Molecular Pathogens 2024, Vol.15, No.3, 142-154 http://microbescipublisher.com/index.php/mp 146 expression of genes involved in photorespiration, peroxisome function, and glycine metabolism. These pathways are linked to the oxidative stress response, highlighting their role in protecting pine trees from pathogen-induced damage (Park et al., 2020). In addition to JA and SA pathways, ethylene signaling is also involved in modulating defense responses. Crosstalk between these signaling pathways ensures a coordinated and effective defense strategy against diverse pathogens. For example, ethylene signaling has been implicated in the regulation of sulfur metabolism and flavonoid biosynthesis, which contribute to the plant's defense arsenal (Visser et al., 2022). 5 Functional Verification of Resistance Genes 5.1 Gene cloning and expression Cloning and expressing resistance genes in pine trees involve isolating the target genes and inserting them into suitable vectors for propagation and study. For example, in western white pine (Pinus monticola), researchers identified multiple novel members of the PR10 gene family through bioinformatic mining and subsequently selected PmPR10-3.1 for further study. This gene was cloned and expressed in Escherichia coli, and the purified recombinant protein exhibited inhibitory effects on spore hyphal growth of various fungal pathogens, demonstrating its potential role in disease resistance (Liu et al., 2021). Another example is the cloning of nucleotide-binding site leucine-rich repeat (NBS-LRR) genes in limber pine (Pinus flexilis), which are associated with resistance to white pine blister rust (WPBR). These genes were cloned and sequenced to understand their structure and function, and their expression was analyzed in resistant and susceptible pine varieties (Weiss et al., 2020) (Figure 1). Cloning and expressing these genes in model systems or heterologous hosts helps validate their function and provides insights into their role in disease resistance mechanisms. Figure 1 Consensus linkage map for sugar pine showing 12 linkage groups and results of the QTL analysis (Adopted from Weiss et al., 2020) Image caption: Several significantly associated gene families were identified through Genome-Wide Association Studies (GWAS) and Quantitative Trait Loci (QTL) analysis, including the LRR gene family related to MGR. These genes are involved in various disease resistance mechanisms such as pathogen detection, necrosis of infected cells, ubiquitin-dependent protein catalysis, response to oxidative stress, and immune effector processes. Additionally, some genes are primarily involved in abiotic stress, highlighting the diversity of quantitative disease resistance responses in sugar pine (Adapted from Weiss et al., 2020). 5.2 Functional assays and testing Functional assays are critical for verifying the role of cloned resistance genes. These assays involve testing the gene's ability to confer resistance when expressed in host plants or model systems. In the case of PmPR10-3.1 from western white pine, in-vitro antifungal assays were conducted where the recombinant protein was tested against various fungal pathogens. The protein exhibited significant antifungal activity, supporting its role in resistance (Liu et al., 2021).
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