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.