Subhas Hajeri | University of Florida (original) (raw)
Papers by Subhas Hajeri
Archives of Virology, Jan 9, 2023
Plant Disease, Dec 1, 2022
Vibrio parahaemolyticus is an important pathogenic bacterium in both food safety management and m... more Vibrio parahaemolyticus is an important pathogenic bacterium in both food safety management and mariculture. Rapid and accurate detection technologies are critical for effective control of its outbreak and spreading. Conventional technologies and polymerase chain reaction (PCR)-based approaches have limited usage because of the requirement of laboratory instruments and trained personnel. Using the isothermal recombinase polymerase amplification (RPA) technology, several detection assays have been developed with added convenience. Combining the lateral flow strip (LFS) test with RPA can further simplify the detection. In this study, an improved RPA assay using LFS for visual detection of V. parahaemolyticus was developed. Primers were designed targeting the virulence genes and screened for amplification efficiency, nonspecific amplification, and primer-dimer formation. Probes were designed for the best primer pairs, and the weakness of LFS tests, being easily affected by primer-dependent artifacts, was overcome by sequence modifications on primers and probe. The RPA-LFS assay took 25 min at 35 to 45°C, and showed excellent specificity. It detected as low as one colony forming unit (CFU) of V. parahaemolyticus per reaction without DNA purification, or 10 CFU/10 g spiked food samples with 2 hr of enrichment. The detection limit was better than the currently available RPA-based detection methods. Application of the RPA-LFS assay for simulated samples or real clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. The RPA-LFS assay provided a rapid, accurate, and convenient V. parahaemolyticus detection method suitable for on-site detection in resource-limited conditions.
Archives of Virology, Mar 17, 2018
Citrus tristeza virus, the most important viral disease of citrus, is reported in Assam and other... more Citrus tristeza virus, the most important viral disease of citrus, is reported in Assam and other NE states of India to infect Khasi mandarin (Citrus reticulata), the most economically important citrus crop of the region. For effective management, an attempt was made to identify a potential mild isolate against virus. Leaf samples were collected from Khasi Mandarin plants expressing differential symptoms from three different locations viz., Tinsukia, Golaghat and Mariani of Upper Brahmaputra Valley Zone of Assam. These were then grouped into three categories based on ELISA OD 405 values. Biological indexing with CTV positive samples from these three serological categories on Mexican lime (Citrus aurantifolia) seedlings resulted in symptom expression within three months post grafting. Visible symptoms of CTV infection were observed in some of the graft successful indicator plants whereas, in Khasi mandarin selection ‗CRS 4', no visible symptom development took place within this period. Based on the results, the plants were grouped into two groups-symptom producing and nonsymptom producing, and were confirmed through Bi-directional RT-PCR with mild and severe strain primers. PCR products for ‗CRS 4' were sequenced. Consensus sequences showed a single nucleotide difference at position 371 for mild isolates (CRS 4), thereby confirming the identity.
bioRxiv (Cold Spring Harbor Laboratory), Nov 3, 2020
105 and is also made available for use under a CC0 license.
PLOS ONE, May 17, 2018
Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production ... more Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.
Plants
Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) i... more Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of ‘Candidatus Liberibacter asiaticus’ (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles h...
Crizotinib treatment significantly prolongs progression-free survival, increases response rates, ... more Crizotinib treatment significantly prolongs progression-free survival, increases response rates, and improves the quality of life in patients with ALK-positive nonesmall-cell lung cancer. Droplet Digital PCR (ddPCR), a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of ALK rearrangements and crizotinib effectiveness. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma. Fluorescent in situ hybridization (FISH) and ddPCR were performed. The results revealed that 14 (13.6%) of the 103 patients were positive by dual-color, break-apart FISH. Three variants (1, 2, and 3) of the EML4-ALK gene rearrangements were detected. Thirteen of 14 ALK-positive cases identified by FISH were confirmed by ddPCR (four with variant 1, two with variant 2, and seven with variant 3). The case missed by ddPCR was identified as KIF5B-ALK gene rearrangement by PCR-based direct sequencing. Sixteen patients were detected with low copy numbers of EML4-ALK gene rearrangement, which failed to meet the positive cutoff point of FISH. Two of them responded well to crizotinib after unsuccessful chemotherapy. Our study indicates that ddPCR can be used as a molecular analytical tool to accurately measure the EML4-ALK rearrangement copy numbers in FFPE samples of lung adenocarcinoma patients.
Additional file 3: Supplementary Table S3. COG functional categories of Spiroplasma citri LB319
Additional file 4: Supplementary Table S4. Summary of PHAge Search Tool Enhanced Release (PHASTER... more Additional file 4: Supplementary Table S4. Summary of PHAge Search Tool Enhanced Release (PHASTER) analysis of Spiroplasma citri strains
PLOS ONE
Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains ... more Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains of CTV have been characterized and their symptomology widely varies, ranging from asymptomatic or mild infections to severe symptomology that results in substantial yield loss or host death. The capacity of the different CTV strains to affect the biochemistry of different citrus species has remained largely unstudied, despite that associated metabolomic shifts would be relevant toward symptom development. Thus, amino acid, sugar, phenolic, and terpenoid levels were assessed in leaves of healthy and CTV-infected grapefruit, lemon, mandarin, and two different sweet orange cultivars. Both mild [VT-negative (VT-)] and severe [VT-positive (VT+)] CTV genotype strains were utilized. When looking at overall totals of these metabolite classes, only amino acid levels were significantly increased by infection of citrus with severe CTV strains, relative to mild CTV strains or healthy plants. No sign...
Methods in Molecular Biology, 2021
Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a sp... more Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.
Methods in molecular biology, 2022
Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-strande... more Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.
Additional file 5: Supplementary Fig. S1. Phylogenetic analysis of Spiroplasma strains. Maximum-l... more Additional file 5: Supplementary Fig. S1. Phylogenetic analysis of Spiroplasma strains. Maximum-likelihood phylogeny of Spiroplasma based on core orthologous genes. In total, 601 orthologous genes were concatenated, and a maximum-likelihood approach was used to generate the phylogeny with 1000 bootstrap replicates. Bootstrap values are indicated at each node. The resulting phylogeny was visualized using FigTree v.1.4.3 [70]. S. citri strains analyzed in this report are underlined.
Additional file 1: Supplementary Table S1. BLASTn similarity search of Spiroplasma citri putative... more Additional file 1: Supplementary Table S1. BLASTn similarity search of Spiroplasma citri putative plasmid (contigs) against available Spiroplasma sequences deposited in NCBI database.
Additional file 7: Supplementary Fig. S3. Predicted prophage sequences in Spiroplasma citri genom... more Additional file 7: Supplementary Fig. S3. Predicted prophage sequences in Spiroplasma citri genomes. Prophage annotation was performed by PHASTER (PHAge Search Tool – Enhanced Release). The program predicts the completeness of the predicted prophages, based on the proportion of phage genes in the identified region. Green bars are scored as intact (score > 90); blue bars are questionable (score 70–90); red bars are incomplete (score
Additional file 6: Supplementary Fig. S2. Dot-Matrix representation of a BLASTn comparison of com... more Additional file 6: Supplementary Fig. S2. Dot-Matrix representation of a BLASTn comparison of complete Spiroplasma citri genome sequences. The chromosome of each newly assembled strain (X-axis) was compared to the reference sequence, strain R8-A2 (Y-axis). A) C189; B) LB 319; C) BR12; D) CC-2; E) C5; F) BLH-13; G) BLH-MB.
Additional file 2: Supplementary Table S2. GenBank accession numbers for the 16S rRNA Spiroplasma... more Additional file 2: Supplementary Table S2. GenBank accession numbers for the 16S rRNA Spiroplasma sequences used for phylogenetic analyses in Fig. 1.
Methods in Molecular Biology, 2021
Viroids are RNA-based infectious agents that are single-stranded, covalently closed circular, non... more Viroids are RNA-based infectious agents that are single-stranded, covalently closed circular, non-coding, and naked. Unlike RNA viruses, which at least encode proteins for replication, encapsidation, and movement, lack of protein-coding capacity of viroids makes them completely reliant on host for replication and movement. The high genetic diversity in viroids is believed to be due to the absence of proofreading activity of the host RNA polymerases, the large population size, and the rapid rate of replication. Protoplasts are viable plant cells that are prepared by enzymatic removal of cell walls. Plant protoplasts provide a synchronous single-cell system for studying early events of viroid infection such as replication and genetic diversity at the cellular level. A simple and efficient method to isolate and transfect citrus protoplasts with transcript RNA of citrus exocortis viroid is described in this chapter.
Archives of Virology, Jan 9, 2023
Plant Disease, Dec 1, 2022
Vibrio parahaemolyticus is an important pathogenic bacterium in both food safety management and m... more Vibrio parahaemolyticus is an important pathogenic bacterium in both food safety management and mariculture. Rapid and accurate detection technologies are critical for effective control of its outbreak and spreading. Conventional technologies and polymerase chain reaction (PCR)-based approaches have limited usage because of the requirement of laboratory instruments and trained personnel. Using the isothermal recombinase polymerase amplification (RPA) technology, several detection assays have been developed with added convenience. Combining the lateral flow strip (LFS) test with RPA can further simplify the detection. In this study, an improved RPA assay using LFS for visual detection of V. parahaemolyticus was developed. Primers were designed targeting the virulence genes and screened for amplification efficiency, nonspecific amplification, and primer-dimer formation. Probes were designed for the best primer pairs, and the weakness of LFS tests, being easily affected by primer-dependent artifacts, was overcome by sequence modifications on primers and probe. The RPA-LFS assay took 25 min at 35 to 45°C, and showed excellent specificity. It detected as low as one colony forming unit (CFU) of V. parahaemolyticus per reaction without DNA purification, or 10 CFU/10 g spiked food samples with 2 hr of enrichment. The detection limit was better than the currently available RPA-based detection methods. Application of the RPA-LFS assay for simulated samples or real clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. The RPA-LFS assay provided a rapid, accurate, and convenient V. parahaemolyticus detection method suitable for on-site detection in resource-limited conditions.
Archives of Virology, Mar 17, 2018
Citrus tristeza virus, the most important viral disease of citrus, is reported in Assam and other... more Citrus tristeza virus, the most important viral disease of citrus, is reported in Assam and other NE states of India to infect Khasi mandarin (Citrus reticulata), the most economically important citrus crop of the region. For effective management, an attempt was made to identify a potential mild isolate against virus. Leaf samples were collected from Khasi Mandarin plants expressing differential symptoms from three different locations viz., Tinsukia, Golaghat and Mariani of Upper Brahmaputra Valley Zone of Assam. These were then grouped into three categories based on ELISA OD 405 values. Biological indexing with CTV positive samples from these three serological categories on Mexican lime (Citrus aurantifolia) seedlings resulted in symptom expression within three months post grafting. Visible symptoms of CTV infection were observed in some of the graft successful indicator plants whereas, in Khasi mandarin selection ‗CRS 4', no visible symptom development took place within this period. Based on the results, the plants were grouped into two groups-symptom producing and nonsymptom producing, and were confirmed through Bi-directional RT-PCR with mild and severe strain primers. PCR products for ‗CRS 4' were sequenced. Consensus sequences showed a single nucleotide difference at position 371 for mild isolates (CRS 4), thereby confirming the identity.
bioRxiv (Cold Spring Harbor Laboratory), Nov 3, 2020
105 and is also made available for use under a CC0 license.
PLOS ONE, May 17, 2018
Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production ... more Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.
Plants
Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) i... more Early detection and prompt response are key factors in the eradication of ‘huanglongbing’ (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of ‘Candidatus Liberibacter asiaticus’ (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles h...
Crizotinib treatment significantly prolongs progression-free survival, increases response rates, ... more Crizotinib treatment significantly prolongs progression-free survival, increases response rates, and improves the quality of life in patients with ALK-positive nonesmall-cell lung cancer. Droplet Digital PCR (ddPCR), a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of ALK rearrangements and crizotinib effectiveness. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma. Fluorescent in situ hybridization (FISH) and ddPCR were performed. The results revealed that 14 (13.6%) of the 103 patients were positive by dual-color, break-apart FISH. Three variants (1, 2, and 3) of the EML4-ALK gene rearrangements were detected. Thirteen of 14 ALK-positive cases identified by FISH were confirmed by ddPCR (four with variant 1, two with variant 2, and seven with variant 3). The case missed by ddPCR was identified as KIF5B-ALK gene rearrangement by PCR-based direct sequencing. Sixteen patients were detected with low copy numbers of EML4-ALK gene rearrangement, which failed to meet the positive cutoff point of FISH. Two of them responded well to crizotinib after unsuccessful chemotherapy. Our study indicates that ddPCR can be used as a molecular analytical tool to accurately measure the EML4-ALK rearrangement copy numbers in FFPE samples of lung adenocarcinoma patients.
Additional file 3: Supplementary Table S3. COG functional categories of Spiroplasma citri LB319
Additional file 4: Supplementary Table S4. Summary of PHAge Search Tool Enhanced Release (PHASTER... more Additional file 4: Supplementary Table S4. Summary of PHAge Search Tool Enhanced Release (PHASTER) analysis of Spiroplasma citri strains
PLOS ONE
Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains ... more Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains of CTV have been characterized and their symptomology widely varies, ranging from asymptomatic or mild infections to severe symptomology that results in substantial yield loss or host death. The capacity of the different CTV strains to affect the biochemistry of different citrus species has remained largely unstudied, despite that associated metabolomic shifts would be relevant toward symptom development. Thus, amino acid, sugar, phenolic, and terpenoid levels were assessed in leaves of healthy and CTV-infected grapefruit, lemon, mandarin, and two different sweet orange cultivars. Both mild [VT-negative (VT-)] and severe [VT-positive (VT+)] CTV genotype strains were utilized. When looking at overall totals of these metabolite classes, only amino acid levels were significantly increased by infection of citrus with severe CTV strains, relative to mild CTV strains or healthy plants. No sign...
Methods in Molecular Biology, 2021
Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a sp... more Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.
Methods in molecular biology, 2022
Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-strande... more Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.
Additional file 5: Supplementary Fig. S1. Phylogenetic analysis of Spiroplasma strains. Maximum-l... more Additional file 5: Supplementary Fig. S1. Phylogenetic analysis of Spiroplasma strains. Maximum-likelihood phylogeny of Spiroplasma based on core orthologous genes. In total, 601 orthologous genes were concatenated, and a maximum-likelihood approach was used to generate the phylogeny with 1000 bootstrap replicates. Bootstrap values are indicated at each node. The resulting phylogeny was visualized using FigTree v.1.4.3 [70]. S. citri strains analyzed in this report are underlined.
Additional file 1: Supplementary Table S1. BLASTn similarity search of Spiroplasma citri putative... more Additional file 1: Supplementary Table S1. BLASTn similarity search of Spiroplasma citri putative plasmid (contigs) against available Spiroplasma sequences deposited in NCBI database.
Additional file 7: Supplementary Fig. S3. Predicted prophage sequences in Spiroplasma citri genom... more Additional file 7: Supplementary Fig. S3. Predicted prophage sequences in Spiroplasma citri genomes. Prophage annotation was performed by PHASTER (PHAge Search Tool – Enhanced Release). The program predicts the completeness of the predicted prophages, based on the proportion of phage genes in the identified region. Green bars are scored as intact (score > 90); blue bars are questionable (score 70–90); red bars are incomplete (score
Additional file 6: Supplementary Fig. S2. Dot-Matrix representation of a BLASTn comparison of com... more Additional file 6: Supplementary Fig. S2. Dot-Matrix representation of a BLASTn comparison of complete Spiroplasma citri genome sequences. The chromosome of each newly assembled strain (X-axis) was compared to the reference sequence, strain R8-A2 (Y-axis). A) C189; B) LB 319; C) BR12; D) CC-2; E) C5; F) BLH-13; G) BLH-MB.
Additional file 2: Supplementary Table S2. GenBank accession numbers for the 16S rRNA Spiroplasma... more Additional file 2: Supplementary Table S2. GenBank accession numbers for the 16S rRNA Spiroplasma sequences used for phylogenetic analyses in Fig. 1.
Methods in Molecular Biology, 2021
Viroids are RNA-based infectious agents that are single-stranded, covalently closed circular, non... more Viroids are RNA-based infectious agents that are single-stranded, covalently closed circular, non-coding, and naked. Unlike RNA viruses, which at least encode proteins for replication, encapsidation, and movement, lack of protein-coding capacity of viroids makes them completely reliant on host for replication and movement. The high genetic diversity in viroids is believed to be due to the absence of proofreading activity of the host RNA polymerases, the large population size, and the rapid rate of replication. Protoplasts are viable plant cells that are prepared by enzymatic removal of cell walls. Plant protoplasts provide a synchronous single-cell system for studying early events of viroid infection such as replication and genetic diversity at the cellular level. A simple and efficient method to isolate and transfect citrus protoplasts with transcript RNA of citrus exocortis viroid is described in this chapter.