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Summary Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescentin situhybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescentin situdetection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetricin situhybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.
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Trans -Species Mobility of RNA Interference between Plants and Associated Organisms
Abstract Trans-species RNA interference (RNAi) occurs naturally when small RNAs (sRNAs) silence genes in species different from their origin. This phenomenon has been observed between plants and various organisms including fungi, animals and other plant species. Understanding the mechanisms used in natural cases of trans-species RNAi, such as sRNA processing and movement, will enable more effective development of crop protection methods using host-induced gene silencing (HIGS). Recent progress has been made in understanding the mechanisms of cell-to-cell and long-distance movement of sRNAs within individual plants. This increased understanding of endogenous plant sRNA movement may be translatable to trans-species sRNA movement. Here, we review diverse cases of natural trans-species RNAi focusing on current theories regarding intercellular and long-distance sRNA movement. We also touch on trans-species sRNA evolution, highlighting its research potential and its role in improving the efficacy of HIGS.
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- Award ID(s):
- 2003315
- PAR ID:
- 10510770
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Plant And Cell Physiology
- Volume:
- 65
- Issue:
- 5
- ISSN:
- 0032-0781
- Format(s):
- Medium: X Size: p. 694-703
- Size(s):
- p. 694-703
- Sponsoring Org:
- National Science Foundation
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