Transcriptomic analysis of resistance mechanisms in peach (Prunus persica) in response to powdery mildew infection

【Objective】Based on field investigations and laboratory inoculation experiments conducted by our research group, the peach cultivar 3Z-1 exhibits a resistant phenotype to powdery mildew, whereas the cultivar 9-25-23 is highly susceptible. Previous studies have shown that, during pathogen infection, changes in the expression of genes related to cuticular wax biosynthesis and metabolism, cell wall remodeling, and plant hormone signaling are closely associated with pathogen attachment, penetration, hyphal expansion, and host defense responses. Therefore, this study aimed to compare the difference in time-course transcriptional responses between 3Z-1 and 9-25-23 during powdery mildew infection. By integrating disease- resistance gene family analysis and co- expression network analysis, we intend to identify candidate resistance- related genes, thereby providing a reproducible transcriptomic evidence for molecular breeding of powdery mildew resistance in peach.【Methods】Leaves of similar size and free from other pathogen infections were collected from peach cultivars 3Z-1 and 9-25-23 and subjected to powdery mildew inoculation, with non-inoculated leaves used as controls. After inoculation, leaves were sealed with plastic film to maintain humidity and incubated at 25 ℃ for 7 days for phenotypic observation. Leaf samples were dynamically collected at 0, 24, 48, and 72 hours post-inoculation. Total RNA was extracted from all samples for transcriptome sequencing and differential expression analysis. In addition, the NBS-LRR gene family was identified in the peach genome, and its expression patterns under powdery mildew infection were analyzed. Weighted gene co-expression network analysis (WGCNA) was further employed to screen candidate genes associated with infection responses, and the expression trends of selected candidate genes were validated by RT-qPCR.【Results】At 7 days post-inoculation, scattered powdery mildew lesions were observed on the leaf surface of 9-25-23, whereas no obvious disease symptoms were detected on 3Z- 1. Transcriptome sequencing and quality control yielded 34.60-57.09 million clean reads per sample, and the sequencing data showed good alignment with the peach reference genome. Time- course comparisons using 0 hours post-inoculation as the baseline revealed that, in the resistant cultivar 3Z-1, 2219 genes were upregulated and 1713 genes were downregulated at 24 hours post-inoculation; 5525 genes were upregulated and 1899 genes were downregulated at 48 hours; and 3231 genes were upregulated and 3271 genes were downregulated at 72 hours. In the susceptible cultivar 9- 25- 23, 2908 genes were upregulated and 2027 genes were downregulated at 24 hours; 2028 genes were upregulated and 1466 genes were downregulated at 48 hours; and 5646 genes were upregulated and 1522 genes were downregulated at 72 hours. Notably, at 48 hours post-inoculation, the resistant cultivar 3Z-1 exhibited a markedly stronger upregulated transcriptional response, with 5525 upregulated genes, which was substantially exceeding the number of upregulated genes (2028) observed in the susceptible cultivar“9-25-23”at the same time point. Moreover, cross-cultivar comparisons at identical time points indicated that transcriptional differences were particularly pronounced at 48 hours. Therefore, 48 hours post-inoculation was selected as the key time point for pathway enrichment and candidate gene screening. KEGG enrichment analysis was subsequently performed on differentially expressed genes at 48 hours post-inoculation using the corresponding 0-hour samples of each cultivar as controls. The results showed that differentially expressed genes in the resistant cultivar 3Z-1 were mainly enriched in plant hormone signal transduction and plant- pathogen interaction pathways, whereas those in the susceptible cultivar 9-25-23 were predominantly enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism pathways. At the resistance gene family level, a total of 367 NBS-LRR genes were identified in the peach genome, among which more than 80% met the criteria for differential expression under powdery mildew infection, indicating an extensive involvement of this gene family in transcriptional responses to infection. Three NBS-LRR genes with significant upregulation during infection were further identified, namely Prupe.8G032400, Prupe.8G109500, and Prupe.8G005200. Among them, Prupe.8G032400 was homologous to the resistance protein RUN1, Prupe.8G109500 was homologous to RPP2B, and Prupe.8G005200 was homologous to RPV1. At the coexpression network level, WGCNA identified 15 candidate genes associated with infection responses. Of these, 10 genes lacked clear functional annotations, while the remaining five genes (Prupe.3G185100.1, Prupe.7G242200.1, Prupe.4G104300.1, Prupe.4G162000.1, and Prupe.1G038900.1) were functionally annotated as being related to cell wall modification, signal transduction, and light- responsive regulation. RT-qPCR validation showed that the expression trends of all 15 candidate genes were consistent with the transcriptome sequencing results, with 11 genes showing an overall upregulated trend and four genes showing an overall downregulated trend.【Conclusion】This study demonstrates that the resistant cultivar 3Z-1 and the susceptible cultivar 9-25-23 exhibit markedly different transcriptional responses during powdery mildew infection. The resistant cultivar displays a stronger upregulated transcriptional response at 48 hours post-inoculation, with differentially expressed genes significantly enriched in pathways related to phytohormone signal transduction and plant-pathogen interactions; in contrast, the susceptible cultivar showed enrichment in pathways associated with phenylpropanoid biosynthesis and starch and sucrose metabolism. Genes belonging to the NBS-LRR family showed a high rate of responsiveness to infection, indicating their extensive involvement in the transcriptional response of peach to powdery mildew; three NBS-LRR genes potentially linked to powdery mildew resistance were identified. By integrating WGCNA- based co-expression analysis, 15 candidate genes potentially associated with powdery mildew resistance were further selected, and their expression patterns were validated via RT- qPCR, providing candidate molecular targets for subsequent functional characterization and resistance breeding.