The Experts below are selected from a list of 3606 Experts worldwide ranked by ideXlab platform
Stuart A. Macfarlane - One of the best experts on this subject based on the ideXlab platform.
-
Newly identified RNAs of raspberry Leaf Blotch virus encoding a related group of proteins
The Journal of general virology, 2015Co-Authors: Wendy J. Mcgavin, Peter J. A. Cock, Esther Schnettler, Fei Yan, Jianping Chen, Stuart A. MacfarlaneAbstract:Members of the genus Emaravirus, including Raspberry Leaf Blotch virus (RLBV), are enveloped plant viruses with segmented genomes of negative-strand RNA, although the complete genome complement for any of these viruses is not yet clear. Currently, wheat mosaic virus has the largest emaravirus genome comprising eight RNAs. Previously, we identified five genomic RNAs for RLBV; here, we identify a further three RNAs (RNA6–8). RNA6–8 encode proteins that have clear homologies to one another, but not to any other emaravirus proteins. The proteins self-interacted in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments, and the P8 protein interacted with the virus nucleocapsid protein (P3) using BiFC. Expression of two of the proteins (P6 and P7) using potato virus X led to an increase in virus titre and symptom severity, suggesting that these proteins may play a role in RLBV pathogenicity; however, using two different tests, RNA silencing suppression activity was not detected for any of the RLBV proteins encoded by RNA2–8.
-
Experimental and bioinformatic evidence that raspberry Leaf Blotch emaravirus P4 is a movement protein of the 30K superfamily
Journal of General Virology, 2013Co-Authors: David Karlin, Jianping Chen, Kathryn M. Wright, Stuart A. MacfarlaneAbstract:Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry Leaf Blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form β-strands, namely β1 and β2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the β1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within β1 without preventing the membrane association or movement function of P4.
-
Raspberry Leaf Blotch virus, a putative new member of the genus Emaravirus, encodes a novel genomic RNA
Journal of General Virology, 2012Co-Authors: Wendy J. Mcgavin, Peter J. A. Cock, Kathryn M. Wright, Carolyn Mitchell, Stuart A. MacfarlaneAbstract:A new, segmented, negative-strand RNA virus with morphological and sequence similarities to other viruses in the genus Emaravirus was discovered in raspberry plants exhibiting symptoms of Leaf Blotch disorder, a disease previously attributed to the eriophyid raspberry Leaf and bud mite (Phyllocoptes gracilis). The virus, tentatively named raspberry Leaf Blotch virus (RLBV), has five RNAs that each potentially encode a single protein on the complementary strand. RNAs 1, 2 and 3 encode, respectively, a putative RNA-dependent RNA polymerase, a glycoprotein precursor and the nucleocapsid. RNA4 encodes a protein with sequence similarity to proteins of unknown function that are encoded by the genomes of other emaraviruses. When expressed transiently in plants fused to green or red fluorescent protein, the RLBV P4 protein localized to the peripheral cell membrane and to punctate spots in the cell wall. These spots co-localized with GFP-tagged tobacco mosaic virus 30K cell-to-cell movement protein, which is itself known to associate with plasmodesmata. These results suggest that the P4 protein may be a movement protein for RLBV. The fifth RLBV RNA, encoding the P5 protein, is unique among the sequenced emaraviruses. The amino acid sequence of the P5 protein does not suggest any potential function; however, when expressed as a GFP fusion, it localized as small aggregates in the cytoplasm near to the periphery of the cell.
José Guerri - One of the best experts on this subject based on the ideXlab platform.
-
Precocious flowering of juvenile citrus induced by a viral vector based on Citrus Leaf Blotch virus: a new tool for genetics and breeding
Plant Biotechnology Journal, 2016Co-Authors: Karelia Velazquez, M. C. Vives, Luis Navarro, Pedro Moreno, Jesus Aguero, Pablo Aleza, José Antonio Pina, José GuerriAbstract:Summary The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus Leaf Blotch virus-based vector (clbvINpr-AtFT and clbvINpr-CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4–6 months, with no alteration of the plant architecture, Leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr-AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.
-
effectiveness of gene silencing induced by viral vectors based on citrus Leaf Blotch virus is different in nicotiana benthamiana and citrus plants
Virology, 2014Co-Authors: Jesus Aguero, M. C. Vives, Luis Navarro, Pedro Moreno, Karelia Velazquez, Jose A Pina, José GuerriAbstract:Virus induced gene silencing (VIGS) is an effective technology for gene function analysis in plants. We assessed the VIGS effectiveness in Nicotiana benthamiana and citrus plants of different Citrus Leaf Blotch virus (CLBV)-based vectors, using insets of the phytoene desaturase (pds) gene. While in N. benthamiana the silencing phenotype was induced only by the construct carrying a 58-nt pds hairpin, in citrus plants all the constructs induced the silencing phenotype. Differences in the generation of secondary small interfering RNAs in both species are believed to be responsible for differential host–species effects. The ability of CLBV-based vectors to silence different endogenous citrus genes was further confirmed. Since CLBV-based vectors are known to be stable and induce VIGS in successive flushes for several months, these vectors provide an important genomic tool and it is expected that they will be useful to analyze gene function by reverse genetics in the long-lived citrus plants.
-
citrus Leaf Blotch virus invades meristematic regions in nicotiana benthamiana and citrus
Molecular Plant Pathology, 2013Co-Authors: Jesus Aguero, M. C. Vives, Luis Navarro, Susana Ruizruiz, Pedro Moreno, Karelia Velazquez, Jose Juarez, José GuerriAbstract:Summary To invade systemically host plants, viruses need to replicate in the infected cells, spread to neighbouring cells through plasmodesmata and move to distal parts of the plant via sieve tubes to start new infection foci. To monitor the infection of Nicotiana benthamiana plants by Citrus Leaf Blotch virus (CLBV), leaves were agroinoculated with an infectious cDNA clone of the CLBV genomic RNA expressing green fluorescent protein (GFP) under the transcriptional control of a duplicate promoter of the coat protein subgenomic RNA. Fluorescent spots first appeared in agroinfiltrated leaves 11–12 days after infiltration, indicating CLBV replication. Then, after entering the phloem vascular system, CLBV was unloaded in the upper parts of the plant and invaded all tissues, including flower organs and meristems. GFP fluorescence was not visible in citrus plants infected with CLBV-GFP. Therefore, to detect CLBV in meristematic regions, Mexican lime (Citrus aurantifolia) plants were graft inoculated with CLBV, with Citrus tristeza virus (CTV), a virus readily eliminated by shoot-tip grafting in vitro, or with both simultaneously. Although CLBV was detected by hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR) in 0.2-mm shoot tips in all CLBV-inoculated plants, CTV was not detected. These results explain the difficulty in eliminating CLBV by shoot-tip grafting in vitro.
-
development of viral vectors based on citrus Leaf Blotch virus to express foreign proteins or analyze gene function in citrus plants
Molecular Plant-microbe Interactions, 2012Co-Authors: Jesus Aguero, M. C. Vives, Luis Navarro, Susana Ruizruiz, Pedro Moreno, Karelia Velazquez, Leandro Pena, José GuerriAbstract:Viral vectors have been used to express foreign proteins in plants or to silence endogenous genes. This methodology could be appropriate for citrus plants that have long juvenile periods and adult plants that are difficult to transform. We developed viral vectors based on Citrus Leaf Blotch virus (CLBV) by duplicating a minimum promoter (92 bp) either at the 3′ untranslated region (clbv3′pr vector) or at the intergenic region between the movement and coat protein (CP) genes (clbvINpr vector). The duplicated fragment (–42/+50) around the transcription start site of the CP subgenomic RNA (sgRNA) had the full promoter activity and induced synthesis of a new sgRNA in infected plants. Agroinoculation with these vectors resulted in systemic infection of Nicotiana benthamiana and the resulting virions systemically infected citrus plants. A clbvINpr vector carrying the green fluorescent protein (GFP) gene expressed GFP in citrus plants and triggered gfp silencing in gfp-transgenic citrus plants, and vectors carry...
-
the citrus Leaf Blotch virus movement protein acts as silencing suppressor
Virus Genes, 2012Co-Authors: Agueda Renovell, Luis Navarro, Mari Carmen Vives, Susana Ruizruiz, Pedro Moreno, José GuerriAbstract:To counteract plant antiviral defense based on RNA silencing, many viruses express proteins that inhibit this mechanism at different levels. The genome of Citrus Leaf Blotch virus (CLBV) encodes a 227-kDa protein involved in replication, a 40-kDa movement protein (MP), and a 41-kDa coat protein (CP). To determine if any of these proteins might have RNA silencing suppressor activities, we have used Agrobacterium-mediated transient assays in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c. Only CLBV MP was able to suppress intracellular GFP silencing induced by expression of either single- or double-stranded (ds) GFP RNA, but not cell-to-cell or long distance spread of the silencing signal. The MP suppressor activity was weak compared to other characterized viral suppressor proteins. Overall our data indicate that MP acts as a suppressor of local silencing probably by interfering in the silencing pathway downstream of the steps of dsRNA and small RNAs generation.
Jari P. T. Valkonen - One of the best experts on this subject based on the ideXlab platform.
-
Occurrence and genetic diversity of Raspberry Leaf Blotch virus (RLBV) infecting cultivated and wild Rubus species in Finland
Annals of Applied Biology, 2015Co-Authors: L. Dong, Anne Lemmetty, Satu Latvala, Olga Samuilova, Jari P. T. ValkonenAbstract:Raspberry Leaf Blotch virus (RLBV) is a recently described (−)ssRNA virus (genus Emaravirus) associated with the long-known, severe raspberry Leaf Blotch disorder (RLBD). The virus is presumably transmitted by the raspberry Leaf and bud mite (Phyllocoptes gracilis; Eriophyidae). Cultivated and wild raspberries (Rubus idaeus) displaying RLBD or yellowing symptoms were sampled in 14 districts in Finland and tested for RNA3 and RNA5 of RLBV by reverse transcription PCR (RT-PCR) and dot blot hybridization. A total of 59 samples were tested for RLBV, including 36 plants of cultivated raspberry, 20 wild raspberry plants, one ornamental purple flowering raspberry plant (Rubus odoratus), and two samples of P. gracilis (20 mites pooled per sample) collected from RLBD-affected Glen Ample. Fifty-three (93%) of the 57 plants tested were RLBV-positive, including seven raspberry cultivars (Balder, Glen Ample, Jenkka, Malling Minerva, Maurin Makea, Muskoka, Ottawa) and purple flowering raspberry, which is a new host for RLBV. RLBV was also detected by RT-PCR in mites (P. gracilis) collected from buds of RLBV-positive raspberry plants. The partial nucleotide (nt) sequence of the RLBV NP gene (nt 835–1284 of RNA3) was determined for 21 isolates obtained from 10 districts in Finland. Identical isolates were detected in distant districts, and distinctly different isolates were found in the same raspberry cultivation. Furthermore, eight different NP sequence variants were detected in the nine plants of a single raspberry cultivar (Glen Ample) tested from two districts. The nt and deduced amino acid sequences of the 21 isolates were 92.0–100% and 89.3–100% identical, respectively. Phylogenetic analysis revealed two main clusters. One cluster included three isolates, of which two isolates were from one farm in Finland and one isolate was previously characterised in Scotland. The other cluster contained the remaining 19 isolates characterised in this study. The results indicate that RLBV is widely distributed in cultivated and wild raspberries in Finland and shows considerable genetic variability.
-
First Report of Raspberry Leaf Blotch virus in Raspberries in Finland.
Plant disease, 2012Co-Authors: K. Artola, Takeshi Kurokura, Timo Hytönen, Jari P. T. ValkonenAbstract:Raspberry (Rubus idaeus L.) is a valuable and widely grown softfruit that is a host for 40 viruses and virus-like agents, of which many are not characterized at the molecular level. Recently, Raspberry Leaf Blotch virus (RLBV, putative emaravirus species) was described from raspberries (cv. Glen Ample) in the United Kingdom and Serbia. Plants displayed conspicuous yellow Blotches on leaves and abnormal development of Leaf hairs in the corresponding areas of the abaxial side (3). Similar symptoms were observed in 'Glen Ample' grown in protective plastic tunnels and open fields in the main berry growing area in eastern Finland in June 2011. In three farms, leaves were sampled from symptomatic and symptomless plants of 'Glen Ample' and also cv. Polka displaying no symptoms. Total RNA was extracted using CTAB reagent. Equal amounts of RNA were pooled from 13 samples and subjected to small-RNA (sRNA) deep sequencing (Fasteris SA, Plan-les-Ouates, Switzerland) to detect viruses without advance information (2). Contigs were built on 21- to 24-nt sRNA reads using Velvet. Contigs larger than 50 nt were used to search homologous sequences in GenBank by BLAST and significant similarity (up to 99%) was observed with RLBV RNA3 and RNA4. Mapping sRNA reads to the genome of RLBV (1) by MAQ resulted in significant coverage of RNA1 (16%), RNA2 (37%), RNA3 (46%), RNA4 (65%), and RNA5 (27%). cDNA was synthesized on RNA of one symptom-expressing plant using random hexamer primers and the cDNA tested by PCR with a forward primer (RLBV-F 5'-TCAAATCCACTTGCATAGAACC-3', nt 723 to 744) and reverse primer (RLBV-R1 5'-CCTCAAACCTTGCAAACACA-3', nt 1,318 to 1,337) designed according to the nucleocapsid (NP) gene of the Scottish RLBV isolate (3). The sequence of the amplified partial NP gene (576 nt; GenBank Accession No. JQ684678) was 92.8% and 94.8% identical to the Scottish isolate at nt and amino acid levels, respectively. The forward primer RLBV-F and a new reverse primer (RLBV-R2 5'-GCCGAAAGTCAAACCTGGTG-3', nt 943 to 962) were used to test additional plants for RBLV and to make a probe (198 nt) to detect RLBV using digoxigen-labeled sense and antisense RNA probes, as described for European mountain ash ringspot associated virus (1). RLBV was detected in all tested plants of 'Glen Ample' with yellow Leaf Blotch symptoms in the three farms, but not in any symptomless plants of 'Glen Ample' and 'Polka.' The sense probes gave strong signals, in contrast to the antisense probes, which gave only weak or no detectable signals in the virus-positive plants, consistent with the negative RNA strand of RLBV being encapsidated in virus particles. The results show RLBV is associated with severe, distinct, and characteristic symptoms in raspberries of cv. Glen Ample grown in plastic tunnels and open fields in Finland and has an apparent negative impact on plant growth and yield. Our observations in 2011 also suggest that the incidence of diseased plants is much higher in plastic tunnels than in open fields, perhaps because the conditions for the vector of RLBV (raspberry Leaf and bud mite, Phyllocoptes gracilis Nalepa) (1) are more favorable in plastic tunnels. These results clarify the etiology of raspberry Leaf Blotch disease in Finland and emphasize the need to inspect raspberry planting materials for RLBV for better control of the disease. References: (1) A. K. Kallinen et al. Phytopathology 99:344, 2009. (2) J. F. Kreuze et al. Virology 388:1, 2009. (3) W. J. McGavin et al. J. Gen. Virol. 93:430, 2012.
Cees Waalwijk - One of the best experts on this subject based on the ideXlab platform.
-
isolation and characterization of the mating type idiomorphs from the wheat septoria Leaf Blotch fungus mycosphaerella graminicola
Fungal Genetics and Biology, 2002Co-Authors: Cees Waalwijk, Maarten A. De Waard, E C P Verstappen, O Mendes, Gert H. J. KemaAbstract:Abstract Both mating-type loci from the wheat septoria Leaf Blotch pathogen Mycosphaerella graminicola have been cloned and sequenced. The MAT1-2 gene was identified by screening a genomic library from the MAT1-2 isolate IPO94269 with a heterologous probe from Tapesia yallundae. The MAT1-2 idiomorph is 2772 bp and contains a single gene encoding a putative high-mobility-group protein of 394 amino acids. The opposite idiomorph was obtained from isolate IPO323, which has the complementary mating type, by long-range PCR using primers derived from sequences flanking the MAT1-2 idiomorph. The MAT1-1 locus is 2839 bp in size and contains a single open reading frame encoding a putative α1-domain protein of 297 amino acids. Within the nonidiomorphic sequences, homology was found with palI, encoding a membrane receptor from Aspergillus nidulans, and a gene encoding a putative component of the anaphase-promoting complex from Schizosaccharomyces pombe and a DNA-(apurinic or apyrimidinic) lyase from S. pombe. For each of the MAT genes specific primers were designed and tested on an F1 mapping population that was generated from a cross between IPO323 and IPO94269. An absolute correlation was found between the amplified allele-specific fragments and the mating type as determined by backcrosses of each F1 progeny isolate to the parental isolates. The primers were also used to screen a collection of field isolates in a multiplex PCR. An equal distribution of MAT1-1 and MAT1-2 alleles was found for most geographic origins examined.
-
avirulence in the wheat septoria tritici Leaf Blotch fungus mycosphaerella graminicola is controlled by a single locus
Molecular Plant-microbe Interactions, 2000Co-Authors: Gert H. J. Kema, E C P Verstappen, Cees WaalwijkAbstract:Segregation of avirulence in Mycosphaerella graminicola, a heterothallic ascomycete that causes wheat septoria tritici Leaf Blotch, was studied in F1, BC1, and F2 populations by inoculation assays on five wheat cultivars in the seedling stage and by amplified fragment length polymorphism and random amplified polymorphic DNA analyses. F1 was generated by crossing isolates IPO323 (avirulent) and IPO94269 (virulent). All F1, BC1, and F2 progeny isolates were virulent on the susceptible check cultivar Taichung 29 and were avirulent on the resistant check cultivar Kavkav-K4500. Avirulence segregation was observed in F1 and in several BC1 and F2 generations on the differential cultivars Shafir, Kavkaz, and Veranopolis at a 1:1 ratio. Avirulence for the three differential cultivars always cosegregated. We conclude that avirulence in isolate IPO323 is controlled by a single, seemingly complex locus.
Gert H. J. Kema - One of the best experts on this subject based on the ideXlab platform.
-
isolation and characterization of the mating type idiomorphs from the wheat septoria Leaf Blotch fungus mycosphaerella graminicola
Fungal Genetics and Biology, 2002Co-Authors: Cees Waalwijk, Maarten A. De Waard, E C P Verstappen, O Mendes, Gert H. J. KemaAbstract:Abstract Both mating-type loci from the wheat septoria Leaf Blotch pathogen Mycosphaerella graminicola have been cloned and sequenced. The MAT1-2 gene was identified by screening a genomic library from the MAT1-2 isolate IPO94269 with a heterologous probe from Tapesia yallundae. The MAT1-2 idiomorph is 2772 bp and contains a single gene encoding a putative high-mobility-group protein of 394 amino acids. The opposite idiomorph was obtained from isolate IPO323, which has the complementary mating type, by long-range PCR using primers derived from sequences flanking the MAT1-2 idiomorph. The MAT1-1 locus is 2839 bp in size and contains a single open reading frame encoding a putative α1-domain protein of 297 amino acids. Within the nonidiomorphic sequences, homology was found with palI, encoding a membrane receptor from Aspergillus nidulans, and a gene encoding a putative component of the anaphase-promoting complex from Schizosaccharomyces pombe and a DNA-(apurinic or apyrimidinic) lyase from S. pombe. For each of the MAT genes specific primers were designed and tested on an F1 mapping population that was generated from a cross between IPO323 and IPO94269. An absolute correlation was found between the amplified allele-specific fragments and the mating type as determined by backcrosses of each F1 progeny isolate to the parental isolates. The primers were also used to screen a collection of field isolates in a multiplex PCR. An equal distribution of MAT1-1 and MAT1-2 alleles was found for most geographic origins examined.
-
avirulence in the wheat septoria tritici Leaf Blotch fungus mycosphaerella graminicola is controlled by a single locus
Molecular Plant-microbe Interactions, 2000Co-Authors: Gert H. J. Kema, E C P Verstappen, Cees WaalwijkAbstract:Segregation of avirulence in Mycosphaerella graminicola, a heterothallic ascomycete that causes wheat septoria tritici Leaf Blotch, was studied in F1, BC1, and F2 populations by inoculation assays on five wheat cultivars in the seedling stage and by amplified fragment length polymorphism and random amplified polymorphic DNA analyses. F1 was generated by crossing isolates IPO323 (avirulent) and IPO94269 (virulent). All F1, BC1, and F2 progeny isolates were virulent on the susceptible check cultivar Taichung 29 and were avirulent on the resistant check cultivar Kavkav-K4500. Avirulence segregation was observed in F1 and in several BC1 and F2 generations on the differential cultivars Shafir, Kavkaz, and Veranopolis at a 1:1 ratio. Avirulence for the three differential cultivars always cosegregated. We conclude that avirulence in isolate IPO323 is controlled by a single, seemingly complex locus.