Extracellular Vesicles From Xylella fastidiosa Carry sRNAs and Genomic Islands, Suggesting Roles in Recipient Cells

Xylella fastidiosa (Xf) is a Gram-negative bacterial plant pathogen responsible for severe diseases in a variety of economically important crops. A critical aspect of its virulence is the production of extracellular vesicles (EVs). In this study, we discovered that DNA-binding proteins and non-ribosomal RNA-binding proteins are abundant in the corona of Xf-EVs. DNA-seq revealed enrichment of three genomic islands (GIS) in EVs, which carry molecular signatures indicative of horizontal gene transfer (HGT). The most abundant GI encodes five homologous small RNAs designated sXFs. RNA sequencing revealed a distinct pattern of non-coding RNAs enriched in EVs, including four island-encoded sXFs. One of the sXF’s stem-loops contains motifs for binding the RNA chaperone Hfq, which is also abundant in EVs. Predicted target analysis suggests that sXFs play a role in regulation of natural competence in bacteria. Additionally, sXF plant target prediction identifies a coiled-coil nucleotide-binding domain leucine-rich repeat receptor (CNL) immune gene that is downregulated following Xf infection and Xf-EV treatment. We propose a model where Xf releases nucleic acid carrying EVs with two functions: one to deliver RNA-related cargo that regulates gene expression in both bacterial and plant cells, and another to deliver DNA-related cargo for the genetic transfer of genomic islands. We highlight island-encoded sXFs as potential virulence factors and vesiduction as a mechanism of horizontal gene transfer of sXFs in Xf. Taken together, our data on Xf-EV cargoes provide a molecular framework for understanding the virulence of Xf.

Xylella fastidiosa（Xf）是一种革兰氏阴性（Gram-negative）植物病原细菌，可引发多种具有重要经济价值的作物严重病害。其致病性的关键特征之一是产生胞外囊泡（extracellular vesicles, EVs）。本研究发现，Xf胞外囊泡（Xf-EVs）的冠层中富集DNA结合蛋白与非核糖体RNA结合蛋白。DNA测序（DNA-seq）结果显示，囊泡中富集了三个基因组岛（genomic islands, GIS），这些基因组岛带有预示水平基因转移（horizontal gene transfer, HGT）的分子特征。丰度最高的基因组岛编码五种同源小RNA，命名为sXFs。RNA测序（RNA sequencing）显示，囊泡中富集的非编码RNA（non-coding RNAs）具有独特的表达模式，其中包括四个由基因组岛编码的sXFs。其中一个sXF的茎环（stem-loop）结构包含可结合RNA分子伴侣（RNA chaperone）Hfq的基序，而Hfq在囊泡中同样富集。预测靶标分析表明，sXFs参与调控细菌的自然感受态（natural competence）。此外，对sXF的植物靶标预测发现，一种卷曲螺旋核苷酸结合域富亮氨酸重复受体（coiled-coil nucleotide-binding domain leucine-rich repeat receptor, CNL）免疫基因在Xf感染及Xf-EVs处理后表达下调。我们提出了如下模型：Xf释放携带核酸的胞外囊泡，具备双重功能——一是递送RNA相关载荷，以调控细菌与植物细胞内的基因表达；二是递送DNA相关载荷，用于基因组岛的基因转移。我们提出，由基因组岛编码的sXFs可作为潜在致病因子，而囊泡介导转移（vesiduction）是Xf中sXFs水平基因转移的机制。综上，本研究关于Xf-EV载荷的相关数据，为解析Xf的致病性提供了分子框架。