Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 73 they have the potential to confer broad-spectrum disease resistance. Studies have already shown that engineering plants with antifungal protein genes can establish new defense mechanisms—like breaking down fungal cell walls or blocking the infection process (Risk et al., 2013; Boni et al., 2017; Milne et al., 2018; Camenzind et al., 2024). So, putting these antifungal protein genes into barley and getting them to express stably is considered a promising strategy for improving barley’s resistance to fungal diseases. This study focuses on transgenic barley lines expressing antifungal proteins and exploring their functions. This study outlines the sources and functions of the antifungal genes used, describes the construction and screening process for the transgenic barley lines, evaluates the effectiveness of these genetic modifications in protecting barley from major fungal diseases, and examines whether they affect plant growth or yield. The implications of these findings for sustainable barley production and future crop improvement are discussed. By developing and analyzing these transgenic barley lines containing antifungal proteins, this study hopes to provide new materials and strategies for breeding disease-resistant barley. 2 Source and Function of Antifungal Genes 2.1 Gene origin Antifungal genes can come from plants themselves or from other organisms that naturally fight off pathogens. For instance, many plants have chitinase genes, which are part of the pathogenesis-related (PR) protein family and are widely present in higher plants. Chitinases break down chitin in fungal cell walls. In one example, barley’s class II chitinase gene was transferred into wheat, and it helped the wheat become more resistant to Fusarium head blight (Selitrennikoff, 2001; Wong et al., 2010). Another group of genes is the β-1,3-glucanase genes (also PR proteins), which break apart the glucan polymers in fungal cell walls and work synergistically with chitinases to enhance disease resistance. Plant antimicrobial peptide (AMP) genes are also drawing a lot of attention. Some of these AMPs are naturally found in plants (such as plant defensins or thionin-like proteins), and others originate from non-plant organisms (for example, insects produce potent AMPs). One notable example is the Metchnikowin (Mtk) gene from the fruit fly, which encodes an antifungal peptide. Researchers have introduced the Mtk gene into barley and overexpressed it, which improved the plant’s resistance to powdery mildew (Yan et al., 2015). There are also antifungal genes derived from plants’ secondary metabolism pathways or detoxification enzymes. For example, the Fhb7 gene, cloned from a wild relative of wheat, encodes a glutathione transferase enzyme that can break down fungal toxins. When Fhb7 was introduced into wheat (through wide hybridization and transgenic methods), it conferred broad resistance to Fusarium head blight by detoxifying the fungus’s toxins. This shows that genes outside the typical PR protein family can also be harnessed to provide disease resistance. 2.2 Antifungal mechanisms Antifungal proteins can attack pathogens in a few main ways: Breaking down the fungal cell wall: Enzymes like chitinases and β-1,3-glucanases directly hydrolyze key components of the fungal cell wall (chitin and glucans, respectively). This weakens or ruptures the cell wall, causing the fungus to collapse or spores to fail. Studies have shown that putting both a chitinase gene and a glucanase gene into plants can significantly boost their fungal resistance (Selitrennikoff, 2001; Yan et al., 2015). Punching holes in the fungal membrane: Some plant-derived antimicrobial peptides and defensins carry positive charges and have both hydrophilic and hydrophobic regions. They can latch onto negatively charged parts of a fungal cell membrane and then form pores, causing the fungal cell to leak its contents or triggering it to die. For example, small proteins like barley defensins and lipid transfer proteins have been found to inhibit fungal spore germination and hyphal growth in vitro (Theis and Stahl, 2004; Van Der Weerden et al., 2013; Struyfs et al., 2021). Blocking the pathogen’s enzymes or starving it of nutrients: A good example is barley’s dimeric α-amylase inhibitor (BDAI), which is a protein in barley seeds. BDAI can inhibit the amylase enzymes that fungi secrete to
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