[Show abstract][Hide abstract] ABSTRACT: Previous attempts to develop RNAi-mediated viroid-resistant transgenic plants using nearly full-length Potato spindle tuber viroid (PSTVd) hairpin RNA (hpRNA) were successful; however unusual phenotypes resembling viroid infection occurred. Therefore, in the present work, transgenic Nicotiana benthamiana lines expressing both partial and truncated versions of PSTVd hpRNA were developed. Specifically, seven partial or truncated versions of PSTVd sequences were selected according to the hotspots of both PSTVd-sRNAs and functional domains of the PSTVd. A total of 21 transgenic lines Nicotiana benthamiana were developed under the control of either the CaMV-35S or the CoYMV promoters. All of the transgenic lines established here were monitored for the induction of phenotypic changes, for PSTVd-sRNA expression and for the resistance against PSTVd infection. Additionally, this study demonstrates the use of inverted repeat construct sequences as short as 26- to -49 nucleotides for both the efficient expression of the PSTVd-sRNA and the inhibition of PSTVd infection.
Full-text · Article · Dec 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: The dahlia isolate of potato spindle tuber viroid (PSTVd) accumulates slowly and induces mild disease symptoms in tomato (Solanum lycopersicum, cv. Rutgers) plants in contrast to the intermediate isolate (PSTVd-I). The dahlia isolate (PSTVd-D) differs from PSTVd-I in eight locations: 42 and 43 in the terminal left (TL); 64/65, 311, and 312/313 in the pathogenicity (P); 118 and 126 in the variable (V); and 201 in the terminal right (TR) domains. To investigate the molecular determinants in the PSTVd-D genome responsible for the attenuation of symptom severity and lower replication/accumulation in tomato plants, a series of mutants between PSTVd-D and PSTVd-I were constructed by focusing first on the mutations in the TL and P domains in the left-hand half of the molecule. Then, more detailed analysis was performed on the three mutations at positions 118, 126, and 201 in the V and TR domains. One of these mutations is located around the boundary of the right border of the RY-motif, a predicted recognition site of Virp1, a viroid-binding protein. Of 14 mutants (seven based on PSTVd-D and the other seven based on PSTVd-I) examined, 11 propagated stably and three lost infectivity. Mutations in the TL and P domains (42U, 43C, 310U/C, and U or UU insertion to 311/312 in PSTVd mild types) majorly influenced the expression of mild-like symptoms. In contrast, when each of the mutations at 118, 126, and 201 in the V and TR domains were exchanged independently, they minimally influenced systemic accumulation and symptom expression. Mutants based on PSTVd-D with PSTVd-I-type mutations at nucleotide positions 118, 126, and/or 201 showed mild symptoms similar to PSTVd-D, but their systemic accumulation was a little faster than PSTVd-D. In contrast, mutants based on PSTVd-I with PSTVd-D-type mutations at 118, 126, and/or 201 nucleotide positions showed severe symptoms similar to PSTVd-I, and the systemic accumulation was similar to or a little slower than PSTVd-I. The nucleotide at position 201 could be changed to U, G, or A, but C was not acceptable for replication. Because introduction of C at the position 201 can change the loop structure at the right boundary of the RY-motif's consensus sequence, the loop structure may influence recognition by Virp1.
[Show abstract][Hide abstract] ABSTRACT: In order to analyze the production of small RNA (sRNA) by viroids upon infecting the plants, the tomato plants (Solanumlycopersicum cultivar Rutgers) were inoculated with the variants of Potato spindle tuber viroid (PSTVd). After 21-days of post inoculation, total RNA was extracted and subjected for deep-sequencing using Illumina HiSeqplatform. The primers were trimmed and only 21- to 24-nt long sRNAs were filtered after quality check of the raw data. The filtered sRNA population was then mapped against both the genomic (+) andantigenomic(−) strands of the respective PSTVd variants using standard pattern-matching algorithm. The profiling of viroid derived sRNA (vd-sRNA) revealed that the viroids are susceptible to host RNA silencing mechanism.High-throughput sequence data linked to this project have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE69225.
[Show abstract][Hide abstract] ABSTRACT: Viroids like Potato spindle tuber viroid (PSTVd) are the smallest known agents of infectious disease - small, highly structured, circular RNA molecules that lack detectable messenger RNA activity yet are able to replicate autonomously and cause disease in susceptible plant species. To better understand the role of different hormone signaling pathways in disease induction, microarray analysis was used to compare changes in tomato gene expression associated with PSTVd infection in two tomato cultivars plus a third transformed line expressing small PSTVd siRNAs in the absence of viroid replication. Changes in mRNA levels for the sensitive cultivar 'Rutgers' were extensive, involving more than half of the ca. 10,000 genes present on the array. Effects on mRNAs encoding enzymes involved in gibberellin and brassinosteroid biosynthesis were accompanied by numerous changes affecting their respective signaling pathways. In the dwarf cultivar 'MicroTom', a marked upregulation of genes involved in response to stress and other stimuli was observed only when exogenous brassinosteroid was applied to infected plants, thereby providing the first evidence for the involvement of brassinosteroid-mediated signaling in viroid disease induction.
No preview · Article · Feb 2015 · Acta horticulturae
[Show abstract][Hide abstract] ABSTRACT: Viroids are noncoding RNA pathogens inducing severe to mild disease symptoms on agriculturally important crop plants. Viroid replication is entirely dependent on host transcription machinery, and their replication/accumulation in the infected cells can activate RNA silencing-a host defense mechanism that targets the viroid itself. RNA silencing produces in the cell large amounts of viroid-specific small RNAs of 21-24-nucleotides by cleaving (or "dicing") entire molecules of viroid RNA. However, viroid replication is resistant to the effects of RNA silencing and disrupts the normal regulation of host gene expression, finally resulting in the development of disease symptoms on infected plant.The molecular mechanisms of biological processes involving RNA silencing and underlying various aspects of viroid-host interaction, such as symptom expression, are of special interests to both basic and applied areas of viroid research. Here we present a method to create infectious viroid cDNA clones and RNA transcripts, the starting material for such analyses, using Hop stunt viroid as an example. Next we describe methods for the preparation and analysis of viroid-specific small RNAs by deep sequencing using tomato plants infected with Potato spindle tuber viroid as an example. Finally we introduce bioinformatics tools and methods necessary to process, analyze, and characterize these viroid-specific small RNAs. These bioinformatic methods provide a powerful new tool for the detection and discovery of both known and new viroid species.
Full-text · Article · Jan 2015 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The dahlia isolate of Potato spindle tuber viroid (PSTVd-D) shares 97 % sequence homology with PSTVd-intermediate (PSTVd-I), but differs at eight positions in the nucleotide sequence from PSTVd-I: five substitutions at positions 42, 43, 127, 202, and 311, two insertions at 63/64 and 312/313, and one deletion at 119. PSTVd-D accumulates slowly and induces very mild symptoms in tomato (cv. Rutgers) plants. In contrast, PSTVd-I propagates faster and induces severe symptoms. Here we used deep-sequencing analysis of PSTVd-specific small RNAs (PSTVd-sRNA) that accumulate in PSTVd-I- and PSTVd-D-infected tomato plants to reveal that the number of PSTVd-sRNA reads extensively decreased in PSTVd-D-infected leaf and stem tissues, especially those derived from the regions containing the nucleotides 119, 127, and 202, in which the nucleotide sequence differed between the severe and mild symptom-inducing isolates. In comparison with healthy controls, relative expression levels (i.e., number of reads by deep sequencing) of various host microRNAs changed after infection with PSTVd-I and PSTVd-D. The relative abundance of miR159 and miR162 in PSTVd-I- and PSTVd-D-infected leaf and stem tissues decreased to nearly 50 % of that in healthy tissues. In PSTVd-I- and PSTVd-D-infected stem tissues, miR319, which is approximately five times more abundant in stem tissues than in leaves, also decreased to 33–63 % of that in healthy controls.
Full-text · Article · Jan 2015 · Journal of General Plant Pathology
[Show abstract][Hide abstract] ABSTRACT: Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of next-generation sequencing and bioinformatics for known and previously unrecognized viroids.
Full-text · Article · Sep 2014 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: Australian grapevine viroid (AGVd) is a viroid specific to grapevine with the least records in the world till date. Here, we report for the first time the presence of AGVd in grapevines in Indian sub-continent. The overall infection rate of AGVd in major grapevine producing areas in India was 9.3 %, which is conspicuously higher than the other regions of the world except for Tunisia and Iran. To understand the AGVd diversity in India, the genetic divergence was examined based on the disparity in the cultivars and the locations. Nucleotide sequence analysis revealed the existence of five major AGVd variants in India besides other 44 minor variants implying the "quasi-species" nature. Further, sequence alignment of all the Indian AGVd variants along with Australian type species underscored the presence of eleven mutation points which are archetypal for Indian AGVd, irrespective of the region, and cultivar of grapevines. Plotting of Indian AGVd sequence variants against Australian type species unveiled that all these eleven mutations are distributed on upper and lower left terminal and pathogenicity regions of the molecule. Phylogenetic analysis divulged all the major Indian AGVd variants formed two distinct clusters, suggesting the two separate evolutionary lineages of AGVd in Indian viticulture.
[Show abstract][Hide abstract] ABSTRACT: Apple scar skin viroid (ASSVd) is a fruit-damaging pathogen that causes significant economic losses to pome fruit trees. In the context of a survey on fruit tree viroids in Greece, ASSVd was initially detected by RTPCR in two sweet cherry trees of cv. Tragana Edessis in an apple orchard in Florina (Macedonia, Greece). This finding was confirmed by direct viroid sequencing of the amplified RT-PCR products. In order to verify this finding, we further examined four sweet cherry trees cvs Tragana Edessis and Biggareau Burlat, two sweet cherry trees of undetermined cultivar, and fifteen neighboring apple trees in the same orchard for possible infection with ASSVd. The viroid assay was done by tissue print hybridization using an ASSVd-specific DIG-labeled probe at stringent hybridization conditions and by RTPCR using two different ASSVd-specific primer pairs. ASSVd was detected in the six sweet cherry trees, including symptomatic samples, but not in any of the 15 apple trees. Purified ASSVd-positive RT-PCR product s from sweet cherries were sequenced either directly or after cloning into pGEM-T or pCR II plasmid vectors. Sixteen ASSVd sequences obtained from five trees were 327-340 nucleotide long and shared 96-99% identity with ASSVd isolates from Indian apples. There was no cherry-specific nucleotide changes in the ASSVd sequences obtained. The viroid was graft transmitted successfully from cherry trees to cherry rootstocks and the newly developed rootstock leaves were ASSVd-positive by RT-PCR. To our knowledge, this is the first molecular and biological analyses of ASSVd infecting sweet cherry trees.
No preview · Article · Jul 2013 · JOURNAL OF PLANT PATHOLOGY