project is to generate a synthetic NLR gene that confers complete SDS resistance.

It has been demonstrated that the NLR receptor proteins Pikm-1 and Pikm-2 conferring resistance against the rice blast fungus, Magnaporthe oryzae can be modified to provide immunity of a wild tobacco species Nicotiana benthamiana against the Potato Virus X (PVX) (Kourelis et al. 2023).

We have applied the same system to generate an NLR receptor conferring complete resistance against F. virguliforme as follows.
To determine if the proposed system can generate single NLR genes for providing complete SDS resistance in transgenic soybean plants, we are developing a transient system in wild-type tobacco N. benthamiana. In this approach, we will transiently express each of the modified 11 Pikm-1 receptors with each of the two FvTox1 proteins encoded by the FvTox1 gene (Brar et al. 2011).
To accomplish our goal, we proposed to modify the two vectors received from Sophien Kamoun, Sainsbury Laboratory, England. If we are successful in showing that one or more of the 11 modified Pikm-1 genes generate HR following co-expression with one or both FvTox1 proteins, we will express that modified Pickm-1 gene in stable transgenic soybean lines. Our lab has recently established the soybean transformation protocol, and we will generate transgenic soybean plants in the Year 2 of this project as proposed in our funded proposal.

In Year 1, the proposed deliverables are:
1. The 11 modified Pikm-1 genes generated and co-expressed with Pikm-2 in N. benthamiana.
2. The modified Pikm-1 genes that do not activate Pikm-2 in N. benthamiana in absence of FvTox1 will be identified.
3. The modified Pikm-1 genes that initiate HR in N. benthamiana in presence of FvTox1 will be identified.

Our progress in the last six months is summarized under each of the above three deliverables:

1. The 11 modified Pikm-1 genes generated and co-expressed with Pikm-2 in N. benthamiana.

The pJK-B2-0529 vector provided by Dr. Kamoun carries the modified Pikm-1 gene containing the anti-GFP pico-antibody (very small antibody raised against GFP) for binding to the GFP protein expressed from the pPVX-001. The pJK-B2-0529 vector is a large plasmid (16 kilo bases). Therefore, more than one site is found for most of the restriction endonuclease enzymes and engineering this plasmid is complex. The strategy to be followed for developing 11 modified pJK-B2-0529 vectors is described below.

The 1 kb NarI-NsiI fragment of pJK-B2-0529 containing this pico-antibody sequence was replaced with a 689 bp synthetic DNA fragment containing AscI-PacI-AvrII sites for incorporation of each of the 11 DNA sequences encoding nine FvTox1-interacting peptides or two anti-FvToxI plant antibodies.

The following six steps were followed in developing the vectors carrying modified Pikm-1 protein genes.
1. We synthesized the 689-base pair NarI-NsiI fragment containing the AscI-PacI-AvrII cloning sites to replace the 1 kb NarI-NsiI fragment containing the anti-GFP piko-antibody in the pJK-B2-0529 vector.
2. The 689-base pair NarI-NsiI fragment was cloned the pBlueScript vector to sequence and confirm the identity of the fragment.
3. The 8 kb SacII-MluI fragment of the pJK-B2-0529 vector was cloned into the modified pBlueScript vector developed by placing an MluI site in between BamHI and EcoRI sites. The 689-base pair NarI-NsiI fragment was then cloned in the 8 kb SacII-MluI fragment in the pBlueScript vector.
4. The 8 kb modified SacII-MluI fragment was used to replace the 8 kb SacII-MluI fragment of the pJK-B2-0529 vector.
5. Each of the 11 synthetic DNA fragments encoding each of the nine FvTox1-interacting peptide and two anti-FvTox1 plant antibodies were individually cloned into the AscI -AvrII sites of the modified pJK-B2-0529 vector.
6. The resultant 11 modified pJK-B2-0529 vectors were transformed into Agrobacterium tumefaciens for transient expression of the fusion genes in N. benthamiana along with each of the two modified pJK-PVX-001 constructs that have been engineered to carry each of two FvTox1 DNA fragments described under the Deliverable # 3.
We have successfully cloned 11 modified Pikm-1 genes. We replaced the DNA sequence encoding integrated domain of Pikm-1 with each of the 11 synthetic DNA sequences encoding nine FvTox1-interacting peptides and two anti-FvTox1 plant antibodies.


2. The modified Pikm-1 genes that do not activate Pikm-2 in Nicotiana benthamiana in absence of FvTox1 will be identified.

Each of the modified Pikm-1 genes were transformed into Agrobacterium tumefaciens; and transformed A. tumefaciens strains carrying each of the 11 modified Pikm-1 genes were infiltrated into the dorsal leaf surfaces of N. benthamiana for transient expression of each of the 11 genes. None of the genes self-activated the Pikm-2 gene since no hypersensitive cell death was recorded. We will investigate if each of the 11 fusion Pikm-1 genes expressed in the infiltrated leaves by conducting western blot analysis.

3. The modified Pikm-1 genes that initiate HR in N. benthamiana in presence of FvTox1 will be identified.

To accomplish this deliverable, we have replaced the green fluorescence protein gene (GFP) from the pJK-PVX-001 construct, obtained from Dr. Kamoun, with either FvTox1-1 or FvTox1-2. FvTox1-1 and FvTox1-2 encode two mature forms of the FvTox1 toxin, respectively (Brar et al. 2011).

Recently, we have cloned both FvTox1-1 and FvTox1-2 genes from RNA samples obtained from the F. virguliforme infected soybean roots and used to replace the GFP gene of the pPVX-001 vector plasmid, developed from Potato Virus X (PVX) as described in the previous report.

The two modified pJK-PVX-001 constructs, pPVX-FvTox1-1 and pPVX-FvTox1-2 were transformed into A. tumefaciens. The A. tumefaciens isolates carrying either pPVX-FvTox1-1 or pPVX-FvTox1-2 were identified by conducting PCR and were used to infect N. benthamiana for transient expression of the pPVX-FvTox1-1 and pPVX-FvTox1-2 toxin. Neither of the FvTox1-1 and FvTox1-2 induced any hypersensitive response (HR) in N. benthamiana following transient expression in multiple experiments and therefore this transient system should be ideal in studying the 11 Pikm-1 fusion genes for inducing Pikm-2 following binding to FvTox1-1 and FvTox1-2.

We have started to study the interactions of FvTox1-1 and FvTox1-2 with each of the 11 fusion Pikm-1 proteins in N. benthamiana leaves following transient expression. There was no change in the chlorophyl color when the leaf was infiltrated with Agrobacterium strains carrying either Pikm-1-Peptide 7 (Peptide 7), FvTox1-1 or FvTox1-2. Interestingly, when Pikm-1-Peptide 5 (Pep5) was co-expressed with either FvTox1-1 or FvTox1-2, the entire infiltrated regions have started to show yellowing due to HR.

Self-evaluation:
Project milestones & deliveries:
By the end of Year 1, it will be known:
1. If any of the 11 modified Pikm-1 genes that do not activate the Pickm-2 NLR protein in absence FvTox1.
2. If any of the modified Pikm-1 genes that do not activate Pikm-2 do activate HR in presence of FvTox1.

Self-evaluation: Our progress is in the right track. We have completed all cloning steps. We have cloned the two forms of the FvTox1 gene and generated 11 modified Pickm-1 genes.
We have demonstrated that none of the 11 modified Pikm-1 genes self-activates Pikm-2.
We have observed that most likely the modified Pikm-1-peptide 5 protein activates Pikm-2 resulting in yellowing and HR.

References
Brar H.K., and Bhattacharyya, M.K. (2012) Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants. Mol. Plant-Microbe Interact. 25:817-824.
Brar H.K., Swaminathan, S., and Bhattacharyya, M.K. (2011) The Fusarium virguliforme toxin FvTox1 causes foliar sudden death syndrome-like symptoms in soybean. Mol. Plant-Microbe Interact. 24:1179-1188.
Gao, H., Narayanan, N., Ellison, L., and Bhattacharyya, M.K. (2005) Two classes of highly similar coiled coil-nucleotide binding-leucine rich repeat genes isolated from the Rps1-k locus encode Phytophthora resistance in soybean. Mol. Plant-Microbe Interact. 18:1035-1045.
Kourelis J., Marchal C., Posbeyikian A., Harant A., Kamoun S. (2023) NLR immune receptor-nanobody fusions confer plant disease resistance. Science. 379:934-939.
Wang, B., Swaminathan, S., and Bhattacharyya, M.K. (2015) Identification of Fusarium virguliforme FvTox1-interacting synthetic peptides for enhancing foliar sudden death syndrome resistance in soybean. PLoS ONE 10: e0145156.

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The modified rice Pikm-1 and Pikm-2 gene mediated SDS resistance is expected to be complete. We should be able to stop the growth of F. virguliforme at the entry points to the soybean roots. The SDS causes annual soybean yield suppression valued at approximately $300 millions. Therefore, development of superior SDS resistant cultivars is a soybean breeding priority. A 10% decrease in soybean yield suppression from SDS because of the cultivation soybean cultivars carrying the modified Pikm-1 and Pikm-2 gene will be translated into increased soybean yield valued at approximately $30 million. Thus, this project will significantly (i) increase the soybean growers’ profitability and (ii) improve the sustainability of soybean industry.