A highly sensitive biotin‐based probe for small RNA northern blot and its application in dissecting miRNA function in pepper

• Published in: The Plant journal : for cell and molecular biology 2024-04, Vol.118 (1), p.263-276
• Main Authors: Wang, Hongzheng, Yu, Ruimin, Zhu, Qiangqiang, Tian, Zhendong, Li, Feng
• Format: Article
• Language: English
• Subjects: Biotin; biotin probe; Capsicum annuum; Digoxigenin; disease resistance; DNA polymerase; DNA probes; DNA-directed DNA polymerase; Environmental regulations; Immunity; Leucine; MicroRNAs; miR6027; miRNA; NLR; northern blot; Nucleic acids; Nucleotides; Phytophthora capsici; Ribonucleic acid; RNA; RNA probes; Sensitivity enhancement; Vegetables
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.16585

SUMMARY Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope‐ and biotin‐ (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope‐based probe is limited by strict environmental regulation and availability in many places in the world while biotin‐based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase‐based method for incorporation of a cluster of 33 biotin‐labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32P‐labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low‐abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus‐based miRNA silencing in pepper, knocking down accumulation of Can‐miR6027a and Can‐miR6149L. Importantly, further analysis showed that knocking‐down Can‐miR6027a led to upregulation of a nucleotide binding‐leucine rich repeat domain protein coding gene (CaRLb1) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici. Significance Statement A highly sensitive and convenient method is developed for synthesis of biotin‐based probe and validated in small RNA northern blot. Can‐miR6027a is identified as a negative regulator of immunity against Phytophthora capsici in pepper using virus‐based miRNA silencing technology in conjunction with newly developed northern blot method.