Wheat blast, caused by the fungus *Magnaporthe oryzae* pathotype triticum (MoT), poses a significant threat to global wheat production. First identified in Brazil in 1985, it has since spread to Bangladesh and Zambia, jeopardizing yields in major wheat-producing regions like India and China. The disease's impact stems from the high host specificity of *M. oryzae* pathotypes, with only a limited number of resistance genes effective against MoT. These include *Rmg7*, *Rmg8*, and the 2NS translocation from *Aegilops ventricosa*. However, limitations exist: the 2NS translocation is not universally effective and some MoT isolates overcome it. *Rmg7* and *Rmg8* recognize the same effector, AVR-Rmg8, but neither gene has been cloned. The recent identification of a clonal lineage (B7) prevalent in Bangladesh and Zambia, all carrying AVR-Rmg8, emphasizes the urgency of identifying the corresponding resistance gene(s). Previous research identified two wheat resistance genes, *Rwt3* (an NLR) and *Rwt4* (a tandem kinase), acting as host specificity barriers against non-MoT pathotypes. This study aimed to identify the wheat gene responsible for recognizing and conferring resistance to MoT isolates carrying AVR-Rmg8, using a large, whole-genome sequenced wheat diversity panel and k-mer-based association mapping.

Extensive research has been conducted on wheat blast resistance. Several resistance genes have been identified, including *Rmg7* and *Rmg8*, which recognize the AVR-Rmg8 effector, and the 2NS translocation from *Aegilops ventricosa*. However, these resistances exhibit limitations, such as incomplete resistance and the emergence of virulent isolates. Studies have explored the genetic basis of resistance, using methods like genome-wide association studies (GWAS). The cloning of resistance genes against MoT has proven challenging. Recent genomic surveillance highlighted the emergence of a pandemic clonal lineage (B7) in Bangladesh and Zambia, which universally carries AVR-Rmg8, emphasizing the need for new resistance sources. The identification of *Rwt3* and *Rwt4* as host specificity barriers against other *M. oryzae* pathotypes suggested the possibility of identifying other genes with broader resistance capabilities.

This study employed a multi-faceted approach. First, two MoT isolates (Py 15.1.018 carrying the *el* allele of AVR-Rmg8 and NO6047 + AVR8, a derivative of NO6047 transformed with the *el* allele) were used to phenotype a panel of 320 wheat lines, including landraces and lines with chromosome-scale assemblies. Seedlings were inoculated with the isolates, and disease scores were recorded. K-mer-based association mapping was performed using the SY-Mattis genome as a reference, identifying an association peak on chromosome 2AL. Haplotype analysis of the 5.3 Mbp association interval across a larger collection of wheat accessions refined the region of interest to a 400 kb interval. RNA-seq data from leaf tissue were used to identify expressed genes within this interval. Sequence comparisons of these genes between resistant and susceptible lines revealed polymorphisms in TraesSYM2A03G00828360. Further investigation identified this gene as *Pm4*, a previously characterized powdery mildew resistance gene. To confirm the role of *Pm4* in wheat blast resistance, near-isogenic lines (NILs) for *Pm4* alleles, EMS-induced mutants, and transgenic lines overexpressing *Pm4b* were inoculated with isogenic MoT transformants differing only in the presence of AVR-Rmg8. Disease scores were recorded for both seedling leaves and spikes. Quantitative real-time PCR was used to assess the expression levels of *Pm4b_V1* and *Pm4b_V2* transcripts in spike tissues. PCR-based assays were designed to detect *Pm4* and investigate its prevalence in landraces and modern varieties. Finally, the relative effectiveness of different *Pm4* alleles against various AVR-Rmg8 alleles was examined by screening wheat lines carrying different *Pm4* alleles for resistance to transformants with different *AVR-Rmg8* alleles (el, ell, ell'). Detached leaf and spike assays were conducted at different temperatures to assess the impact of temperature on resistance.

The study identified *Pm4*, a previously known powdery mildew resistance gene, as a key factor in conferring resistance to wheat blast. K-mer-based association mapping pinpointed the resistance locus to a 5.3 Mbp region on chromosome 2A, subsequently narrowed down to a 400 kb interval using haplotype analysis. Within this interval, TraesSYM2A03G00828360 was identified as a strong candidate gene, showing strong homology to *Pm4*. Functional analyses using NILs, EMS mutants, and transgenic lines confirmed that *Pm4* confers resistance to MoT isolates carrying AVR-Rmg8, particularly in seedling leaves. However, the resistance was stage-specific, with adult plants showing susceptibility. Different *Pm4* alleles showed varying levels of effectiveness against different AVR-Rmg8 alleles, with some providing broader resistance than others. The *Pm4f* allele demonstrated strong resistance in both seedling leaves and spikes against a Bangladeshi isolate. The prevalence of *Pm4* varies across different wheat collections, being rare in landraces but more common in modern European varieties, highlighting potential selection by breeders. The study also revealed new *Pm4* alleles (*Pm4i* and *Pm4j*), one of which shows differential effectiveness against various AVR-Rmg8 alleles.

The discovery that *Pm4*, a gene previously known for its role in powdery mildew resistance, also confers resistance to wheat blast is a significant finding. This dual functionality highlights the potential for pleiotropic effects of resistance genes and opens avenues for broadening disease resistance in wheat breeding. The varying effectiveness of different *Pm4* alleles underscores the importance of allele selection for durable resistance. The identification of *Pm4f* as a robust source of resistance against the pandemic clonal lineage in Bangladesh and Zambia, particularly in spikes, is highly relevant for disease management strategies. The finding challenges conventional approaches of searching for resistance genes in regions where the disease originated, as wheat blast's recent emergence makes such an approach less feasible. The study's results suggest that focusing on regions with high powdery mildew pressure might reveal additional genes with broad-spectrum resistance against wheat blast. Future research should explore the mechanisms underlying *Pm4*'s dual resistance against powdery mildew and wheat blast, understanding how this single gene can recognize two distinct pathogens with different infection strategies.

This research successfully cloned a wheat blast resistance gene, unexpectedly revealing it to be *Pm4*, a previously characterized powdery mildew resistance gene. Different *Pm4* alleles exhibit variable efficacy against diverse AVR-Rmg8 alleles, with *Pm4f* showing exceptional resistance in spikes against a Bangladeshi isolate. The findings emphasize the importance of considering multiple diseases in breeding programs and suggest exploring regions with high powdery mildew pressure for additional broad-spectrum resistance genes. Future work should investigate the molecular mechanisms of *Pm4*'s dual functionality and evaluate its effectiveness at higher temperatures.

The study focused primarily on seedling and detached leaf/spike assays, which might not fully reflect the complexities of whole-plant infection. The temperature-dependent nature of resistance also needs further investigation in field settings, particularly in the hotter environments of Bangladesh and Zambia. The limited number of *Pm4* alleles analyzed might not represent the complete allelic diversity, and further exploration of wild relatives could uncover novel alleles with enhanced resistance.