Abstract
Despite extensive works on miRNA's role during plant–oomycete interaction, its role in Capsicum annuum–Phytophthora capsici pathosystem is not fully explored. Therefore, the present study was designed to identify known and novel miRNAs along with their target genes in two contrasting chili peppers genotypes, i.e., GojamMecha_9086 (resistant) and Dabat_80045 (susceptible) under P. capsici infection associated with modulating the defense response during disease pathogenesis. The result demonstrated 79 known miRNAs corresponding to 24 miRNAs families and 477 novel miRNAs along with 22,895 potential targets, including 30 defense-related target genes against P. capsici infection. The expression analysis of 29 known and 157 novel miRNAs in resistant and 30 known and 177 novel miRNAs in susceptible genotypes revealed differential accumulation patterns. qRT-PCR analysis of 8 defense-related miRNAs representing 4 novels (Pz-novel-miR428-1, Pz-novel-miR160-1, Pz-novel-miR1028-1, Pz-novel-miR204-1) and 4 known miRNAs (Pz-known-miR803-1, Pz-known-miR2059-1, Pz-known-miR2560-1, Pz-known-miR1872-1) revealed differential accumulation pattern in both resistant and susceptible genotypes. Additionally, validation of eight target genes of miRNAs using regional amplification quantitative RT-PCR (RA-PCR), a superior technique to 5′-RNA Ligase-Mediated-rapid amplification of cDNA ends (5′ RLM-RACE), revealed expression of six target genes positively correlated with their corresponding miRNAs in RC versus RI leaf, while five target genes observed an inverse correlation with their corresponding miRNAs in SC versus SI leaf, suggesting their key role during disease response. The Pz-known-miR1872-PODs pair showed perfect inverse relation in all four samples. The significant findings of the current study provide comprehensive genome-wide information about the repertoire of miRNAs and their target genes expressed in resistant and susceptible chili pepper genotypes, which can serve as a valuable resource for better understanding the post-transcriptional regulatory mechanism during C. annuum–P. capsici pathosystem.
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Data availability
The generated raw reads were submitted to the Sequence Read Archive (SRA) of NCBI under Accession No. PRJNA720526 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA720526/ and accession number SAMN18653142: RC_Mi, SAMN18653145: RI_Mi, SAMN18653146: SC_Mi, SAMN18653149: SI_Mi. The raw reads for the target were submitted to the Sequence Read Archive (SRA) of NCBI under number PRJNA665332 with accession number RC: SAMN16251280; RI: SAMN16251798; SC: SAMN16251797; SI: SAMN16251799 at the link https://www.ncbi.nlm.nih.gov/biosample?LinkName=bioproject_biosample_all&from_uid=665332.
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Acknowledgements
TR is gratefully acknowledging the Ministry of Education and Wolkite University, Ethiopia, for sponsoring a fellowship program. Besides, appreciation and many thanks go out to the Department of Bio and Nanotechnology, Guru Jambheshwar University of Science and Technology, Hisar, India, for providing all necessary laboratory facilities. Special thanks also go to all our lab mates who gave invaluable advice and feedback at lab work.
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Conceptualization (TR, VC); methodology: (VC, TR, OPG); formal analysis and investigation: (VC, TR); writing—original draft preparation: (TR]; writing—review and editing: (VC, OPG); resources: (VC); supervision: (VC).
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Fig. S1
: Bioinformatics data analysis workflow for small RNA sequencing and data analysis from the four leaves of C. annuum L. exposed to P. capsici infection. Fig. S2: Length distribution of the miRNAs final cleaned reads (15–29 nts) for the four-leaf samples of chili pepper. (DOCX 291 KB)
Table S1
: List of miRNAs, RTQ, RTQ-UNIr, and U6snRNA primers designed to validate miRNA by qRT-PCR analysis. Table S2: List of target genes primers and exogenous actin-7-like primer for qRT-PCR validation analysis. Table S3: List of commonly expressed known and novel miRNAs and their fold change value in C. annuum L. under P. capsici infection. Table S4: List of all the novel miRNA and their fold change value in RC versus RI and SC versus SI sample of C. annuum L. under P. capsici infection. Table S5: List of differentially expressed genes of known and novel miRNA with corresponding target genes associated with P. capsici infection in resistance and susceptible C. annuum L (in RC vs. RI, and SC vs. SI leaf). Table S6: List of some of the target genes associated with defense against P. capsici infection along with their potential corresponding miRNAs. (DOCX 85 KB)
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Rabuma, T., Gupta, O.P. & Chhokar, V. Genome-wide comprehensive analysis of miRNAs and their target genes expressed in resistant and susceptible Capsicum annuum genotypes during Phytophthora capsici infection. Mol Genet Genomics 298, 273–292 (2023). https://doi.org/10.1007/s00438-022-01979-y
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DOI: https://doi.org/10.1007/s00438-022-01979-y