Evaluation of Disease Severity and Global Transcriptome Response Induced by Citrus bark cracking viroid, Hop latent viroid, and Their Co-Infection in Hop (Humulus lupulus L.).

Nataša Štajner,Sebastjan Radišek,Jernej Jakše,Vishnu Sukumari Nath,Jaroslav Matoušek,Ajay Kumar Mishra

doi:10.3390/ijms20133154

Abstract

Viroids are small non-capsidated, single-stranded, covalently-closed circular noncoding RNA replicons of 239–401 nucleotides that exploit host factors for their replication, and some cause disease in several economically important crop plants, while others appear to be benign. The proposed mechanisms of viroid pathogenesis include direct interaction of the genomic viroid RNA with host factors and post-transcriptional or transcriptional gene silencing via viroid-derived small RNAs (vd-sRNAs) generated by the host defensive machinery. Humulus lupulus (hop) plants are hosts to several viroids among which Hop latent viroid (HLVd) and Citrus bark cracking viroid (CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop. Additionally, the effect of HLVd on the aggressiveness of CBCVd that underlies severe stunting in hop in a mixed infection was studied by transcriptomic analysis. Our analysis revealed that CBCVd infection resulted in dynamic changes in the activity of genes as compared to single HLVd infection and their mixed infection. The differentially expressed genes that are involved in defense, phytohormone signaling, photosynthesis and chloroplasts, RNA regulation, processing and binding; protein metabolism and modification; and other mechanisms were more modulated in the CBCVd infection of hop. Nevertheless, Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in the proteolysis mechanism is more active in a mixed infection as compared to a single one, suggesting co-infecting viroids may result in interference with host factors more prominently. Collectively, our results provide a deep transcriptome of hop and insight into complex single HLVd, CBCVd, and their coinfection in hop-plant interactions

Highlights

Viroids are the smallest known non-encapsidated infectious pathogens, consisting of covalently closed single-stranded RNA molecules ranging in size from 239 to 401 nucleotides, depending on the particular viroid species [1]
Humulus lupulus plants are hosts to several viroids among which Hop latent viroid (HLVd) and Citrus bark cracking viroid (CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop
Emerging evidence shows that viroid-specific small RNAs accumulate during viroid infection and are involved in transcriptional gene silencing via gene methylation [10], direct interaction with plant proteins or/and by viroid-induced RNA interference (RNAi) based post-transcriptional gene silencing (PTGS) [11]

Summary

Introduction

Viroids are the smallest known non-encapsidated infectious pathogens, consisting of covalently closed single-stranded RNA molecules ranging in size from 239 to 401 nucleotides (nt), depending on the particular viroid species [1]. HLVd is considered a common denominator in CBCVd-infected hop plants [19], which raises important questions about the interactions between these two viroids and the etiology of the disease, especially because single CBCVd infections of hop are not present in nature or hop fields In this context, knowledge of the impact of potential interaction of HLVd and CBCVd with hop immune responses is essential for the development of disease management strategies and the mechanisms that underlie severe stunting in hop in the mixed infection of HLVd and CBCVd. In this study, we employed comparative transcriptome profiling to investigate genome-wide changes in gene expression associated with individual HLVd and CBCVd hop infections. We have extended our study to investigate global changes in gene expression patterns in the association of HLVd-CBCVd coinfection, which could help in understanding the mechanisms that underlie the induction of more severe disease symptoms in mixed HLVd-CBCVd infection as compared to CBCVd single infection in hop

Results

Plant Inoculation and Disease Assessment

Transcriptome Assembly and Identification of Differentially Expressed Genes

Functional Annotation and Gene Enrichment Analysis