Evaluation of varietal resistance and transcriptomic characterization of defense- related responses to Phakopsora euvitis in Korean wild grapevines

다국어 초록 (Multilingual Abstract)

Grapevine leaf rust （GLR） caused by Phakopsora euvitis diminishes fruit quality and reduces yield in viticulture, making it one of the world’s most significant fruit crop threats. To develop GLR-resistant grape varieties, substantial efforts have been made to select rust-resistant genes and determine effective strategies for achieving durable resistance in grapevines. This study aims to identify genetic resources resistant to GLR by investigating disease incidence in vineyards and symptom development in grapevines inoculated with pathogens. Fifty-seven genotypes from Ampelopsis brevipedunculata, Vitis acerifolia, V. aestivalis, V. amurensis, V. cinerea, V. coignetiae, V. flexuosa, V. labrusca, V. labruscana, V. rotundifolia, and V. vinifera were evaluated for rust disease incidence over four years （2021–2024） at Yeungnam University. Three plants per genotype were potted and sprayed with a pathogen suspension to assess disease incidence on their adaxial leaf surfaces in a greenhouse. Variation in resistance to GLR was observed among genotypes within each species. Resistant genotypes of V. coignetiae exhibited no symptoms on their leaves, while severe infections were noted in the leaves of susceptible genotypes of A. brevipedunculata, V. amurensis, V. flexuosa, V. labruscana, and V. vinifera. None of the tested V. rotundifolia genotypes displayed yellow pustules and remained unaffected for three weeks after artificial inoculation. These findings highlight the potential of V. rotundifolia and V. coignetiae as valuable genetic resources for breeding rust- resistant grapevines. Chlorophyll content was comparable across all uninfected genotypes. While resistant genotypes maintained relatively stable chlorophyll levels, the average chlorophyll content in the majority of susceptible genotypes was significantly lower following inoculation than before. This negative relationship suggests a general decline in photosynthetic capacity due to disease development in grapevines. Identifying resistant genotypes among both resistant and susceptible genotypes across different Vitis species provides crucial insights for developing new grape varieties with improved resistance to GLR. Keywords: Chlorophyll, Genotypes, Germplasm, Grapevine leaf rust, Phakopsora euvitis, Vitis species.

목차 (Table of Contents)

• CHAPTER I 1
• General Introduction 1
• CHAPTER II. 7
• Evaluation of varietal resistance to grapevine leaf rust and change of chlorophyll contents in grapevine genotypes by infection with Phakopsora euvitis 7
• Abstract 7
• Introduction 8
• Materials and methods 10
• Plant materials and pathogens 10
• Inoculation of pathogen. 10
• Evaluation of rust disease incidence in field and greenhouse environments 10
• Measurement of leaf chlorophyll content following artificial inoculation. 11
• Results and discussion 12
• Assessment of rust disease severity across grapevine species and genotypes under greenhouse and field conditions 12
• Correlation between disease incidence of field infection and greenhouse inoculation 14
• Correlation between disease incidence of greenhouse inoculation and leaf chlorophyll content in grapevine genotypes 15
• Conclusions 17
• Tables 18
• Figures 21
• CHAPTER III. 25
• Mining and expression annotation of transcriptome-derived NLR- type rust resistance genes in Korean wild grapevine, Vitis coignetiae 25
• Abstract 25
• Introduction 26
• Materials and methods. 29
• Plant materials and pathogens. 29
• Inoculation of pathogens 29
• RNA extraction and real-time PCR analysis 30
• Identification of NLR genes in V. coignetiae 30
• Pairwise sequence identity analysis of VcNLRs 31
• Bioinformatics and phylogenetic analysis of candidate VcNLR proteins 31
• Chromosomal distribution, synteny, and gene duplication of candidate VcNLRs 32
• Protein-protein interaction （PPI） network. 33
• Results and discussion 34
• Identification of candidate NLR genes in the V. coignetiea transcriptome 34
• Pairwise sequence identity of VcNLRs 34
• Structural characterization and phylogenetic analysis of candidate NLR proteins in V. coignetiae. 35
• Genomic distribution and evolutionary expansion of the VcNLR genes. 38
• Protein-protein interaction （PPI） network analysis of candidate VcNLRs. 39
• Expression analysis of VcNLRs genes after inoculation with P. euvitis, E. ampelina, and R. vitis 40
• Conclusions 41
• Tables 44
• Figures 47
• CHAPTER IV. 53
• Characterization of pathogenesis-related protein 1 genes expressed in Korean wild grapevine genotypes inoculated with Phakopsoraeuvitis 53
• Abstract 53
• Introduction 54
• Materials and methods. 56
• Plant materials and pathogens. 56
• Salicylic acid （SA） treatment 56
• Total RNA extraction and real-time PCR synthesis 57
• Gene sequence analysis 58
• Results and discussion 59
• Identification and characterization of grapevine VcPR1 genes 59
• Multiple sequence alignment and conserved motif analysis of five VcPR1 genes 62
• Expression analysis of VcPR1 genes 65
• Conclusions 68
• Tables 70
• Figures 75
• CHAPTER V 81
• Transcriptomic mining and structural analysis of Lr34-related ATP-binding cassette transporter genes in Korean wild grapevines reveals their potential role in grapevine leaf rust response. 81
• Abstract 81
• Introduction 82
• Materials and methods. 85
• Plant materials and pathogens. 85
• RNA extraction and real-time PCR analysis 85
• Gene sequence analysis 87
• Results and discussion 88
• Identification and characterization of VcLr34R genes in rapevine 88
• Multiple sequence alignment and conserved domains and motifs analysis of five VcLr34R genes 90
• Expression analysis of VcLr34R genes 95
• Expression analysis of ABA- and defense-related genes 96
• Conclusion 99
• Tables 100
• Figures 105
• CHAPTER VI. 113
• Conclusion 113
• References 117
• Abstract （Korean） 133