Document Type : Research Articles
Authors
1 Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.
2 PhD student of Agricultural Biotechnology, Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.
Abstract
Wheat stem rust, a devastating fungal disease, threatens global food security through new highly virulent races like Ug99 and TKTTF that overcome plant resistance. Understanding the molecular mechanisms of infection and immune evasion is essential for developing sustainable control strategies. This study elucidates the wheat-Pgt stem rust interaction by identifying pathogen effectors and analyzing genomic variations among three virulent isolates (TKTTF, Ug99, PTRTF). At 96 hours post-inoculation, total RNA was extracted in triplicate from infected leaves of susceptible wheat (cv. Morocco) inoculated with three distinct P. graminis isolates for RNA-seq analysis. Integrated RNA-seq transcriptomics and bioinformatic analyses were employed to characterize effector gene expression profiles and functional attributes. Differential expression analysis revealed a significant upregulation of virulence-associated and metabolic genes in the TKTTF and Ug99 isolates compared to the PTRTF reference isolate. Using Effector P (v5.0) prediction software, we identified 196 high-confidence effector candidates (prediction score >0.5), including both core effectors conserved across isolates and strain-specific variants. Comparative analysis revealed distinct effector repertoires: the Ug99 isolate showed significant enrichment for secreted proteins containing N-terminal signal peptides (SPs), whereas TKTTF predominantly encoded membrane-anchored effectors. Domain analysis identified conserved virulence-associated motifs, including RNA Recognition Motif (RRM) and CDC_Septin domains, implicating their functional importance in pathogen adaptation and host manipulation. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) showed significant representation (FDR <0.05) across three fundamental categories as biological processes, cellular components and molecular functions, reflecting isolate-specific pathogenic strategies. This study establishes that genomic variation among Puccinia graminis f. sp. tritici (Pgt) isolates drives the evolution of divergent effector profiles and virulence strategies. These findings provide a molecular framework for developing next-generation control measures, including precision engineering of disease resistance in wheat, by identifying effector proteins as critical targets for breeding programs.
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