Virus-mediated export of chromosomal DNA in plants

Viruses are potential vectors for horizontal gene transfer. Here, studying viral infection of sugar beet plants, the authors report the generation of virus-host circular DNA hybrids and provide a picture of the initial steps in virus-mediated horizontal transfer of chromosomal DNA between plant species

Characterisation of DNA replication of tomato leaf curl geminivirus / Seyyed Ali Akbar Behjatnia.

Bibliography: leaves 133-152.xi, 152 leaves : ill. (some col.), col. map ; 30 cm.Studies biological relatedness of strains of tomato leaf curl virus and cross-interaction with the replication-associated protein requireed for DNA replication.Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 199

The full-length genome characterization and diversity of faba bean necrotic stunt virus in Iran

Faba bean necrotic stunt virus (FBNSV) (genus Nanovirus; family Nanoviridae) is a multipartite virus comprising eight circular single-stranded DNA components, each about 1 kb in length. Also, two alphasatellites have often been reported to be associated with FBNSV infection. The only full-length genome sequence of an Iranian FBNSV isolate, FBNSV-[IR;Ka:FB], so far reported is one infecting faba bean (Vicia faba L.). To obtain more information about Iranian isolates of FBNSV (FBNSV-IRs) and their diversity, the full-length genome of four FBNSV isolates and associated alphasatellites obtained from faba bean (two isolates), common bean (Phaseolus vulgaris L., one isolate) and chickpea (Cicer arietinum L., one isolate) were characterized. All genomic components except U4 had the expected size in accordance with the FBNSV genomic sequences published in the GenBank. The component U4 had an additional stretch of 16 nucleotides (nts 765\u2013780) in its non-coding region (NCR). Alphasatellites of the aforementioned isolates showed 98\u2013100% sequence identity. Intra-component sequence analyses of FBNSV-IRs indicated a range of 98 to 100% nt identity between each single FBNSV-IRs component or between their associated alphasatellites. DNA-M and DNA-U2 exhibited the highest value of diversity, sharing 98\u201399% identity. Phylogenetic analyses indicated that FBNSV-IRs were clustered with a Moroccan isolate and some Azerbaijani isolates of FBNSV forming a clade distinct from Ethiopian isolates. Inter-component sequence analyses of NCRs revealed the DNA-S and DNA-U4 subgrouped together in all FBNSV-IRs isolates except the chickpea isolate in which NCRs of DNA-S and DNA\u2013U4 formed separate subgroups. \ua9 2020, Koninklijke Nederlandse Planteziektenkundige Vereniging

Expression of Human Immunodeficiency Virus type 1 (HIV-1) coat protein genes in plants using cotton leaf curl Multan betasatellite-based vector.

It has already been demonstrated that a betasatellite associated with cotton leaf curl Multan virus (CLCuMB) can be used as a plant and animal gene delivery vector to plants. To examine the ability of CLCuMB as a tool to transfer coat protein genes of HIV-1 to plants, two recombinant CLCuMB constructs in which the CLCuMB βC1 ORF was replaced with two HIV-1 genes fractions including a 696 bp DNA fragment related to the HIV-1 p24 gene and a 1501 bp DNA fragment related to the HIV-1 gag gene were constructed. Gag is the HIV-1 coat protein gene and p24 is a component of the particle capsid. Gag and p24 are used for vaccine production. Recombinant constructs were inoculated to Nicotiana glutinosa and N. benthamiana plants in the presence of an Iranian isolate of Tomato yellow leaf curl virus (TYLCV-[Ab]) as a helper virus. PCR analysis of inoculated plants indicated that p24 gene was successfully replicated in inoculated plants, but the gag gene was not. Real-time PCR and ELISA analysis of N. glutinosa and N. benthamiana plants containing the replicative forms of recombinant construct of CLCuMB/p24 indicated that p24 was expressed in these plants. This CLCuMB-based expression system offers the possibility of mass production of recombinant HIV-1 p24 protein in plants

The schematic map of (A) pBinβΔC1/p24 and (B) pBinβΔC1/gag constructs.

<p>In (A) the CLCuMBΔC1 DNA with a direct repeat of 282 bp flanked the 696 bp DNA fragment of HIV-1 p24 and in (B) flanked the 1501 bp DNA fragment of HIV-1 gag. The direct repeat of the CLCuMB DNA is represented by the dotted rods. The restriction enzymes used in the construction of the cassettes and their nucleotide positions in CLCuMB DNA (Acc. No. AJ298903) are shown by the vertical bars. The positions of the polyadenylation signal and the TATA box in βC1 promoter, upstream of the initiation codon of the βC1 ORF are shown by the A and TATA signals on the diagram, respectively. The one sided arrows are used to show the position of the primers used in the construction of the constructs and further analysis of the resulted DNAs in inoculated plants. The two sided arrows are used to show the expected size of the amplified fragments. These constructs cloned separately as <i>Kpn</i>I/<i>Sna</i>BI fragments into the pBin20 binary vector were used in agroinoculation experiments.</p

Optical density values at 405 nm of the ELISA plate readings of protein samples from inoculated plants.

<p>Plants, as indicated at the top of the columns, inoculated with the constructs as indicated at the bottom of the columns. The value represented for each treatment is the mean of three replicates. Bars with different letter are significantly (<i>p</i> ≤ 0.05) different. Quantitation of HIV-1 p24 protein in plant extracts was estimated by ELISA using the positive antigen (Ag) control of the ELISA kit with a definite (100 pgml-1) concentration. The p24 protein concentration was estimated to be 407.1 ngml^-1 in a <i>N</i>. <i>bethamiana</i> plant extract (corresponding to a yield of 814.2 ng per g fresh plant tissue) whereas the concentration of the same protein in four <i>N</i>. <i>glutinosa</i> plant extracts ranged from 39.4 to 231.3 ngml^-1 (corresponding to a yield of 78.8 to 462.6 ng per g fresh plant tissue). This showed that <i>N</i>. <i>bethamiana</i> was a more suitable host for production of HIV-1 <i>p24</i> protein using the CLCuMB-based expression vector.</p

Transreplicon of recombinant βΔC1/p24 DNA (1735 bp) in <i>N</i>. <i>glutinosa</i> and <i>N</i>. <i>benthamiana</i> plants.

<p>The DNA fragments were obtained from plants, as indicated at the bottom of the lanes, co-agroinoculated with the pBinβΔC1/p24 construct and the helper virus TYLCV-[Ab] (lanes 1–8) by PCR using specific adjacent beta1285^V/1290^C primer pair (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190403#pone.0190403.t001" target="_blank">Table 1</a>). No DNA was amplified from the extracts of plants inoculated with the helper virus alone (lanes 9–10) and plants co-inoculated with the pBinβΔC1/gag construct and the helper virus (lanes 11–15) using the same primer pair. M, marker DNA ladder (Fermentas).</p

Amplification of the 142 bp p24 DNA fragments (A) and 112 bp tobacco elongation factor (Ef) 1-alpha DNA fragments (B).

<p>The DNA fragments were obtained by RT-PCR using the total RNA extracted from <i>N</i>. <i>glutinosa</i> plants inoculated with the constructs as shown at the top of the lanes using a pair of either p24 (A) or tobacco EF1 (B) specific primer pairs as shown at the bottom of each panel. The p24 142 bp DNA fragment and 112 bp tobacco EF1 DNA fragment were absent (lane 7 of each panel) in PCR control reactions without RT from the extracted RNA of <i>N</i>. <i>glutinosa</i> plants co-inculated with βΔC1/p24 construct in the presence of TYLCV-[Ab].</p

Seed Transmission of Beet Curly Top Virus and Beet Curly Top Iran Virus in a Local Cultivar of Petunia in Iran

Beet curly top virus (BCTV) and beet curly top Iran virus (BCTIV) are known as the causal agents of curly top disease in beet and several other dicotyledonous plants in Iran. These viruses are transmitted by Circulifer species, and until now, there has been no confirmed report of their seed transmission. A percentage (38.2–78.0%) of the seedlings developed from the seeds of a petunia local cultivar under insect-free conditions showed stunting, interveinal chlorosis, leaf curling, and vein swelling symptoms, and were infected by BCTV when tested by PCR. Presence of BCTV in seed extracts of petunia local cultivar was confirmed by PCR and IC-PCR, followed by sequencing. Agroinoculation of curly top free petunia plants with a BCTV infectious clone resulted in BCTV infection of plants and their developed seeds. These results show the seed infection and transmission of BCTV in a local cultivar of petunia. Similar experiments performed with BCTIV showed that this virus is also seed transmissible in the same cultivar of petunia, although with a lower rate (8.8–18.5%). Seed transmission of curly top viruses may have significant implications in the epidemiology of these viruses