세균성 시들음병균 비병원성 이펙터 단백질 RipAZ1의 특성 구명

DoctorRalstonia solanacearum is a destructive pathogen causing bacterial wilt in important solanaceous crops as well as many other plant species. Despite a few identified NLRs conferring bacterial wilt resistance, it has been a challenge to breed durable resistance to R. solanacearum species complex due to the high genetic diversity of the pathogen. Solanum americanum, a wild species of black nightshade, has been reported to harbor a disease resistance gene against potato late blight. The gene can confer resistance when transferred into another Solanaceae species, suggesting black nightshades as a promising disease resistance source. Avirulence effectors are recognized by corresponding disease resistance proteins so they have been often utilized for disease resistance gene mining. Hence, towards the identification of avirulence effector and disease resistance genes, various black nightshade accessions were investigated. Firstly, a bacterial wilt-resistant S. americanum accession was identified. To identify bacterial wilt resistance source, soil-drenching infection assay using R. solanacearum was established in various Solanaceae plant species. 37 accessions of wild species of black nightshades were then screened for bacterial wilt phenotypes against multiple R. solanacearum strains. Consequently, S. americanum accession SP2273 was identified as a bacterial wilt-resistance source. Secondly, R. solanacearum type III effector RipAZ1 (Ralstonia-injected protein AZ1) was identified as an avirulence effector in the resistant S. americanum accession SP2273. RipAZ1 induced accession-specific cell death upon Agrobacterium-mediated transient expression in S. americanum SP2273. Furthermore, loss of ripAZ1 in an avirulent R. solanacearum strain resulted in acquired virulence while ripAZ1-complemented strain recovered avirulence, indicating a role of RipAZ1 as an avirulence determinant in S. americanum SP2273. RipAZ1 natural polymorphism across phylotypes was additionally investigated. Results show that C-terminal truncation leads to loss of RipAZ1-triggered cell death. It was also shown that the 213 amino acid central region of RipAZ1 is sufficient to induce cell death in S. americanum SP2273. Finally, next-generation sequencing-based bulked segregant analysis was conducted to identify RipAZ1-recognizing candidate resistance genes. F2 mapping populations were generated between S. americanum SP2273 and SP2275. RipAZ1-triggered cell death was then screened in the F2 populations. It resulted in a ratio converging on 1 (cell death) : 3 (no cell death), indicating that the corresponding resistance gene may be located at a single locus in S. americanum SP2273. Genomic DNA from F2 individuals showing RipAZ1-triggered cell death was bulked and sequenced in addition to the whole genome sequencing of the two parental accessions SP2273 and SP2275. These findings shall contribute towards the identification of RipAZ1-recognizing resistance gene which in turn shall pave the way for durable bacterial wilt resistance