It is becoming clear that nearly all single resistance genes that confer complete resistance against certain races of a pathogen encode nucleotide binding site (NBS) – leucine-rich repeat region (LRR) receptors, abbreviated as NLR. In this project, we proposed engineer a rice NLR protein to induce complete SDS resistance, a serious soybean disease caused by Fusarium virguliforme. In rice, a pair of NLR receptor proteins, Pikm-1 and Pikm-2, confer resistance against the rice blast fungus, Magnaporthe oryzae. Pikm-1 recognizes the M. oryzae Avr-Pik effector protein through a unique integrated domain (ID) that lies between CC and NBS domains. The Pikm-1 ID is amenable to mutations for recognizing new M. oryzae effectors. It was demonstrated that by manipulating ID of Pikm-1 a novel resistance NLR gene can be created. We proposed to bioengineer ID of Pikm-1 for creating a novel NLR gene that will confer complete SDS resistance in soybean. In 2011, we isolated the F. virguliforme FvTox1 toxin gene. Subsequently, we have shown that mutation of the FvTox1 gene eliminates about 80% of the foliar SDS symptoms. We have also shown that overexpression of anti-FvTox1 plant antibodies enhances foliar SDS resistance. Subsequently, we created nine synthetic genes that encode small FvTox1-interacting peptides. We proposed to replace ID of Pikm-1 with each of the nine FvTox1-interacting peptides and two anti-FvTox1 antibodies for recognition of FvTox1 and activating Pikm-2 for mediating complete SDS resistance. The modified Pikm-1 genes will be transiently co-expressed with FvTox1 in wild tobacco (Nicotiana benthamiana). The modified genes will also be expressed in absence of FvTox1 to determine if any of the 11 modified Pikm-1 genes can activate the resistance responses. Those will be not considered for any further studies. The modified Pikm1-1 genes that can activated resistance responses only in presence of FvTox1 will be investigated further in transgenic soybean plants.

Two research objectives to be carried out are:
Objective 1. Determine if any one of the 11 modified Pikm-1 genes activates the Pikm-2 gene in presence, but not in absence of FvTox1.
Objective 2. Determine if any of the modified Pikm-1 genes activating the Pikm-2 gene in presence but not in absence of FvTox1 can confer robust SDS resistance following F. virguliforme infection.

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 co-expressed 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 and exhibit resistance response 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 under growth chamber and field conditions.

Updated September 4, 2025:
Iowa Soybean Association Contract Research Project Report
Half-yearly Report (November 15, 2024 – April 15, 2025)
Investigator: Madan K. Bhattacharyya, G303 Agronomy Hall, Iowa State University, 515-294-2505, mbhattac@iastate.edu, Department of Agronomy, Iowa State University
Project Title: Bioengineering of an NLR gene for Creating Robust SDS resistance in Soybean

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 proteins (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 protein 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 developed a transient system in wild-type tobacco N. benthamiana. In this approach, we have transiently co-expressed each of the modified 11 Pikm-1 receptors with FvTox1 toxin encoded by the FvTox1 gene (Brar et al. 2011).

The two vectors for this project were obtained from Sophien Kamoun, Sainsbury Laboratory, England. We have shown that at least three modified Pickm-1 genes induced hypersensitive cell death response (HR) in presence of FvTox1 in a transient system in wild-type tobacco N. benthamiana. The three modified Pickm-1 genes will be expressed in stable transgenic soybean lines. Our lab has recently established the soybean transformation protocol, and we will initiate the generation of transgenic soybean plants for these three selected three modified Pickm-1 genes.

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The modified rice Pikm-1 genes together with the native Pikm-2 gene are expected to confer complete SDS resistance. We should be able to stop the growth of F. virguliforme in the infected roots. A 10% decrease in soybean yield suppression from SDS among the soybean cultivars carrying the modified Pikm-1 and Pikm-2 genes 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.