Phytophthora diseases, caused by oomycetes of the genus *Phytophthora*, pose a significant threat to global agriculture. *Phytophthora sojae*, in particular, causes substantial yield losses in soybean crops worldwide, estimated at ~$200 million annually in the United States and billions globally. Chemical control methods are often ineffective, making the development of resistant soybean varieties a crucial strategy for disease management. Previous research has mapped several resistance-to-*P. sojae* (*Rps*) loci to genomic regions rich in nucleotide-binding site leucine-rich repeat (NLR) genes. However, many of these loci have lost effectiveness against newly evolved pathogen isolates, highlighting the need for new, durable resistance genes. This study focuses on identifying and characterizing a novel *Rps* gene, *Rps11*, that provides broad-spectrum resistance to *P. sojae*. The researchers hypothesized that a large NLR gene, possibly resulting from recombination events, would be responsible for the observed resistance.

The literature extensively documents the economic impact of *Phytophthora* diseases, particularly *P. sojae* on soybean production. Several *Rps* loci have been identified and mapped in soybean, but many have become ineffective due to the evolution of new *P. sojae* races. The complex genetic architecture of resistance, involving NLR genes and their dynamic evolution through duplication and recombination, is well-established. However, the functional validation and detailed characterization of specific resistance genes remain limited. The existing knowledge emphasizes the need for new resistance sources and a deeper understanding of the evolutionary dynamics of NLR gene clusters.

The study used a combination of genetic mapping, genome sequencing, and gene expression analysis to identify and functionally characterize *Rps11*. Initially, the *Rps11* locus was mapped to a 348 kb region on soybean chromosome 7 using a population of F2:3 families derived from a cross between a resistant landrace (PI 594527) and a susceptible variety (Williams). To overcome challenges posed by gaps in existing reference genome assemblies, researchers sequenced and assembled the genome of PI 594527 using a combination of PacBio, Bionano optical maps, and 10x Genomics sequencing. This generated a high-quality genome assembly that allowed for gap-free fine mapping and gene identification. Fine mapping narrowed the *Rps11* locus to a 151 kb region containing four NLR genes. To pinpoint the candidate gene, researchers compared gene expression levels in resistant and susceptible recombinant lines after inoculation with *P. sojae*. They found that only one gene (R6) was expressed in resistant lines and its expression was responsive to pathogen inoculation. This gene was identified as the candidate gene, *Rps11*. The complete gene model of *Rps11* was determined using 5'-RACE. To validate the function of *Rps11*, the researchers performed a complementation test using an *Arabidopsis* ubiquitin promoter to drive the expression of the *Rps11* CDS in a susceptible soybean variety (93Y21). The transgenic lines exhibited resistance to multiple *P. sojae* races, confirming the role of *Rps11* in conferring resistance. Comparative genomic analysis was conducted on the *Rps11* region across 28 soybean accessions to examine the evolutionary dynamics of the NLR gene cluster, using high-quality genome assemblies. Phylogenetic analysis and dot plots were used to infer the evolutionary history of the *Rps11* cluster and the origin of the large NLR genes through unequal recombination events, including tandem duplications, deletions, inversions, and promoter fusion. Copy number variations and expression levels were also analyzed.

The study successfully identified *Rps11*, a 27.7 kb NLR gene conferring broad-spectrum resistance to *P. sojae* in soybean. The gene's unusually large size is attributed to multiple rounds of unequal recombination events, leading to expansion of the leucine-rich repeat (LRR) domain and promoter fusion. The *Rps11* locus is located within a cluster of large NLR genes sharing a common ancestor. This cluster exhibits substantial structural diversification and copy number variation (ranging from 5 to 23 copies) across diverse soybean varieties. The absence of allelic copies of *Rps11* in the examined non-*Rps11*-donor varieties highlights the unique nature of this resistance gene. Functional validation through transgenic complementation demonstrated that the *Rps11* gene alone is sufficient to confer broad-spectrum resistance to *P. sojae*. The expression level of the transgene in the transgenic lines was found to be lower than that observed in the resistant line from the mapping population, ruling out the possibility of autoimmunity caused by overexpression. The genomic region containing *Rps11* showed significant structural variation across different soybean accessions, mainly driven by unequal recombination. The study provides a detailed account of the evolutionary plasticity of this NLR gene cluster, including gain, loss, and reinforcement of resistance through recombination.

The identification of *Rps11* provides a valuable new source of resistance against *P. sojae* in soybean. The gene's broad-spectrum resistance is likely linked to its expanded LRR domain, enabling the recognition of multiple pathogen effectors. The substantial structural and copy number variations in the NLR gene cluster highlight the role of unequal recombination in shaping the evolution of resistance. The absence of allelic copies in other varieties underscores the unique evolutionary trajectory of *Rps11*. This research expands our understanding of the evolution and function of NLR genes in plant immunity. The finding has direct implications for breeding programs aiming to develop disease-resistant soybean varieties. The availability of *Rps11*-based markers will facilitate marker-assisted selection for durable resistance.

This study successfully cloned and characterized *Rps11*, a novel giant NLR gene conferring broad-spectrum resistance to *P. sojae* in soybean. The unique structure and evolutionary history of *Rps11*, arising from unequal recombination events, provides valuable insights into the dynamic evolution of plant disease resistance genes. The findings have direct implications for developing more durable resistance in soybean through breeding and genetic engineering. Future research should focus on identifying the specific pathogen effectors recognized by *Rps11* and investigating the functional roles of the expanded LRR domains and 5'-UTR introns.

The complementation test used an *Arabidopsis* ubiquitin promoter rather than the native *Rps11* promoter, which might not perfectly reflect the natural gene expression pattern. The study's focus was on a specific geographic region, and the broad-spectrum resistance might vary in other regions with different *P. sojae* populations. While the study provides compelling evidence of *Rps11*'s role in resistance, future studies could further explore the mechanisms by which it confers resistance and the interactions between *Rps11* and other resistance genes.