Construction of Full-length Infectious cDNA Clones of Apple chlorotic leaf spot virus and Their Agroinoculation to Woody Plants by a Novel Method of Vacuum Infiltration

Construction and agroinoculation of full-length infectious cDNA clones of plant RNA viruses have been used in plant virology to prove Koch’s postulates and for development of viruses as vectors for expressing foreign genes in plants. Four full-length cDNA clones (pIF3-12, pIF3-14, pIF3-15, and pIF3-19) of Apple chlorotic leaf spot virus (ACLSV) isolate 38/85 were produced. Two of the four full-length cDNA clones (pIF3-15 and pIF3-19) proved to be infectious on Nicotiana occidentalis 37B test plants by agroinoculation and were then mechanically transmissible to healthy N. occidentalis 37B. The genomic cDNAs of ACLSV pIF3-15 and pIF3-19 shared nucleotide identity of 77.5%, demonstrating mixed infections of multiple strains of ACLSV in the source tree of isolate 38/85. The two full-length cDNA clones were agroinoculated to apple seedlings by a newly developed vacuum infiltration method. The success rate of agroinoculation was greater than 78%, defined as the number of PCR positive seedlings to the number of apple seedlings that survived. ACLSV was transmissible from agroinoculated seedlings by cleft grafting. The results of this study will be useful for construction of infectious cDNA clones of plant viruses from full-length PCR fragments and agroinoculating woody host plants using the vacuum infiltration method outlined here.

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Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

Virology Journal ◽

10.1186/1743-422x-8-488 ◽

2011 ◽

Vol 8 (1) ◽

pp. 488 ◽

Cited By ~ 23

Author(s):

Fater Youssef ◽

Armelle Marais ◽

Chantal Faure ◽

Pascal Gentit ◽

Thierry Candresse

Keyword(s):

Leaf Spot ◽

Full Length ◽

Spot Virus ◽

Chlorotic Leaf

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Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity

Journal of General Virology ◽

10.1099/vir.0.82984-0 ◽

2007 ◽

Vol 88 (9) ◽

pp. 2611-2618 ◽

Cited By ~ 29

Author(s):

Hajime Yaegashi ◽

Masamichi Isogai ◽

Hiroko Tajima ◽

Teruo Sano ◽

Nobuyuki Yoshikawa

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Coat Protein ◽

Leaf Spot ◽

Amino Acid Sequences ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Cdna Clones ◽

Spot Virus ◽

Chlorotic Leaf

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala40-Val59-Phe75-Ser130-Met184 or Ser40-Leu59-Tyr75-Thr130-Leu184) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala40 to Ser40 or Phe75 to Tyr75) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

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The RT-PCR identification and sequence analysis of Apple chlorotic leaf spot virus from apple cultivars in Jiaodong Peninsula, China

Biotechnology & Biotechnological Equipment ◽

10.1080/13102818.2014.908005 ◽

2014 ◽

Vol 28 (2) ◽

pp. 238-241 ◽

Cited By ~ 1

Author(s):

Dechang Hu ◽

Lei Wang ◽

Xiaoman Jiang ◽

Ning Wang ◽

Liang Gu

Keyword(s):

Sequence Analysis ◽

Leaf Spot ◽

Rt Pcr ◽

Spot Virus ◽

Jiaodong Peninsula ◽

Pcr Identification ◽

Chlorotic Leaf ◽

Apple Cultivars

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STUDIES ON SEVERAL STRAINS OF APPLE CHLOROTIC LEAF SPOT VIRUS (ACLSV) ISOLATED FROM DIFFERENT FRUIT TREE SPECIES.

Acta Horticulturae ◽

10.17660/actahortic.1995.386.5 ◽

1995 ◽

pp. 63-71 ◽

Cited By ~ 12

Author(s):

M. Cieslinska ◽

T. Malinowski ◽

B.J. Zawadzka

Keyword(s):

Tree Species ◽

Leaf Spot ◽

Fruit Tree ◽

Spot Virus ◽

Chlorotic Leaf

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Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein

Journal of General Virology ◽

10.1099/vir.0.80493-0 ◽

2005 ◽

Vol 86 (1) ◽

pp. 225-229 ◽

Cited By ~ 12

Author(s):

Masamichi Isogai ◽

Nobuyuki Yoshikawa

Keyword(s):

Leaf Spot ◽

Rna Binding ◽

Movement Protein ◽

Binding Domain ◽

Sequence Specificity ◽

Nucleic Acid Binding ◽

Spot Virus ◽

Uv Crosslinking ◽

Chlorotic Leaf ◽

Rna Binding Domain

The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin–UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82–126 and 127–287 potentially contain two independently active, single-stranded nucleic acid-binding domains.

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Cloning and characterization of full-length cDNA clones encoding chalcone synthase from the orchid Bromheadia finlaysoniana

Plant Physiology and Biochemistry ◽

10.1016/s0981-9428(98)80013-2 ◽

1998 ◽

Vol 36 (9) ◽

pp. 647-656 ◽

Cited By ~ 18

Author(s):

Chye-Fong Liew ◽

Chong-Jin Goh ◽

Chiang-Shiong Loh ◽

Saw-Hoon Lim

Keyword(s):

Chalcone Synthase ◽

Full Length ◽

Cdna Clones ◽

Full Length Cdna

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Molecular Characterization and Distribution of Apple Chlorotic Leaf Spot Virus on Apple in China

Journal of Phytopathology ◽

10.1111/jph.12188 ◽

2013 ◽

Vol 162 (5) ◽

pp. 284-290

Author(s):

Hao Duan ◽

Zhirui Ji ◽

Shutong Wang ◽

Tongle Hu ◽

Yanan Wang ◽

...

Keyword(s):

Molecular Characterization ◽

Leaf Spot ◽

Spot Virus ◽

Chlorotic Leaf

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Infectious Transcripts from Cloned Genome-Length cDNA of Porcine Reproductive and Respiratory Syndrome Virus

Journal of Virology ◽

10.1128/jvi.72.1.380-387.1998 ◽

1998 ◽

Vol 72 (1) ◽

pp. 380-387 ◽

Cited By ~ 82

Author(s):

J. J. M. Meulenberg ◽

J. N. A. Bos-de Ruijter ◽

R. van de Graaf ◽

G. Wensvoort ◽

R. J. M. Moormann

Keyword(s):

Cdna Clone ◽

De Novo ◽

Infectious Clone ◽

Full Length ◽

Respiratory Syndrome Virus ◽

Cdna Clones ◽

Genome Length ◽

Full Length Cdna ◽

Lung Macrophages ◽

Growth Properties

ABSTRACT The 5′-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5′-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

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