Tree Genetics and Molecular Breeding 2024, Vol.14, No.2, 81-94 http://genbreedpublisher.com/index.php/tgmb 85 Notably, the resistant Eucalyptus trees exhibited a significant defense response 24 hours post-infection, effectively inhibiting pathogen growth. These findings suggest that the early activation and regulation of defense-related genes are crucial for Eucalyptus trees in combating fungal pathogens. Figure 2 Venn diagram of differentially expressed genes (DEGs) at different time points during infection with resistant and susceptible clones of Eucalyptus (Adapted from Santos et al., 2020b) Image caption: A and B show the distribution of differentially expressed genes during three infection stages. In susceptible clones, six DEGs were continuously expressed throughout all infection stages, with five genes downregulated and only one gene upregulated. In the resistant clones, nine DEGs were expressed in all stages, and all showed a down-regulation trend. C shows the comparison of differentially expressed genes at 12 hours and 24 hours after inoculation. Three commonly up-regulated genes were observed in susceptible clones, while there were no unique up-regulated genes in resistant clones. These data illustrate that there are significant differences in gene expression patterns between clones during infection, particularly in gene responses between resistance and susceptibility (Adapted from Santos et al., 2020b) 4.2 Experimental approaches to verify gene functionality Verifying the functionality of identified resistance genes requires a combination of molecular biology, genetic engineering, and plant pathology techniques. One common approach is gene knockout or knockdown using CRISPR/Cas9 technology, where specific genes are inactivated to observe changes in disease susceptibility. For instance, CRISPR/Cas9 has been used to knock out the CCR1 gene in Eucalyptus, leading to alterations in lignin content and providing evidence of its role in disease resistance (Dai et al., 2020). Another experimental approach involves overexpressing candidate resistance genes in transgenic Eucalyptus lines to determine whether increased expression confers enhanced resistance to specific pathogens. This method was employed to verify the function of NBS-LRR genes, where overexpression resulted in a heightened defense response and reduced pathogen proliferation (Candotti et al., 2022). Functional assays, such as pathogen inoculation and subsequent phenotypic analysis, are also essential. By challenging genetically modified Eucalyptus plants with pathogens and assessing disease symptoms, researchers can directly link gene function to resistance phenotypes. These assays provide robust evidence for the role of specific genes in disease resistance. 4.3 Impact of verified genes on disease resistance phenotypes The functional verification of resistance genes has had a significant impact on the development of Eucalyptus varieties with enhanced disease resistance. Verified genes have been shown to directly influence the phenotypic expression of resistance traits, leading to reduced disease incidence and severity in genetically modified
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