Department of Pest-Management and Conservation, Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln, Christchurch, 7647, New Zealand.
The New Zealand Institute for Plant and Food Research Limited, Lincoln, 7608, New Zealand.
Microb Ecol. 2024 Oct 9;87(1):124. doi: 10.1007/s00248-024-02436-6.
Understanding host-microbe interactions in planta is an expanding area of research. Amplicon sequencing of the 16S rRNA gene is a powerful and common method to study bacterial communities associated with plants. However, the co-amplification of mitochondrial and plastid 16S rRNA genes by universal primers impairs the sensitivity and performance of 16S rRNA sequencing. In 2020, a new method, Cas-16S-seq, was reported in the literature to remove host contamination for profiling the microbiota in rice, a well-studied domestic plant, by engineering RNA-programmable Cas9 nuclease in 16S rRNA sequencing. For the first time, we tested the efficiency and applicability of the Cas-16S-seq method on foliage, flowers, and seed of a non-domesticated wild plant for which there is limited genomic information, Leptospermum scoparium (mānuka). Our study demonstrated the efficiency of the Cas-16S-seq method for L. scoparium in removing host contamination in V4-16S amplicons. An increase of 46% in bacterial sequences was found using six guide RNAs (gRNAs), three gRNAs targeting the mitochondrial sequence, and three gRNAs targeting the chloroplast sequence of L. scoparium in the same reaction. An increase of 72% in bacterial sequences was obtained by targeting the mitochondrial and chloroplast sequences of L. scoparium in the same sample at two different steps of the library preparation (DNA and 1st step PCR). The number of OTUs (operational taxonomic units) retrieved from soil samples was consistent when using the different methods (Cas-16S-seq and 16S-seq) indicating that the Cas-16S-seq implemented for L. scoparium did not introduce bias to microbiota profiling. Our findings provide a valuable tool for future studies investigating the bacterial microbiota of L. scoparium in addition to evaluating an important tool in the plant microbiota research on other non-domesticated wild species.
在植物体内研究宿主-微生物相互作用是一个不断发展的研究领域。扩增子测序 16S rRNA 基因是研究与植物相关的细菌群落的一种强大而常用的方法。然而，通用引物对线粒体和质体 16S rRNA 基因的共扩增会降低 16S rRNA 测序的灵敏度和性能。2020 年，文献中报道了一种新方法 Cas-16S-seq，通过在 16S rRNA 测序中工程化 RNA 可编程 Cas9 核酸酶，用于去除宿主污染，从而对水稻等研究充分的家养植物中的微生物组进行分析。我们首次在一种非驯化的野生植物——Leptospermum scoparium（麦卢卡）的叶片、花朵和种子上测试了 Cas-16S-seq 方法的效率和适用性，该植物基因组信息有限。我们的研究表明，Cas-16S-seq 方法在去除宿主污染方面对 L. scoparium 的 V4-16S 扩增子是有效的。在相同反应中使用靶向 L. scoparium 线粒体序列的 6 个向导 RNA（gRNA）、靶向质体序列的 3 个 gRNA，以及靶向线粒体和质体序列的 3 个 gRNA，发现细菌序列增加了 46%。在文库制备的两个不同步骤（DNA 和第 1 步 PCR）中靶向 L. scoparium 的线粒体和质体序列，细菌序列增加了 72%。使用不同方法（Cas-16S-seq 和 16S-seq）从土壤样本中检索到的 OTU（操作分类单元）数量是一致的，这表明针对 L. scoparium 实施的 Cas-16S-seq 没有对微生物组分析引入偏差。我们的研究结果为未来研究 L. scoparium 的细菌微生物组提供了有价值的工具，此外还评估了其他非驯化野生物种植物微生物组研究中的一个重要工具。
