Abstract
The oomycete Phytophthora capsici Leonian is one of the most economically important pathogens limiting pepper (Capsicum annuum) production in many regions around the world. Insights into the defense mechanisms of pepper against P. capsici would be helpful in the breeding of resistant cultivars. However, little is still known about the defense system in pepper against P. capsici. We used suppression subtractive hybridization (SSH) followed by macro-array screening to isolate the putative candidate defense genes (PSH, Phytophthora Subtractive Hybridization) in pepper that are differentially expressed between the resistant cultivar (CM334) and the susceptible cultivar (Chilsungcho) following P. capsici infection. A total of 72 PSH genes were identified and categorized based on their putative functions. Semi-quantitative RT-PCR analyses using 11 selected genes confirmed their differential expressions between the resistant and susceptible cultivars along the time course of infection with P. capsici. Furthermore, RNA-seq analyses were performed to understand the possible roles of PSH genes in the defense response to P. capsici as well as three viruses, including two tobacco mosaic virus strains and one Pepper Mottle Virus strain. We found that 37 genes out of 72 displayed differential expression in our RNA-seq-based heatmap between ‘CM334’ and ‘Chilsungcho’ upon pathogen infection. In particular, two genes, CA00g99220 and CA00g96010, and one gene, CA12g16620, were shown to be strongly and uniquely expressed in the resistant cultivar, CM334, against P. capsici and two viruses, respectively. Thus, we consider that this combined approach using SSH/macro-array screening and RNA-seq analyses is a relevant tool for isolation of candidate defense-related genes upon pathogen infection. Data in this study provide a good source for further study on the defense mechanisms against pathogens in chili pepper.
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Acknowledgements
This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2015R1A6A1A03031413) to W-HK and S-IY and supported by the National Research Foundation of Korea Grant (NRF-2018R1D1A1B07049214) to D-HK.
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D-HK, B-DK, and W-HK planned and prepared plant materials and performed molecular experiments; D-HK and S-IY performed RT-PCR experiments. W-HK and S-IY planned and performed RNA-seq analyses; D-HK and S-IY analyzed the data and wrote the manuscript.
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Supplementary Table 1
Survival rate of the plants used in the P. capsici inoculation tests using the magenta box method (XLSX 10 kb)
Supplementary Table 2
Strategy for construction of suppression subtractive libraries (SSH) (XLSX 10 kb)
Supplementary Table 3
Primer sequences used in this study (XLSX 10 kb)
Supplementary Table 4
RPKM data of 73 pepper genes from SSH and macro-array screening (XLSX 30 kb)
Supplementary Fig. 1
RT-PCR result of the 7 selected PSH genes using cDNAs from root samples inoculated with P. capsici (DOCX 259 kb)
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Kim, DH., Kang, WH., Yeom, SI. et al. Isolation of putative pepper defense-related genes against the pathogen Phytophthora capsici using suppression subtractive hybridization/macroarray and RNA-sequencing analyses. Hortic. Environ. Biotechnol. 60, 685–699 (2019). https://doi.org/10.1007/s13580-019-00157-1
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DOI: https://doi.org/10.1007/s13580-019-00157-1