Integrative time-scale and multi-omic analysis of host-responses to Hop stunt viroid infection

Constricted by an extreme biological simplicity, viroids are compelled to subvert host regulatory networks in order to accomplish their infectious process. Most of the studies focused on the response to viroid infection have only addressed a specific host regulatory level and considered a unique infection time. Thus, much remains to be done if we want to understand the temporal evolution and complex nature of viroid-host interactions. Here we present an integrative analysis of the timing and intensity of the genome-wide alterations in cucumber plants infected with Hop stunt viroid (HSVd). Differential host transcriptome, sRNAnome and methylome were integrated to determine the temporal response to viroid-infection. Our results support that HSVd promotes a dynamic redesign of the cucumber regulatory pathways predominantly affecting specific regulatory layers at different infection phases. Remarkably, the initial response was characterized by a reconfiguration of the host transcriptome by differential exon usage, followed by a predominant down-regulation of the transcriptional activity possibly modulated by the host epigenetic changes associated to infection and characterized by an increased hypermethylation. The silencing of at least three cucumber transcripts potential targets of vd-sRNAs was also observed. The alteration in host sRNA and miRNA metabolism was marginal. We expect that these data constituting the first comprehensive map of the cucumber-response to HSVd could contribute to elucidate the molecular basis of the host alterations triggered by viroid infection.

Virus and Viroid-Derived Small RNAs as Modulators of Host Gene Expression: Molecular Insights Into Pathogenesis

Virus-derived siRNAs (vsiRNAs) generated by the host RNA silencing mechanism are effectors of plant’s defense response and act by targeting the viral RNA and DNA in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) pathways, respectively. Contrarily, viral suppressors of RNA silencing (VSRs) compromise the host RNA silencing pathways and also cause disease-associated symptoms. In this backdrop, reports describing the modulation of plant gene(s) expression by vsiRNAs via sequence complementarity between viral small RNAs (sRNAs) and host mRNAs have emerged. In some cases, silencing of host mRNAs by vsiRNAs has been implicated to cause characteristic symptoms of the viral diseases. Similarly, viroid infection results in generation of sRNAs, originating from viroid genomic RNAs, that potentially target host mRNAs causing typical disease-associated symptoms. Pathogen-derived sRNAs have been demonstrated to have the propensity to target wide range of genes including host defense-related genes, genes involved in flowering and reproductive pathways. Recent evidence indicates that vsiRNAs inhibit host RNA silencing to promote viral infection by acting as decoy sRNAs. Nevertheless, it remains unclear if the silencing of host transcripts by viral genome-derived sRNAs are inadvertent effects due to fortuitous pairing between vsiRNA and host mRNA or the result of genuine counter-defense strategy employed by viruses to enhance its survival inside the plant cell. In this review, we analyze the instances of such cross reaction between pathogen-derived vsiRNAs and host mRNAs and discuss the molecular insights regarding the process of pathogenesis.

Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection

Citrus exocortis viroid (CEVd) is known to cause different symptoms in citrus trees, and its mechanism of infection has been studied in tomato as an experimental host, producing ribosomal stress on these plants. Some of the symptoms caused by CEVd in tomato plants resemble those produced by the phytohormone ethylene. The present study is focused on elucidating the relationship between CEVd infection and ethylene on disease development. To this purpose, the ethylene insensitive Never ripe (Nr) tomato mutants were infected with CEVd, and several aspects such as susceptibility to infection, defensive response, ethylene biosynthesis and ribosomal stress were studied. Phenotypic characterization revealed higher susceptibility to CEVd in these mutants, which correlated with higher expression levels of both defense and ethylene biosynthesis genes, as well as the ribosomal stress marker SlNAC082. In addition, Northern blotting revealed compromised ribosome biogenesis in all CEVd infected plants, particularly in Nr mutants. Our results indicate a higher ethylene biosynthesis in Nr mutants and suggest an important role of this phytohormone in disease development and ribosomal stress caused by viroid infection.

Analysis of Virome by High-Throughput Sequencing Revealed Multiple Infection and Intra-Virus Diversity in a Single Grapevine Plant

The ribosomal-depleted total RNA from white-berry grapevine (Vitis vinifera, SK933) plant showing severe chlorosis and downrolling of leaves was used for the high-throughput sequencing (HTS) analysis in order to unravel the potential contribution of the viral pathogens to the symptomatology observed. The combination of de novo assembly and mapping of ca. 1.1 millions of HTS reads enabled to identify and characterise a complex viral/viroid infection involving Grapevine leafroll-associated virus-2 (GLRaV-2), Grapevine leafroll-associated virus-3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Syrah virus-1 (GSyV-1) and Hop stunt viroid (HSVd). The determined nearly complete genomes of GLRaV-2 SK933 showed its high genetic divergence from previously characterised isolates. In case of GRSPaV, two variants representing different evolutionary lineages have been identified in the plant. The results further pinpoint the complexity of grapevine viral diseases and show that mixed virus infection of grapevine is rather a rule than an exception.

Mapping the Gene Expression Spectrum of Mediator Subunits in Response to Viroid Infection in Plants

The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five viroid species (Apple fruit crinkle viroid (AFCVd), Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), Hop stunt viroid (HSVd), and Potato spindle tuber viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different viroid species, suggesting that the MED expression is viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.

Identification and Characterization of Stress-Responsive TAS3-Derived TasiRNAs in Melon

Small interfering RNAs (siRNA) are key regulators of gene expression that play essential roles in diverse biological processes. Trans-acting siRNAs (tasiRNAs) are a class of plant-endogenous siRNAs that lead the cleavage of nonidentical transcripts. TasiRNAs are usually involved in fine-tuning development. However, increasing evidence supports that tasiRNAs may be involved in stress response. Melon is a crop of great economic importance extensively cultivated in semiarid regions frequently exposed to changing environmental conditions that limit its productivity. However, knowledge of the precise role of siRNAs in general, and of tasiRNAs in particular, in regulating the response to adverse environmental conditions is limited. Here, we provide the first comprehensive analysis of computationally inferred melon-tasiRNAs responsive to two biotic (viroid-infection) and abiotic (cold treatment) stress conditions. We identify two TAS3-loci encoding to length (TAS3-L) and short (TAS3-S) transcripts. The TAS candidates predicted from small RNA-sequencing data were characterized according to their chromosome localization and expression pattern in response to stress. The functional activity of cmTAS genes was validated by transcript quantification and degradome assays of the tasiRNA precursors and their predicted targets. Finally, the functionality of a representative cmTAS3-derived tasiRNA (TAS3-S) was confirmed by transient assays showing the cleavage of ARF target transcripts.

Global Transcriptomic Analysis Reveals Insights into the Response of ‘Etrog’ Citron (Citrus medica L.) to Citrus Exocortis Viroid Infection

Citrus exocortis viroid (CEVd) is the causal agent of citrus exocortis disease. We employed CEVd-infected ‘Etrog’ citron as a system to study the feedback regulation mechanism using transcriptome analysis in this study. Three months after CEVd infection, the transcriptome of fresh leaves was analyzed, and 1530 differentially expressed genes were detected. The replication of CEVd in citron induced upregulation of genes encoding key proteins that were involved in the RNA silencing pathway such as Dicer-like 2, RNA-dependent RNA polymerase 1, argonaute 2, argonaute 7, and silencing defective 3, as well as those genes encoding proteins that are related to basic defense responses. Many genes involved in secondary metabolite biosynthesis and chitinase activity were upregulated, whereas other genes related to cell wall and phytohormone signal transduction were downregulated. Moreover, genes encoding disease resistance proteins, pathogenicity-related proteins, and heat shock cognate 70 kDa proteins were also upregulated in response to CEVd infection. These results suggest that basic defense and RNA silencing mechanisms are activated by CEVd infection, and this information improves our understanding of the pathogenesis of viroids in woody plants.