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Viroids are circular, highly structured, single-stranded, non-coding RNA pathogens known to infect and cause disease in several plant species. They are known to trigger the host plant’s RNA silencing machinery. The detection of viroid-derived small RNAs (vd-sRNA) in viroid-infected host plants opened a new avenue of study in host–viroid pathogenicity. Since then, several viroid research groups have studied the vd-sRNA retrieved from different host–viroid combinations. Such studies require the segregation of 21- to 24-nucleotide long small RNAs (sRNA) from a deep-sequencing databank, followed by separating the vd-sRNA from any sRNA within this group that showed sequence similarity with either the genomic or the antigenomic strands of the viroid. Such mapped vd-sRNAs are then profiled on both the viroid’s genomic and antigenomic strands for visualization. Although several commercial interfaces are currently available for this purpose, they are all programmed for linear RNA molecules. Hence, viroid researchers must develop a computer program that accommodates the sRNAs derived from the circular viroid genome. This is a laborious process, and consequently, it often creates a bottleneck for biologists. In order to overcome this constraint, and to help the research community in general, in this study, a python-based pattern matching interface was developed so as to be able to both profile and map sRNAs on a circular genome. A “matching tolerance” feature has been included in the program, thus permitting the mapping of the sRNAs derived from the quasi-species. Additionally, the “topology” feature allows the researcher to profile sRNA derived from both linear and circular RNA molecules. The efficiency of the program was tested using previously reported deep-sequencing data obtained from two independent studies. Clearly, this novel software should be a key tool with which to both evaluate the production of sRNA and to profile them on their target RNA species, irrespective of the topology of the target RNA molecule.
Charith Adkar-Purushothama; Pavithran Iyer; Teruo Sano; Jean-Pierre Perreault. sRNA Profiler: A User-Focused Interface for Small RNA Mapping and Profiling. Cells 2021, 10, 1771 .
AMA StyleCharith Adkar-Purushothama, Pavithran Iyer, Teruo Sano, Jean-Pierre Perreault. sRNA Profiler: A User-Focused Interface for Small RNA Mapping and Profiling. Cells. 2021; 10 (7):1771.
Chicago/Turabian StyleCharith Adkar-Purushothama; Pavithran Iyer; Teruo Sano; Jean-Pierre Perreault. 2021. "sRNA Profiler: A User-Focused Interface for Small RNA Mapping and Profiling." Cells 10, no. 7: 1771.
Quebec is the third largest wine grape producer in Canada in acreage, tonnage, and wine grape sales (Carisse et al. 2017; Ben Moussa et al. 2019). To evaluate the diversity of viruses infecting grapevine in Quebec, a total of 77 leaf tissue samples (cv. Vidal) were collected from July to October in 2020 in three different vineyards located in Frelighsburg, Hemmingford and Saint-Jacques-le-Mineur in Quebec, Canada. Double-stranded RNA was extracted from each sample and used for cDNA library preparation with the Nextera XT DNA Library Preparation Kit (Illumina) as described previously (Kesanakurti et al. 2016). High-throughput sequencing (HTS, 2x300 bp) was conducted on dual-indexed libraries in a v3 flow cell using the Illumina MiSeq platform (Adkar-Purushothama et al. 2020). The obtained raw FASTQ data was de-multiplexed into 154 separate sequence files, and the adapters and barcode sequences were trimmed. The quality of the sequences was verified using Trimmomatic V.0.32 and the “clean” sequences were analyzed using Virtool and VirFind virus detection pipelines described elsewhere (Ho and Tzanetakis 2014; Rott et al. 2017) to screen for all possible viruses in the databases. Over 100,000 reads per sample were obtained with a percentage of mapped viral reads ranging from 1.47 to 19.43% of total number of reads. Out of 77 samples, 16 revealed the sequence of grapevine yellow speckle viroid 1 (GYSVd-1), for which the length coverage ranged from 98.5 to 99.1%; the depth ranged from 2X to 856X. The GYSVd-1 positive sequence files were subjected to whole genome assembly on CLC genomics Workbench v20.0.4 with the isolate SY-BR from Brazil (KU880715) used as reference. Seven complete genomes of GYSVd-1 of 366-368 nucleotides (nt) in size were deposited (GenBank Acc. MW732682 to MW732688). BLASTN analysis of the sequences showed 98-100% nt identities with isolate SY-BR. Other viruses and viroids such as Grapevine fleck virus, Grapevine rupestris stem pitting-associated virus, Grapevine rupestris vein feathering virus and Hop stunt viroid were also detected. To confirm GYSVd-1 presence in Quebec vineyards, seven of the 16 HTS-positive grapevine leaf tissue samples were subjected to total RNA extraction, followed by RT-PCR assay as before (Adkar-Purushothama et al. 2015; Sahana et al. 2013); all were positive by RT-PCR. The PCR products were directly Sanger-sequenced, and they showed 100% nt identity to the HTS derived sequences. Three of the seven GYSVd-1 positive grapevines exhibited yellow leaf spots and flecks and tiny yellow leaves, but their mixed infection status makes definitive symptoms association difficult to determine. Previously, Hop stunt viroid was reported from grapevines in Canada (Xiao et al. 2019; Fall et al. 2020) but to the best of our knowledge, this is the first report of GYSVd-1 infecting grapevines in Canada, specifically in the province of Quebec. Further research is required to assess the GYSVd-1 related yield loss. Monitoring and testing for GYSVd-1 infection is necessary to prevent propagation of infected materials, spread, and potential negative impact for the Canadian grapevine industry.
Dong Xu; Charith Raj Adkar-Purushothama; Pierre Lemoyne; Jean Pierre Perreault; Mamadou Fall. First report of Grapevine yellow speckle viroid 1 infecting grapevine (Vitis vinifera L.) in Canada. Plant Disease 2021, 1 .
AMA StyleDong Xu, Charith Raj Adkar-Purushothama, Pierre Lemoyne, Jean Pierre Perreault, Mamadou Fall. First report of Grapevine yellow speckle viroid 1 infecting grapevine (Vitis vinifera L.) in Canada. Plant Disease. 2021; ():1.
Chicago/Turabian StyleDong Xu; Charith Raj Adkar-Purushothama; Pierre Lemoyne; Jean Pierre Perreault; Mamadou Fall. 2021. "First report of Grapevine yellow speckle viroid 1 infecting grapevine (Vitis vinifera L.) in Canada." Plant Disease , no. : 1.
Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen.
Shusuke Kawakubo; Fangluan Gao; Shifang Li; Zhongyang Tan; Ying-Kun Huang; Charith Raj Adkar-Purushothama; Chennappa Gurikar; Phoowanarth Maneechoat; Pissawan Chiemsombat; Seint San Aye; Naruto Furuya; Oleksiy Shevchenko; Josef Špak; Dijana Škorić; Simon Y. W. Ho; Kazusato Ohshima. Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road. Proceedings of the National Academy of Sciences 2021, 118, 1 .
AMA StyleShusuke Kawakubo, Fangluan Gao, Shifang Li, Zhongyang Tan, Ying-Kun Huang, Charith Raj Adkar-Purushothama, Chennappa Gurikar, Phoowanarth Maneechoat, Pissawan Chiemsombat, Seint San Aye, Naruto Furuya, Oleksiy Shevchenko, Josef Špak, Dijana Škorić, Simon Y. W. Ho, Kazusato Ohshima. Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road. Proceedings of the National Academy of Sciences. 2021; 118 (12):1.
Chicago/Turabian StyleShusuke Kawakubo; Fangluan Gao; Shifang Li; Zhongyang Tan; Ying-Kun Huang; Charith Raj Adkar-Purushothama; Chennappa Gurikar; Phoowanarth Maneechoat; Pissawan Chiemsombat; Seint San Aye; Naruto Furuya; Oleksiy Shevchenko; Josef Špak; Dijana Škorić; Simon Y. W. Ho; Kazusato Ohshima. 2021. "Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road." Proceedings of the National Academy of Sciences 118, no. 12: 1.
While the potato spindle tuber viroid (PSTVd) variant, PSTVd-Dahlia (PSTVd-D or PSTVd-Dwt) induces very mild symptoms in tomato cultivar ‘Rutgers’, PSTVd-Intermediate (PSTVd-I or PSTVd-Iwt) induces severe symptoms. These two variants differ by nine nucleotides, of which six mutations are located in the terminal left (TL) to the pathogenicity (P) domains. To evaluate the importance of mutations located in the TL to the P domains, ten types of point mutants were created by swapping the nucleotides between the two viroid variants. Bioassay in tomato plants demonstrated that two mutants created on PSTVd-Iwt at positions 42 and 64 resulted in symptom attenuation. Phenotypic and RT-qPCR analysis revealed that mutation at position 42 of PSTVd-Iwt significantly reduced disease severity and accumulation of the viroid, whereas mutation at position 64 showed a significant reduction in stunting when compared to the PSTVd-Iwt infected plant. RT-qPCR analysis on pathogenesis-related protein 1b1 and chalcone synthase genes showed a direct correlation with symptom severity whereas the expansin genes were down-regulated irrespective of the symptom severity. These results indicate that the nucleotides at positions 42 and 64 are in concert with the ones at positions 43, 310, and 311/312, which determines the slower and stable accumulation of PSTVd-D without eliciting excessive host defense responses thus contributing in the attenuation of disease symptom.
Shoya Kitabayashi; Daiki Tsushima; Charith Raj Adkar-Purushothama; Teruo Sano. Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid. International Journal of Molecular Sciences 2020, 21, 7352 .
AMA StyleShoya Kitabayashi, Daiki Tsushima, Charith Raj Adkar-Purushothama, Teruo Sano. Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid. International Journal of Molecular Sciences. 2020; 21 (19):7352.
Chicago/Turabian StyleShoya Kitabayashi; Daiki Tsushima; Charith Raj Adkar-Purushothama; Teruo Sano. 2020. "Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid." International Journal of Molecular Sciences 21, no. 19: 7352.
The early 1970s marked two breakthroughs in the field of biology: (i) The development of nucleotide sequencing technology; and, (ii) the discovery of the viroids. The first DNA sequences were obtained by two-dimensional chromatography which was later replaced by sequencing using electrophoresis technique. The subsequent development of fluorescence-based sequencing method which made DNA sequencing not only easier, but many orders of magnitude faster. The knowledge of DNA sequences has become an indispensable tool for both basic and applied research. It has shed light biology of viroids, the highly structured, circular, single-stranded non-coding RNA molecules that infect numerous economically important plants. Our understanding of viroid molecular biology and biochemistry has been intimately associated with the evolution of nucleic acid sequencing technologies. With the development of the next-generation sequence method, viroid research exponentially progressed, notably in the areas of the molecular mechanisms of viroids and viroid diseases, viroid pathogenesis, viroid quasi-species, viroid adaptability, and viroid–host interactions, to name a few examples. In this review, the progress in the understanding of viroid biology in conjunction with the improvements in nucleotide sequencing technology is summarized. The future of viroid research with respect to the use of third-generation sequencing technology is also briefly envisaged.
Charith Raj Adkar-Purushothama; Jean-Pierre Perreault. Impact of Nucleic Acid Sequencing on Viroid Biology. International Journal of Molecular Sciences 2020, 21, 5532 .
AMA StyleCharith Raj Adkar-Purushothama, Jean-Pierre Perreault. Impact of Nucleic Acid Sequencing on Viroid Biology. International Journal of Molecular Sciences. 2020; 21 (15):5532.
Chicago/Turabian StyleCharith Raj Adkar-Purushothama; Jean-Pierre Perreault. 2020. "Impact of Nucleic Acid Sequencing on Viroid Biology." International Journal of Molecular Sciences 21, no. 15: 5532.
RNA-dependent RNA polymerase 6 (RDR6) is one of the key factors in plant defense responses and suppresses virus or viroid invasion into shoot apical meristem (SAM) in Nicotiana benthamiana. To evaluate the role of Solanum lycopersicum (Sl) RDR6 upon viroid infection, SlRDR6-suppressed (SlRDR6i) ‘Moneymaker’ tomatoes were generated by RNA interference and inoculated with intermediate or lethal strain of potato spindle tuber viroid (PSTVd). Suppression of SlRDR6 did not change disease symptoms of both PSTVd strains in ‘Moneymaker’ tomatoes. Analysis of PSTVd distribution in shoot apices by in situ hybridization revealed that both PSTVd strains similarly invade the basal part but not apical part including pluripotent stem cells of SAM in SlRDR6i plants at a low rate unlike a previous report in N. benthamiana. In addition, unexpectedly, amount of PSTVd accumulation was apparently lower in SlRDR6i plants than in control tomatoes transformed with empty cassette in early infection especially in the lethal strain. Meanwhile, SlRDR6 suppression did not affect the seed transmission rates of PSTVd. These results indicate that RDR6 generally suppresses PSTVd invasion into SAM in plants, while suppression of RDR6 does not necessarily elevate amount of PSTVd accumulation. Additionally, our results suggest that host factors such as RDR1 other than RDR6 may also be involved in the protection of SAM including pluripotent stem cells from PSTVd invasion and effective RNA silencing causing the decrease of PSTVd accumulation during early infection in tomato plants.
Takashi Naoi; Syoya Kitabayashi; Atsushi Kasai; Kohei Sugawara; Charith Raj Adkar-Purushothama; Mineo Senda; Tatsuji Hataya; Teruo Sano. Suppression of RNA-dependent RNA polymerase 6 in tomatoes allows potato spindle tuber viroid to invade basal part but not apical part including pluripotent stem cells of shoot apical meristem. PLOS ONE 2020, 15, e0236481 .
AMA StyleTakashi Naoi, Syoya Kitabayashi, Atsushi Kasai, Kohei Sugawara, Charith Raj Adkar-Purushothama, Mineo Senda, Tatsuji Hataya, Teruo Sano. Suppression of RNA-dependent RNA polymerase 6 in tomatoes allows potato spindle tuber viroid to invade basal part but not apical part including pluripotent stem cells of shoot apical meristem. PLOS ONE. 2020; 15 (7):e0236481.
Chicago/Turabian StyleTakashi Naoi; Syoya Kitabayashi; Atsushi Kasai; Kohei Sugawara; Charith Raj Adkar-Purushothama; Mineo Senda; Tatsuji Hataya; Teruo Sano. 2020. "Suppression of RNA-dependent RNA polymerase 6 in tomatoes allows potato spindle tuber viroid to invade basal part but not apical part including pluripotent stem cells of shoot apical meristem." PLOS ONE 15, no. 7: e0236481.
Viroids are non-coding RNA plant pathogens that are characterized by their possession of a high mutation level. Although the sequence heterogeneity in viroid infected plants is well understood, shifts in viroid population dynamics due to mutations over the course of infection remain poorly understood. In this study, the ten most abundant sequence variants of potato spindle tuber viroid RG1 (PSTVd) expressed at different time intervals in PSTVd infected tomato plants were identified by high-throughput sequencing. The sequence variants, forming a quasi-species, were subjected to both the identification of the regions favoring mutations and the effect of the mutations on viroid secondary structure and viroid derived small RNAs (vd-sRNA). At week 1 of PSTVd infection, 25% of the sequence variants were similar to the “master” sequence (i.e., the sequence used for inoculation). The frequency of the master sequence within the population increased to 70% at week 2 after PSTVd infection, and then stabilized for the rest of the disease cycle (i.e., weeks 3 and 4). While some sequence variants were abundant at week 1 after PSTVd infection, they tended to decrease in frequency over time. For example, the variants with insertions at positions 253 or 254, positions that could affect the Loop E as well as the metastable hairpin I structure that has been shown important during replication and viroid infectivity, resulted in decreased frequency. Data obtained by in silico analysis of the viroid derived small RNAs (vd-sRNA) was also analyzed. A few mutants had the potential of positively affecting the viroid’s accumulation by inducing the RNA silencing of the host’s defense related genes. Variants with mutations that could negatively affect viroid abundance were also identified because their derived vd-sRNA were no longer capable of targeting any host mRNA or of changing its target sequence from a host defense gene to some other non-important host gene. Together, these findings open avenues into understanding the biological role of sequence variants, this viroid’s interaction with host components, stable and metastable structures generated by mutants during the course of infection, and the influence of sequence variants on stabilizing viroid population dynamics.
Charith Raj Adkar-Purushothama; François Bolduc; Pierrick Bru; Jean-Pierre Perreault. Insights Into Potato Spindle Tuber Viroid Quasi-Species From Infection to Disease. Frontiers in Microbiology 2020, 11, 1235 .
AMA StyleCharith Raj Adkar-Purushothama, François Bolduc, Pierrick Bru, Jean-Pierre Perreault. Insights Into Potato Spindle Tuber Viroid Quasi-Species From Infection to Disease. Frontiers in Microbiology. 2020; 11 ():1235.
Chicago/Turabian StyleCharith Raj Adkar-Purushothama; François Bolduc; Pierrick Bru; Jean-Pierre Perreault. 2020. "Insights Into Potato Spindle Tuber Viroid Quasi-Species From Infection to Disease." Frontiers in Microbiology 11, no. : 1235.
Viroids are one of the most enigmatic highly structured, circular, single‐stranded RNA phytopathogens. Although they are not known to code for any peptide, viroids induce visible symptoms in susceptible host plants that resemble those associated with many plant viruses. It is known that viroids induce disease symptoms by direct interaction with host factors; however, the precise mechanism by which this occurs remains poorly understood. Studies on the host's responses to viroid infection, host susceptibility and nonhost resistance have been underway for several years, but much remains to be done in order to fully understand the complex nature of viroid–host interactions. Recent progress using molecular biology techniques combined with computational algorithms, in particular evidence of the role of viroid‐derived small RNAs in the RNA silencing pathways of a disease network, has widened the knowledge of viroid pathogenicity. The complexity of viroid–host interactions has been revealed in the past decades to include, but not be limited to, the involvement of host factors, viroid structural complexity, and viroid‐induced ribosomal stress, which is further boosted by the discovery of long noncoding RNAs (lncRNAs). In this review, the current understanding of the viroid–host interaction has been summarized with the goal of simplifying the complexity of viroid biology for future research. This article is categorized under: RNA in Disease and Development > RNA in Disease
Charith Raj Adkar‐Purushothama; Jean‐Pierre Perreault. Current overview on viroid–host interactions. WIREs RNA 2019, 11, e1570 .
AMA StyleCharith Raj Adkar‐Purushothama, Jean‐Pierre Perreault. Current overview on viroid–host interactions. WIREs RNA. 2019; 11 (2):e1570.
Chicago/Turabian StyleCharith Raj Adkar‐Purushothama; Jean‐Pierre Perreault. 2019. "Current overview on viroid–host interactions." WIREs RNA 11, no. 2: e1570.
Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.
Patrick Cottilli; Borja Belda-Palazon; Charith Raj Adkar-Purushothama; Jean-Pierre Perreault; Enrico Schleiff; Ismael Rodrigo; Alejandro Ferrando; Purificación Lisón. Citrus exocortis viroid causes ribosomal stress in tomato plants. Nucleic Acids Research 2019, 47, 8649 -8661.
AMA StylePatrick Cottilli, Borja Belda-Palazon, Charith Raj Adkar-Purushothama, Jean-Pierre Perreault, Enrico Schleiff, Ismael Rodrigo, Alejandro Ferrando, Purificación Lisón. Citrus exocortis viroid causes ribosomal stress in tomato plants. Nucleic Acids Research. 2019; 47 (16):8649-8661.
Chicago/Turabian StylePatrick Cottilli; Borja Belda-Palazon; Charith Raj Adkar-Purushothama; Jean-Pierre Perreault; Enrico Schleiff; Ismael Rodrigo; Alejandro Ferrando; Purificación Lisón. 2019. "Citrus exocortis viroid causes ribosomal stress in tomato plants." Nucleic Acids Research 47, no. 16: 8649-8661.
To date, two plant genes encoding RNA-dependent RNA polymerases (RdRs) that play major roles in the defense against RNA viruses have been identified: (i) RdR1, which is responsible for the viral small RNAs (vsRNAs) found in virus-infected plants, and, (ii) RdR6, which acts as a surrogate in the absence of RdR1. In this study, the role of RdR6 in the defense against viroid infection was examined by knock-down of RdR6 followed by potato spindle tuber viroid (PSTVd) infection. The suppression of RdR6 expression increased the plant’s growth, as was illustrated by the plant’s increased height. PSTVd infection of RdR6 compromised plants resulted in an approximately three-fold increase in the accumulation of viroid RNA as compared to that seen in control plants. Additionally, RNA gel blot assay revealed an increase in the number of viroids derived small RNAs in RdR6 suppressed plants as compared to control plants. These data provide a direct correlation between RdR6 and viroid accumulation and indicate the role of RDR6 in the plant’s susceptibility to viroid infection.
Charith Raj Adkar-Purushothama; Jean-Pierre Perreault. Suppression of RNA-Dependent RNA Polymerase 6 Favors the Accumulation of Potato Spindle Tuber Viroid in Nicotiana Benthamiana. Viruses 2019, 11, 345 .
AMA StyleCharith Raj Adkar-Purushothama, Jean-Pierre Perreault. Suppression of RNA-Dependent RNA Polymerase 6 Favors the Accumulation of Potato Spindle Tuber Viroid in Nicotiana Benthamiana. Viruses. 2019; 11 (4):345.
Chicago/Turabian StyleCharith Raj Adkar-Purushothama; Jean-Pierre Perreault. 2019. "Suppression of RNA-Dependent RNA Polymerase 6 Favors the Accumulation of Potato Spindle Tuber Viroid in Nicotiana Benthamiana." Viruses 11, no. 4: 345.
Viroid infection often leads to early flowering in the host plant. This report describes the targeting of the FRIGIDA‐Like protein 3 (FRL3) mRNA in tomato plants by a small RNA derived from the conserved left terminal region of the potato spindle tuber viroid. This targeting lead to the silencing of the FRL3 mRNA. Viroid infection assays using a severe variant of PSTVd induced early flowering in tomato plants by down‐regulating greater amounts of the target than did a mild PSTVd variant. The targeting of the FRL3 mRNA by RNA silencing was validated by both an artificial microRNA experiment transiently expressing viroid derived small RNAs in tomato plants, and by 5’ RNA ligase mediated RACE. These data unambiguously demonstrated the role of small RNAs in the early flowering seen in the viroid infected plants. This article is protected by copyright. All rights reserved.
Charith Raj Adkar-Purushothama; Teruo Sano; Jean-Pierre Perreault. Viroid-derived small RNA induces early flowering in tomato plants by RNA silencing. Molecular Plant Pathology 2018, 19, 2446 -2458.
AMA StyleCharith Raj Adkar-Purushothama, Teruo Sano, Jean-Pierre Perreault. Viroid-derived small RNA induces early flowering in tomato plants by RNA silencing. Molecular Plant Pathology. 2018; 19 (11):2446-2458.
Chicago/Turabian StyleCharith Raj Adkar-Purushothama; Teruo Sano; Jean-Pierre Perreault. 2018. "Viroid-derived small RNA induces early flowering in tomato plants by RNA silencing." Molecular Plant Pathology 19, no. 11: 2446-2458.
Understanding in intimate details how the viroid interaction with host's defense genes is a cornerstone for developing viroid resistant plants. In this present study, small RNAs (sRNA) derived from Potato spindle tuber viroid (PSTVd) were studied in silico in order to detect any interactions with the serine threonine kinase receptor, a transmembrane protein that plays a role in disease resistance in plants. Using molecular biology techniques, it was determined that PSTVd infection negatively affects at least three serine threonine kinase receptors as well as with three other genes that are known to be involved in the overall development of the tomato plants. The transient expression of these putative PSTVd-sRNAs, using the microRNA sequence as a backbone, in tomato plants induced phenotypes similar to viroid infection. Mutants created by altering the sequence of PSTVd in these regions failed to infect the tomato plant. The data presented here illustrates the importance of these regions in viroid survival, and suggests a possible avenue of exploration for the development of viroid resistant plants.
Charith Raj Adkar-Purushothama; Jean-Pierre Perreault. Alterations of the viroid regions that interact with the host defense genes attenuate viroid infection in host plant. RNA Biology 2018, 15, 955 -966.
AMA StyleCharith Raj Adkar-Purushothama, Jean-Pierre Perreault. Alterations of the viroid regions that interact with the host defense genes attenuate viroid infection in host plant. RNA Biology. 2018; 15 (7):955-966.
Chicago/Turabian StyleCharith Raj Adkar-Purushothama; Jean-Pierre Perreault. 2018. "Alterations of the viroid regions that interact with the host defense genes attenuate viroid infection in host plant." RNA Biology 15, no. 7: 955-966.
5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) is a widely-accepted method for the validation of direct cleavage of a target gene by a microRNA (miRNA) and viroid-derived small RNA (vd-sRNA). However, this method cannot be used if cleavage takes place in the 3' extremity of the target RNA, as this gives insufficient sequence length to design nested PCR primers for 5' RLM RACE. To overcome this hurdle, we have developed 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE). In this method, an oligonucleotide adapter having 5' adenylated and 3' blocked is ligated to the 3' end of the cleaved RNA followed by PCR amplification using gene specific primers. In other words, in 3' RLM RACE, 3' end is mapped using 5' fragment instead of small 3' fragment. The method developed here was verified by examining the bioinformatics predicted and parallel analysis of RNA ends (PARE) proved cleavage sites of chloride channel protein CLC-b-like mRNA in Potato spindle tuber viroid infected tomato plants. The 3' RLM RACE developed in this study has the potential to validate the miRNA and vd-sRNA mediated cleavage of mRNAs at its 3' untranslated region (3' UTR).
Charith Raj Adkar-Purushothama; Pierrick Bru; Jean-Pierre Perreault. 3′ RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3′ extremity of the host mRNA. Journal of Virological Methods 2017, 250, 29 -33.
AMA StyleCharith Raj Adkar-Purushothama, Pierrick Bru, Jean-Pierre Perreault. 3′ RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3′ extremity of the host mRNA. Journal of Virological Methods. 2017; 250 ():29-33.
Chicago/Turabian StyleCharith Raj Adkar-Purushothama; Pierrick Bru; Jean-Pierre Perreault. 2017. "3′ RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3′ extremity of the host mRNA." Journal of Virological Methods 250, no. : 29-33.
It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (−) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3′ UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection.
Charith Raj Adkar-Purushothama; Pavithran Sridharan Iyer; Jean-Pierre Perreault. Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants. Scientific Reports 2017, 7, 1 -12.
AMA StyleCharith Raj Adkar-Purushothama, Pavithran Sridharan Iyer, Jean-Pierre Perreault. Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants. Scientific Reports. 2017; 7 (1):1-12.
Chicago/Turabian StyleCharith Raj Adkar-Purushothama; Pavithran Sridharan Iyer; Jean-Pierre Perreault. 2017. "Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants." Scientific Reports 7, no. 1: 1-12.
Association of Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) with the Chrysanthemum plants exhibiting severe stunting, distinct yellow leaf mottling, and chlorosis was detected in the main chrysanthemum-growing regions of India. Sequence analysis of 90 cDNA clones obtained for CSVd and CChMVd, representing the chrysanthemum-growing regions of India, revealed the high degree of sequence variation throughout the genome under natural conditions. Additionally, all the analyzed CChMVd clones revealed the presence of UUUC in the tetraloop, a signature of symptomatic variants in susceptible cultivars. Phylogenetic analysis revealed that Indian CSVd is closely related to European isolates from ornamentals, whereas CChMVd clustered along with the isolates reported from the East Asian countries.
C. R. Adkar-Purushothama; G. Chennappa; K. Poornachandra Rao; M. Y. Sreenivasa; P. K. Maheshwar; M. N. Nagendra Prasad; T. Sano. Molecular diversity among viroids infecting chrysanthemum in India. Virus Genes 2017, 53, 636 -642.
AMA StyleC. R. Adkar-Purushothama, G. Chennappa, K. Poornachandra Rao, M. Y. Sreenivasa, P. K. Maheshwar, M. N. Nagendra Prasad, T. Sano. Molecular diversity among viroids infecting chrysanthemum in India. Virus Genes. 2017; 53 (4):636-642.
Chicago/Turabian StyleC. R. Adkar-Purushothama; G. Chennappa; K. Poornachandra Rao; M. Y. Sreenivasa; P. K. Maheshwar; M. N. Nagendra Prasad; T. Sano. 2017. "Molecular diversity among viroids infecting chrysanthemum in India." Virus Genes 53, no. 4: 636-642.
G Chennappa; M K Naik; C R Adkar-Purushothama; Y S Amaresh; M Y Sreenivasa. PGP potential, abiotic stress tolerance and antifungal activity of Azotobacter strains isolated from paddy soils. Indian journal of experimental biology 2016, 54, 1 .
AMA StyleG Chennappa, M K Naik, C R Adkar-Purushothama, Y S Amaresh, M Y Sreenivasa. PGP potential, abiotic stress tolerance and antifungal activity of Azotobacter strains isolated from paddy soils. Indian journal of experimental biology. 2016; 54 (5):1.
Chicago/Turabian StyleG Chennappa; M K Naik; C R Adkar-Purushothama; Y S Amaresh; M Y Sreenivasa. 2016. "PGP potential, abiotic stress tolerance and antifungal activity of Azotobacter strains isolated from paddy soils." Indian journal of experimental biology 54, no. 5: 1.
In the present study, fumonisin producing Fusarium verticillioides was specifically detected in pure cultures, cereal samples and plant materials by multiplex PCR using one forward VERTF-1 and two reverse primers VERTR and VERTF-2. A total of 326 Fusarium isolates were obtained from maize, sorghum, paddy wheat and pearl millet samples collected from different districts of Karnataka, India. All Fusarium species were subjected to single round of PCR with species specific and fumonisin specific primers which recorded 59.50% of F. verticillioides and 53.98% of fumonisin producing F. verticillioides. Maize samples recorded highest frequency 34.42% of fumonisin producing F. verticillioides followed by paddy 28.57% and sorghum 16.66%. Sensitivity of multiplex PCR experiment was conducted by whole grain experiment of the collected cereals, roots and leaves of the cereal samples by diluting the DNA 10 to 100 times in which 1:50, 1:75 and 1:100 diluted samples recorded positive. The developed multiplex PCR assay provided a powerful tool for the accurate detection, identification and discrimination of potential fumonisin producing F. verticillioides strains among the population. The present study is the first report of developing the multiplex PCR method for early detection of fumonisin producing F. verticillioides from cereal samples, pure cultures and plant parts.
Deepa Nagaraj; Charith Raj Adkar-Purushothama; Sreenivasa Marikunte Yanjarappa. Multiplex PCR for the early detection of fumonisin producing Fusarium verticillioides. Food Bioscience 2016, 13, 84 -88.
AMA StyleDeepa Nagaraj, Charith Raj Adkar-Purushothama, Sreenivasa Marikunte Yanjarappa. Multiplex PCR for the early detection of fumonisin producing Fusarium verticillioides. Food Bioscience. 2016; 13 ():84-88.
Chicago/Turabian StyleDeepa Nagaraj; Charith Raj Adkar-Purushothama; Sreenivasa Marikunte Yanjarappa. 2016. "Multiplex PCR for the early detection of fumonisin producing Fusarium verticillioides." Food Bioscience 13, no. : 84-88.
Fumonisin-producing Fusarium verticillioides was detected in cereal samples by semi-nested PCR using one forward, VERTF-1, and two reverse primers, VERTR and VERTF-2. A total of 326 Fusarium species isolated from maize, paddy, sorghum, wheat and pearl-millet samples were subjected to a first round of n-PCR with the species specific VERTF-1 and VERTR set of primers which recorded 59.50% of F. verticillioides. Further, second round of n-PCR scored 53.98% of fumonisin-producing F. verticillioides with fumonisin specific VERTF-1 and VERTF-2 set of primers. Maize samples recorded the highest frequency of fumonisin-producing F. verticillioides 40.98%, followed by paddy 33.33%, and sorghum 12.50%. Sensitivity of nested PCR was conducted by whole grain experiment of the cereals, roots and leaves of the cereals by diluting the DNA 10-100 times, in which 1:50, 1:75, and 1:100 diluted samples recorded positive. This method can be used for the early detection of fumonisin producing F. verticillioides occurring on cereals.
Deepa N.; Charith Raj Adkar-Purushothama; Sreenivasa M. Y.. Nested PCR Method for Early Detection of Fumonisin ProducingFusarium verticillioidesin Pure Cultures, Cereal Samples and Plant Parts. Food Biotechnology 2016, 30, 18 -29.
AMA StyleDeepa N., Charith Raj Adkar-Purushothama, Sreenivasa M. Y.. Nested PCR Method for Early Detection of Fumonisin ProducingFusarium verticillioidesin Pure Cultures, Cereal Samples and Plant Parts. Food Biotechnology. 2016; 30 (1):18-29.
Chicago/Turabian StyleDeepa N.; Charith Raj Adkar-Purushothama; Sreenivasa M. Y.. 2016. "Nested PCR Method for Early Detection of Fumonisin ProducingFusarium verticillioidesin Pure Cultures, Cereal Samples and Plant Parts." Food Biotechnology 30, no. 1: 18-29.
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.
Charith Raj Adkar-Purushothama; Atsushi Kasai; Kohei Sugawara; Hideki Yamamoto; Yuto Yamazaki; Ying-Hong He; Nobuyuki Takada; Hideki Goto; Sahori Shindo; Takeo Harada; Teruo Sano. RNAi mediated inhibition of viroid infection in transgenic plants expressing viroid-specific small RNAs derived from various functional domains. Scientific Reports 2015, 5, 17949 .
AMA StyleCharith Raj Adkar-Purushothama, Atsushi Kasai, Kohei Sugawara, Hideki Yamamoto, Yuto Yamazaki, Ying-Hong He, Nobuyuki Takada, Hideki Goto, Sahori Shindo, Takeo Harada, Teruo Sano. RNAi mediated inhibition of viroid infection in transgenic plants expressing viroid-specific small RNAs derived from various functional domains. Scientific Reports. 2015; 5 (1):17949.
Chicago/Turabian StyleCharith Raj Adkar-Purushothama; Atsushi Kasai; Kohei Sugawara; Hideki Yamamoto; Yuto Yamazaki; Ying-Hong He; Nobuyuki Takada; Hideki Goto; Sahori Shindo; Takeo Harada; Teruo Sano. 2015. "RNAi mediated inhibition of viroid infection in transgenic plants expressing viroid-specific small RNAs derived from various functional domains." Scientific Reports 5, no. 1: 17949.
Chrysanthemum chlorotic mottle viroid (CChMVd, genus Pelamoviroid, family Avsunviroidae) has been reported to infect chrysanthemum (Dendranthema x grandiflorum). CChMVd was first reported in 1967 in the cultivar Yellow Delaware in New York State, USA (Dimock et al. 1971). Presence of CChMVd has previously been recorded, based on the symptoms and bioassay, in India (Singh et al. 1978). Karnataka State of India is the prominent chrysanthemum-growing region, with 19,161 ha cultivated under this crop and accounting for more than 40% of country’s flower production. During late summer 2010 (May and June), chrysanthemum leaves with yellow-green mottling and chlorotic spots were noted, indicating the possible association with CChMVd in Karnataka. Total RNA was extracted using a 2% CTAB buffer from leaf samples of symptomatic plants (Adkar-Purushothama et al. 2013). For the detection of CChMVd, reverse transcription PCR (RT-PCR) was performed using PIII/PIV as described previously (De la Peña et al. 1999). PCR products of the expected sizes (∼398 to 401 bp) were cloned and sequenced. BLAST analysis of the obtained sequences confirmed the presence of CChMVd in Karnataka. In order to estimate the incidence of CChMVd, the same RT-PCR assay was performed on 80 randomly selected chrysanthemum leaf samples of four different cultivars (Redgold, Bangalore, Sarval, and CO-1) collected from four chrysanthemum-growing districts of Karnataka. A PCR product of the size expected for CChMVd was detected in 8 of 80 samples analyzed, accounting for the infection incidence of 10%. To identify the prominent CChMVd variants, 10 clones obtained from the four representative samples were sequenced. To confirm the sequence of the primer regions, an additional set of primers CCh1/CCh2 were used for RT-PCR (Hosokawa et al. 2005) with high-fidelity LA-Taq polymerase (Takara-Bio, Japan). Alignment of all 40 sequences revealed the presence of at least two major sequence variants of CChMVd: CChMVd-Ind-1 (GenBank Accession No. KP262531) and CChMVd-Ind-3 (KP262533). Both the sequence variants had UUUC sequences (nucleotide position 82 to 85) in the tetraloop of the CChMVd, which is often associated with induction of symptoms in susceptible chrysanthemum cultivars (De la Peña et al. 1999). CChMVd-Ind-1was found to differ from CChMVd-Ind-3 by 17 nucleotide substitutions. BLAST analysis of CChMVd-Ind-1 showed 98% sequence identity with the CChMVd strain reported from Spain (GenBank Accession No. FJ647515) and from Himachal Pradesh state of India (FN646404), whereas CChMVd-Ind-3 showed 96% sequence identity with CChMVd strain W2-4, isolated from China (HQ891014). To our knowledge, this is the first molecular evidence for the presence of CChMVd infecting chrysanthemum in Karnataka State, India. The high degree of sequence diversity reported in the Karnataka isolates of CChMVd will help to clarify the evolution of this viroid and its possible threat to important crops
C. R. Adkar-Purushothama; G. Chennappa; K. Poornachandra Rao; M. Y. Sreenivasa; M. N. Nagendra Prasad; P. K. Maheshwar; T. Sano. Molecular Identification of Chrysanthemum chlorotic mottle viroid Infecting Chrysanthemum in Karnataka, India. Plant Disease 2015, 99, 1868 .
AMA StyleC. R. Adkar-Purushothama, G. Chennappa, K. Poornachandra Rao, M. Y. Sreenivasa, M. N. Nagendra Prasad, P. K. Maheshwar, T. Sano. Molecular Identification of Chrysanthemum chlorotic mottle viroid Infecting Chrysanthemum in Karnataka, India. Plant Disease. 2015; 99 (12):1868.
Chicago/Turabian StyleC. R. Adkar-Purushothama; G. Chennappa; K. Poornachandra Rao; M. Y. Sreenivasa; M. N. Nagendra Prasad; P. K. Maheshwar; T. Sano. 2015. "Molecular Identification of Chrysanthemum chlorotic mottle viroid Infecting Chrysanthemum in Karnataka, India." Plant Disease 99, no. 12: 1868.