Abstract
Phytoplasmas are cell wall-less plant pathogenic bacteria responsible for major crop losses throughout the world. In grapevine they cause grapevine yellows, a detrimental disease associated with a variety of symptoms. The high economic impact of this disease has sparked considerable interest among researchers to understand molecular mechanisms related to pathogenesis. Increasing evidence exist that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation during plant development and responses to biotic and abiotic stresses. Thus, we aimed to dissect complex high-throughput small RNA sequencing data for the genome-wide identification of known and novel differentially expressed miRNAs, using read libraries constructed from healthy and phytoplasma-infected Chardonnay leaf material. Furthermore, we utilised computational resources to predict putative miRNA targets to explore the involvement of possible pathogen response pathways. We identified multiple known miRNA sequence variants (isomiRs), likely generated through post-transcriptional modifications. Sequences of 13 known, canonical miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a unique genomic location, were predicted, of which 23 were differentially expressed. A homology search revealed that some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of randomly selected known and novel miRNAs was determined with real-time RT-qPCR analysis, thereby validating the trend of expression seen in the normalised small RNA sequencing read count data. Among the putative miRNA targets, we identified genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. Our results may assist in understanding the role that miRNA pathways play during plant pathogenesis, and may be crucial in understanding disease symptom development in aster yellows phytoplasma-infected grapevines.
Highlights
Phytoplasmas are known to infect hundreds of plant species worldwide and are responsible for devastating yield losses of many economically important crops, fruit trees, and ornamental plants [1]
Our study employed different computational tools to provide the first report on the identification of differentially expressed miRNAs in grapevine leaves infected with aster yellows (AY) phytoplasma
Some of the novel miRNAs shared a high degree of homology with other known plant miRNAs, and were classified as newlyidentified members of existing miRNA families
Summary
Phytoplasmas are known to infect hundreds of plant species worldwide and are responsible for devastating yield losses of many economically important crops, fruit trees, and ornamental plants [1] They are obligate cell wall-less bacterial pathogens (class Mollicutes), and rely on plants and homopterous phloem-sucking insects for biological dispersal. Typical symptoms caused by GY disease include discolouration and necrosis of leaf veins and laminae, downward curling of leaves, abnormal leaf shape and size, incomplete lignification, stunting and necrosis of shoots, flower abortion and berry withering These symptoms eventually lead to reduced plant vitality and fruit yield that may hold devastating consequences for the wine and table grape industries [1,7]. The only available control strategies include early eradication of infected crops, early eradication of infected source plants (weed control), and chemical control of vectors through regular insecticide treatments [8]
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