In planta protein-protein interactions assessed using a nanovirus-based replication and expression system

The multipartite genome of the nanovirus Faba bean necrotic yellows virus, which consists of one gene on each DNA component, was exploited to construct a series of virus-based episomal vectors designed for transient replication and gene expression in plants. This nanovirus based expression system yields high levels of protein which allows isolation of recombinant protein and protein complexes from plant tissues. As examples, we demonstrated in planta interaction between the nanovirus F-box protein Clink and SKP1, a constituent of the ubiquitin-dependent protein turnover pathway. Thus, replicative nanovirus vectors provide a simple and efficient means for in planta characterization of protein-protein interaction.     

Detection and Partial Characterization of Milk vetch dwarf virus Isolates from Faba Bean (Vicia faba L.) in Yunnan Province, China

A virus disease of faba bean ( Vicia faba L.) in China, characterized by leaf yellowing and rolling and plant stunting, was shown to be caused by a virus of the genus Nanovirus based on serological reactions to nanovirus-specific monoclonal antibodies and the generation of polymerase chain reaction amplicons using nanovirus-specific primers. To identify the faba bean-infecting nanovirus, regions of the DNA components encoding the master replication initiator protein and capsid protein of two nanovirus isolates from China were cloned, sequenced and compared with those of other members of the genus Nanovirus . The two Chinese virus isolates shared nucleotide sequence identities ranging from 95 to 98% with the type isolate of Milk vetch dwarf virus (MDV) from Japan. They were thus identified as isolates of MDV, a virus so far known to cause important diseases of legumes in Japan. This is the first record of MDV-infecting faba bean in China.     

Faba Bean Necrotic Yellows Virus Naturally Infects Phaseolus Bean and Cowpea in the Coastal Area of Syria

In an attempt to identify possible summer hosts of the faba bean necrotic yellows virus (FBNYV), a field survey was conducted in the coastal area of Syria. Using a monoclonal antibody to FBNYV in indirect ELISA, FBNYV was detected in a large number of samples from Phaseolus vulgaris L. and in a few samples from Vigna unguiculata (L.) Walp. in which it caused severe symptoms. This is the first report of natural infection of P. vulgaris and V. unguiculata with FBNYV.     

Gold Nanoparticle Interference Study during the Isolation,Quantification, Purity and Integrity Analysis of RNA

Investigations have been conducted regarding the interference of nanoparticles (NPs) with different toxicological assay systems, but there is a lack of validation when conducting routine tests for nucleic acid isolation, quantification, integrity, and purity analyses. The interference of citrate-capped gold nanoparticles (AuNPs) was investigated herein. The AuNPs were added to either BEAS-2B bronchial human cells for 24 h, the isolated pure RNA, or added during the isolation procedure, and the resultant interaction was assessed. Total RNA that was isolated from untreated BEAS-2B cells was spiked with various concentrations (v/v%) of AuNPs and quantified. A decrease in the absorbance spectrum (220–340 nm) was observed in a concentration-dependent manner. The 260 and 280 nm absorbance ratios that traditionally infer RNA purity were also altered. Electrophoresis was performed to determine RNA integrity, but could not differentiate between AuNP-exposed samples. However, the spiked post-isolation samples did produce differences in spectra (190–220 nm), where shifts were observed at a shorter wavelength. These shifts could be due to alterations to chromophores found in nucleic acids. The co-isolation samples, spiked with 100 µL AuNP during the isolation procedure, displayed a peak shift to a longer wavelength and were similar to the results obtained from a 24 h AuNP treatment, under non-cytotoxic test conditions. Moreover, hyperspectral imaging using CytoViva dark field microscopy did not detect AuNP spectral signatures in the RNA isolated from treated cells. However, despite the lack of AuNPs in the final RNA product, structural changes in RNA could still be observed between 190–220 nm. Consequently, full spectral analyses should replace the traditional ratios based on readings at 230, 260, and 280 nm. These are critical points of analyses, validation, and optimization for RNA-based techniques used to assess AuNPs effects.     

Cell wall changes during the expansion and senescence of carnation (Dianthus caryophyllus) petals

Senescence of carnation (Dianthus caryophyllus L. ev. White Sim) petals coincided with a decrease on a per flower basis in the yield of cell wall and ethanol-insoluble solids. The decrease in cell wall yield per flower was due largely to a loss of neutral sugars, primarily galactose (45%) and arabinose (23%). On a per flower basis, water-and chelator-soluble pectins increased throughout development, comprising in senescent petals 18 and 58%, respectively, of total pectin. Alkali-soluble pectins ranged from 35 to 45% of the total pectin and decreased during senescence. Gel chromatography of chelator- and alkali-soluble pectins revealed no change in molecular size and polygalacturonase activity was not detected. Large-molecular-size hemicelluloses decreased during development, an observation reminiscent of the changes affecting hemicelluloses during the ripening of a number of fruit types. Compositional analysis of the large hemicellulosic polymers revealed a decrease in xylose and galactose content.     

Characterization of Peanut Stripe Virus Isolates from Soybean in Taiwan

Potyvirus isolates were obtained in Taiwan from soybean showing crinkle, mottle, mosaic or blistering. They were identified as peanut stripe virus (PStV) on the basis of host range, serology, molecular weight of the capsid proteins and morphology of cytoplasmic cylindrical inclusions. PStV was found to be closely related serologically to adzuki bean mosaic virus (AzMV), blackeye cowpea mosaic virus (BICMV), and the bean common mosaic virus (BCMV) strain NY 15. A clear differentiation of PStV from these related viruses was possible on the basis of the cylindrical inclusion morphology. Only the peanut isolate of PStV from the USA and the three soybean isolates of PStV from Taiwan produced pinwheels, scrolls and curved laminated aggregates whereas the other serologically related viruses produced scrolls only. Whilst the peanut isolate of PStV infected all nine peanut cvs tested, the soybean isolate PN of PStV infected two peanut cvs only. AzMV, BICMV and two strains of soybean mosaic virus did not infect any of the peanut cultivars tested. On the other hand, nineteen and three of the 27 soybean cvs were susceptible to the soybean isolate PN and the peanut isolate of PStV, respectively. The capsid proteins of the peanut and the three soybean isolates of PStV and of AzMV appeared to be proteolytically undegraded and to have nearly identical molecular weights of 35 kD. Based upon results of virus surveys in soybean plantings in Taiwan, the incidence of soybean isolates of PStV in soybean is similar to that of soybean mosaic virus, suggesting that these PStV strains might be economically significant to soybean production m Taiwan.     

Properties of Johnsongrass Chlorotic Stripe Mosaic Virus

Physicochemical, biological, and cytopathological properties of Johnsongrass chlorotic stripe mosaic virus (JCSMV) found in Iran were investigated. Virus particles were polyhedral, showed a knobbed surface structure, were c. 30 nm in diameter and had a buoyant density of 1. 359 g/ml in cesium chloride. Virions contained one major protein with a molecular weight of 41 kd and a single species of ssRNA with a molecular weight of 1. 43 × 10⁶ d. Acid hydrolysis of the virus followed by thin-layer electrophoresis gave the following molar percentages of the bases: A: 23. 5, G: 27. 5, C: 26 and U: 23. Separation of nucleotides of the viral RNA using alkaline hydrolysis was not successful. Mechanical inoculation of freshly purified virus or isolated RNA failed to infect Johnsongrass or maize plants. The virus was readily detected by ELISA in seeds from infected plants and young seedlings raised from such seeds, but not in later stages of growth. Ultrathin sections of infected cells showed high concentrations of virus particles in the cytoplasm and vacuoles. Virus-like particles also occurred in the stroma of chloroplasts and mitochondria. Cisternae of endoplasmic reticulum (ER) were often extremely inflated and filled by a fine fibrillar material. Small membrane-associated vesicles were frequently found in ER elements and occurred also in the permuclear space. Based on particle structure, properties of the nucleic acid, molecular weight of the coat protein and cytopathology, the virus resembles carmoviruses. However, lack of mechanical transmissibility is not known for any virus classified with this group. No serological reaction was detected with a total of 30 antisera to carmoviruses and other isometric viruses.     

Graphical genotyping as a method to map <Emphasis Type="Italic">Ny</Emphasis><Subscript><Emphasis Type="Italic">(o,n)sto</Emphasis></Subscript> and <Emphasis Type="Italic">Gpa5</Emphasis> using a reference panel of tetraploid potato cultivars

Key message

The method of graphical genotyping is applied to a panel of tetraploid potato cultivars to visualize haplotype sharing. The method allowed to map genes involved in virus and nematode resistance. The physical coordinates of the amount of linkage drag surrounding these genes are easily interpretable.

Abstract

Graphical genotyping is a visually attractive and easily interpretable method to represent genetic marker data. In this paper, the method is extended from diploids to a panel of tetraploid potato cultivars. Application of filters to select a subset of SNPs allows one to visualize haplotype sharing between individuals that also share a specific locus. The method is illustrated with cultivars resistant to Potato virus Y (PVY), while simultaneously selecting for the absence of the SNPs in susceptible clones. SNP data will then merge into an image which displays the coordinates of a distal genomic region on the northern arm of chromosome 11 where a specific haplotype is introgressed from the wild potato species S. stoloniferum (CPC 2093) carrying a gene (Ny _(o,n)sto ) conferring resistance to two PVY strains, PVY^O and PVY^NTN. Graphical genotyping was also successful in showing the haplotypes on chromosome 12 carrying Ry-f _sto , another resistance gene derived from S. stoloniferum conferring broad-spectrum resistance to PVY, as well as chromosome 5 haplotypes from S. vernei, with the Gpa5 locus involved in resistance against Globodera pallida cyst nematodes. The image also shows shortening of linkage drag by meiotic recombination of the introgression segment in more recent breeding material. Identity-by-descent was found to be a requirement for using graphical genotyping, which is proposed as a non-statistical alternative method for gene discovery, as compared with genome-wide association studies. The potential and limitations of the method are discussed.