Identification of unknown leaf and stripe rust resistances in adult and seedlings stages of Fe and Zn biofortified spring wheat mutant lines using microscopy, KASP markers and expression dynamics of pathogenesis-related (PR) proteins genes

Wheat fungal diseases, particularly leaf rust (LR) and yellow rust (YR), significantly reduce grain yield and quality worldwide. In this study, newly developed M 7 -generation spring wheat mutant lines with the various Fe- and Zn-biofortified properties, derived from cv. Erythrospermum-35 through gamma irradiation (100- and 200-Gy doses) were evaluated for adult plant resistance (APR) and seedling resistance (SR) to race-specific isolates of LR and YR under field and greenhouse conditions, respectively. Thirteen mutant lines exhibiting APR were selected for microscopic SR assessment based on fungal haustorial mother cells (HMCs) development at specific infection time points. All selected mutant lines showed resistance to YR at both developmental stages, and the majority (69.2%) demonstrated combined APR and SR to LR and YR over extended periods of fungal infection. KASP analysis classified twelve out of thirteen mutant lines (92.3) as carriers of “ a ” allele of Lr2a and Lr14 genes despite on variations in HMCs development. Among the thirteen mutant lines, eight (69.2%) displayed a high level of resistance, the remaining four (30.8%) exhibited disease symptoms, indicating the possible involvement of additional genes in the resistance to LR. The Lr1 , Lr9 , Lr10 , Lr17 , and Lr19 were unlikely efficient, all thirteen mutant lines were monomorphic in allele “ b” . Notably, significant up regulation of PR2 and PR4 genes in SR mutant lines with higher grain Fe content, indicates a time-dependent expression pattern in response to LR infection. The sequential involvement of chitinase (encoded by PR4 ) at LR earlier infection stages and b -1,3-glucanases at later stages likely enhances the plant’s defense system, contributing to reinforced resistance against pathogen invasion by LR in the newly developed spring wheat mutant lines exhibiting both APR and SR to LR. The findings suggest that elevated grain Fe content may support a more robust and temporally coordinated defense response in SR lines by modulating PR2 and PR4 gene expression during rust infection. This observed association between Fe levels and PR genes activation underscores the important role of micronutrients in enhancing disease resistance pathways in wheat. These new developed mutant spring wheat lines combining APR and SR to both LR and YR with enhanced Fe and Zn grain biofortification, represent promising genotypes capable of balancing productivity, grain quality, and durable rust resistance. • Thirteen new spring wheat mutant lines (M7), derived from cv. Erythrospermum-35 via gamma irradiation at 100 and 200 Gy, exhibited an increased resistance (APR) to both leaf rust (LR) and yellow rust (YR) in an infectious field, with various levels of Fe/Zn biofortification. • Most lines (69.2%) exhibited combined APR and SR to both rusts, as confirmed by microscopic analysis of haustorial mother cell (HMC) formation. • KASP genotyping revealed that 92.3% of the lines carried the 'a' alleles of Lr2a and Lr14, which are associated with LR resistance. • Significant time-dependent expression of the PR2 and PR4 genes in resistant lines with elevated Fe content indicated a sequential defence response involving early activation of chitinase (PR4), followed by β-1,3-glucanase (PR2).