DNA-A of a highly pathogenic Indian cassava mosaic virus isolated from Jatropha curcas causes symptoms in Nicotiana benthamiana

Jatropha curcas mosaic disease (JcMD) is a newly emerging disease that has been reported in Africa and India. Here, we report the complete nucleotide sequence of a new Indian cassava mosaic virus isolate (ICMV-SG) from Singapore. Infection of ICMV-SG showed more severe JcMD in Jatropha curcas and Nicotiana benthamiana than the other ICMV isolates reported previously, though ICMV-SG shares high sequence identity with the other ICMV isolates. Agroinfectious DNA-A alone sufficiently induced systemic symptoms in N. benthamiana, but not in J. curcas. Results from agroinfection assays showed that systemic infection of ICMV-SG in J. curcas required both DNA-A and DNA-B components.     

Silencing of NbXrn4 facilitates the systemic infection of Tobacco mosaic virus in Nicotiana benthamiana

The 5'-3' exoribonucleases (Xrns) play key roles in degradation and processing pathways of several classes of RNAs including mRNA, rRNA, miRNA and other small RNAs. Recent work revealed that the cytoplasmic Xrn (Xrn1p in yeast and Xrn4 in plants) affected the stability of the viral RNA of tombusviruses in yeast and plants, which indicates that the cytoplasmic Xrn might be involved in plant defense against virus by degrading viral RNA. Here, we demonstrated that silencing of Nicotiana benthamiana cytoplasmic Xrn4 facilitated both local and systemic infection of Tobacco mosaic virus (TMV) in N. benthamiana. The results support the suggestion that cytoplasmic Xrn4 participates in the viral defense system of plants.     

Properties of the F' factors formed in crosses between escherichia coli Hfr donor cells and recombination defective recipients

As a result of crossing between Hfr H, KL-96, and KL-99 donor cells with AB 2463 rec A as the recipient, merodiploids carrying factors of different structure (different length) and different activity were isolated: 1) typical F' factors with incorporated proximal chromosomal markers; 2) long F' factors of different structure, defective for genes controlling sensitivity to phage f₂; 3) long F' factors of different structure defective for genes controlling transfer. Chromosomal markers can be incorporated into the factor regardless of their position relative to sex factor in the original Hfr cells. Defects of the sex factor proper are accompanied by loss of some of its incorporated chromosomal genes, whereas the typical F' factors preserve their structure completely.Department of Biology and General Genetics, Patrice Lumumba Peoples' Friendship University, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 79, No. 4, pp. 104–107, April, 1975.     

Analysis of meiotic recombination events near a recombination hotspot in the yeast Saccharomyces cerevisiae

The region of yeast chromosome III between the HIS4 and LEU2 genes has an unusually high frequency of meiotic recombination. In order to determine the pattern of cross-over and gene conversion events, we constructed a strain with a number of heterozygous markers in this 25-kb interval. We found that very high levels of reombination are localized to regions of DNA near HIS4. In addition, analysis of the patterns of co-conversion of adjacent markers suggests that there is more than one initiation site contributing to recombination of HIS4.     

Mutant MHC class I molecules define interactions between components of the peptide-loading complex

Class I histocompatibility molecules, consisting of a heavy chain, beta2-microglobulin and peptide, are assembled in the endoplasmic reticulum (ER) with the assistance of several molecular chaperones and accessory proteins. Peptide binding occurs when assembling class I molecules associate with a loading complex consisting of the transporter associated with antigen processing (TAP) peptide transporter, tapasin, ERp57 and calreticulin (CRT)/calnexin. To assess the physical organization of this complex, we generated a series of mutants in the murine H-2Dd heavy chain and assessed their association with components of the complex. Seven mutations, clustered between amino acids 122 and 136 in the heavy chain alpha2 domain plus one mutation at position 222 in the alpha3 domain, resulted in loss of interaction with tapasin. Association with TAP was always lost simultaneously, supporting the view that tapasin acts as an obligatory bridge between class I molecules and TAP. Compared with previous studies on the HLA-A2 molecule, some differences in points of tapasin interaction were observed. Failure of the H-2Dd mutants to bind tapasin resulted in low cell-surface expression and altered intracellular transport. Most mutants retained a substantial degree of peptide loading, consistent with the view that although tapasin may promote peptide binding to class I, it is not required. A surprising observation was that all mutants lacking tapasin interaction retained normal association with CRT. This contrasts with previous observations on other class I molecules and, combined with differences in tapasin interaction, suggests that the organization of the ER peptide-loading complex can vary depending on the specific class I molecule examined.     

Molecular characterization of the interaction between the N-terminal region of Potato virus X (PVX) coat protein (CP) and Nicotiana benthamiana PVX CP-interacting protein, NbPCIP1

Using yeast two-hybrid assays and a Nicotiana benthamiana cDNA library, we previously identified an N. benthamiana protein, NbPCIP1, that interacts with Potato virus X (PVX) coat protein (CP). We also previously determined that NbPCIP1 enhances PVX replication in plants. To determine the domains and/or amino acid residues required for PVX CP and NbPCIP1 interaction, here we used yeast two-hybrid and β-galactosidase filter assays to test the effects of deletion and site-directed mutations on the interaction. Truncation analysis revealed that the N-terminal region of PVX CP interacts with NbPCIP1. To identify which N-terminal region PVX CP amino acid(s) interact with NbPCIP1, we substituted the 12 charged amino acids on the PVX CP N-terminal region to alanine. Yeast two-hybrid, β-galactosidase filter, and bimolecular fluorescence complementation (BiFC) assays confirmed that ten of the 12 alanine-substituted mutations blocked the interaction with NbPCIP1. The results suggest that the N-terminal region of PVX CP including its helical structure is important for interaction with NbPCIP1.     

Characterization of tobacco geminiviruses in the Old and New World

Summary.  Biological differences and molecular variability between six phenotypically distinct tobacco-infecting geminivirus isolates from southern Africa (Zimbabwe) and Mexico were investigated. Host range studies conducted with tobacco virus isolates ZIM H from Zimbabwe and MEX 15 and MEX 32 from Mexico indicated all had narrow host ranges restricted to the Solanaceae. Alignment of coat protein gene (CP) and common region (CR) sequences obtained by PCR, and phylogenetic analysis of the CP sequences indicated Zimbabwean isolates were distantly related to those from Mexico and that geographically proximal isolates shared their closest affinities with Old and New World geminiviruses, respectively. Zimbabwean isolates formed a distinct cluster of closely related variants (>98% sequence identity) of the same species, while MEX 15 segregated independently from MEX 32, the former constituting a distinct species among New World geminiviruses, and the latter being a variant, Texas pepper virus-Chiapas isolate (TPV-CPS) with 95% sequence identity to TPV-TAM. Results collectively indicated a geographic basis for phylogenetic relationships rather than a specific affiliation with tobacco as a natural host. MEX 15 is provisionally described as a new begomovirus, tobacco apical stunt virus, TbASV, whose closest CP relative is cabbage leaf curl virus, and ZIM isolates are provisionally designated as tobacco leaf curl virus, TbLCV-ZIM, a new Eastern Hemisphere begomovirus, which has as its closest relative, chayote mosaic virus from Nigeria. Received August 6 1998 Accepted October 22, 1998     

Intracellular events in the replication of defective interfering particles of vesicular stomatitis virus.

During a synchronous infection with both wild-type vesicular stomatitis virus (VSV) and the MS-T defective interfering (DI) particle at levels of high interference, total RNA synthesis was both reduced by 75% and shut off early as compared to the VSV infection alone. In the interfered infection the production of both VSV intracellular nucleocapsids and progeny virus was suppressed by 90% while there was a dramatic increase in intracellular MS-T nucleocapsids. There was a linear accumulation of MS-T nucleocapsids for only 5 hr with the maximum rate of synthesis being observed at 3 hr postinfection. The MS-T nucleocapsids contained genome-length, single-stranded 19 S DI RNA and were 40% of the positive (+)- and 60% of the negative (?)-strand sense. Neither the (+)- nor (?)strand of MS-T nucleocapsid RNA contained poly(A) sequences since none of the RNA or its ribonuclease digestion products bound to oligo(dT)-cellulose. We can detect no methylation of the nucleocapsid RNA and the 5′-termini were not blocked since both the (+)- and (?)-strands initiated with pppA.     

On the use of excision repair defective cells in the wing somatic mutation and recombination test in Drosophila melanogaster

Ten chemical mutagens were tested in the wing somatic mutation and recombination test in Drosophila melanogaster. This assay makes use of genetic markers expressed on the wing of adult flies. Larvae which are trans-heterozygous for mwh (multiple wing hairs) and flr (flare) were fed with the compounds either acutely (2, 4, or 6 hr) or chronically (48 or 72 hr), or were treated by inhalation (1 hr). Genetic changes induced in the somatic cells of the wing imaginal discs lead to the formation of mutant clones on the wing (mwh and/or flr). Single spots are produced by point mutation, chromosome breakage, and mitotic recombination; twin spots are produced exclusively by mitotic recombination. All 10 mutagens belonging to different chemical classes were clearly positive in this assay. However, the choice of the optimal treatment conditions (concentration of compound, duration of treatment, age of larvae at treatment) is essential. Eight of the compounds were also tested in excision repair defective cells by introducing the mei-9L1 mutation into the test system. This seems not to improve the detection capacity of the assay, mainly because only small spots are found in excision repair defective wings. In addition, the frequencies of spots in these wings are enhanced four to five times, which makes the scoring more tedious. For these and other practical reasons the use of this specific cross is not recommended in the wing spot test for routine screening purposes.     

Expression of the B subunit of the heat-labile enterotoxin of Escherichia coli in tobacco mosaic virus-infected Nicotiana benthamiana plants and its characterization as mucosal immunogen and adjuvant

We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coli in tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants. We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal. The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA. Eighty-nine percent of N. benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments. The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots. rLTB from plant extracts of TMV-infected N. benthamiana leaves was purified to give 75 microg rLTB pentamers per gram fresh plant material and was capable of binding G(M)1 ganglioside. The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 microg per mouse) induced LTB-specific IgG1 antibodies. To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production. The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen. In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity.     

Genetic variability and potential recombination events in the HC-Pro gene of sugarcane streak mosaic virus

Sugarcane streak mosaic virus (SCSMV), a member of the family Potyviridae, is an important viral pathogen affecting sugarcane production in India. The variability in the nucleotide (nt) and amino acid (aa) sequences of helper component proteinase (HC-Pro) of SCSMV isolates from India was investigated and compared with those of previously published virus isolates from different Asian countries. Comparison of all of the sequenced virus isolates revealed a high level of diversity in the HC-Pro gene (72-97% nt sequence identity; 83-99% aa sequence identity), and the Indian isolates were found to be the most divergent (up to 12% variation at the amino acid level). Phylogenetic analysis revealed clustering of 16 SCSMV isolates into two groups. Group I included isolates from India and Pakistan, and group II consisted of isolates from Japan and Indonesia. Recombination analysis revealed nine potentially significant recombination events, and putative recombination sites were identified throughout the HC-Pro gene. Analysis of selection pressure indicated that the HC-Pro gene of SCSMV is under strong negative selection. It is likely that recombination, along with strong negative selection, enhances the speed of elimination of deleterious mutations in the HC-Pro gene.     

Deletion of flanking ARS elements does not affect meiotic recombination at the HIS4 locus in yeast

The HIS4-BIK1 interval on chromosome III of Saccharomyces cerevisiae contains a hotspot for meiotic recombination. Previous reports demonstrated that the initiating lesion is a double-stranded break which is subsequently processed in an asymmetric manner. Data presented here show that the efficiency of initiation of meiotic recombination is unaffected by the deletion of flanking ARS elements, and that the distribution of recombinants is not altered in strains heterozygous for these deletions. These results suggest that the initiation of recombination is not affected by the time of replication of the hotspot at HIS4. The data also indicate that altering the direction of replication-fork movement through the HIS4 region does not affect meiotic recombination. Received: 18 June / 24 September 1996     

Temperature-sensitive mutants of type 12 adenovirus defective in a late function: protein synthesis and evidence for recombination between mutants in complementation group D.

Four temperature sensitive mutants of adenovirus type 12, classified into complementation group D, synthesize most or all of the late virus-specific polypeptides at the restrictive temperature, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Infections by these mutants under restrictive conditions apparently result in some over production of the virion hexon polypeptide compared to wild type infection. Genetic recombination between the four mutants has been demonstrated. A partial linear genetic map of the D cistron is presented.     

A novel variant of the infectious bronchitis virus resulting from recombination events in Italy and Spain

Infectious bronchitis is considered to be one of the most devastating diseases in poultry. Control of its spread is typically attempted through biosecurity measures and extensive vaccination. However, the remarkable genetic and antigenic variability of the virus, which originate from both mutations and recombination events, represents an unsolved challenge for this disease. The present study reports on the emergence and spread of recombinant clusters detected in Italy and Spain between 2012 and 2014. A total of 36 Spanish and Italian infectious bronchitis virus (IBV) field strains were investigated and genetically characterized using phylogenetic, molecular, recombination and selection pressure analyses of the complete S1 gene. Based on the partial S1 sequencing, 27 IBV strains originating from Spain and nine from Italy were initially classified as being closely related to the Guandong/Xindadi (XDN) genotype. Phylogenetic analysis of the complete S1 gene revealed that the XDN strains formed a homogeneous clade with the Spanish IBV isolates within the QX genotype, whereas there was higher variability within the Italian strains. Recombination analysis determined that these strains belonged to four groups, which originated from independent recombination events between the QX and 793B IBV genotypes. Our data support the hypothesis of two different scenarios: firstly, in Spain, the large and homogeneous clade probably originated from a single offspring of the recombinant founder, which became dominant and spread throughout the country. Secondly, the nine Italian recombinants, which are characterized by three different recombination patterns, probably represent less fitted strains, because they were less viable with respect to their recombinant parents.     

Sequence diversity and potential recombination events in the coat protein gene of Apple stem pitting virus

The variability of the Apple stem pitting virus (ASPV) coat protein (CP) gene was investigated. The CP gene of ten virus isolates from apple and pear trees was sequenced. Comparison of all sequenced virus isolates revealed high diversity of the CP gene (70.7-93.5% at the nucleotide level and 77.8-98.7% at the amino acid level). Additionally, one or two deletions in the N-terminal part of the coat protein gene of the studied virus isolates were identified. The ratio of nonsynonymous to synonymous polymorphic sites indicated that purifying selection has acted to eliminate deleterious mutations in coding sites. Moreover, the evidences for recombination in analyzed sequences were provided. It is likely that recombination, along with selection, enhances the speed of elimination of deleterious mutations in ASPV, following the mutational deterministic hypothesis of Kondrashov.