Development of breed identification markers based on a bovine 50K SNP array.

In Japan, Japanese Black and Holstein cattle are appreciated as popular sources of meat, and imported beef from Australia and the United States is also in demand in the meat industry. Since the BSE outbreak, the problem of false sales has arisen: imported beef has sometimes been mislabeled as domestic beef due to consumer concerns. A method is needed to correctly discriminate between Japanese and imported cattle for food safety. The objective of this study was to develop breed discrimination markers between Japanese and US cattle using a 50K SNP array. As a result, five US-specific markers (BISNP7, BISNP15, BISNP21, BISNP23, and BISNP26) were developed with allelic frequencies that ranged from 0.102 (BISNP15) to 0.250 (BISNP7) and averaged 0.184. The combined use of the five markers would permit discrimination between Japanese and US cattle with a probability of identification of 0.858. This result indicates the potential of the bovine 50K SNP array as a powerful tool for developing breed identification markers. These markers would contribute to the prevention of falsified beef displays in Japan.

Mapping the virus and host genes involved in the resistance response in cucumber mosaic virus-Infected Arabidopsis thaliana.

A yellow strain of cucumber mosaic virus (CMV) [CMV(Y)] induces a resistance response characterized by inhibition of virus systemic movement with development of necrotic local lesions in the virus-inoculated leaves of Arabidopsis thaliana ecotype C24. In this report, the avirulence determinant in the virus genome was defined and the resistance gene (RCY1) of C24 was genetically mapped. The response of C24 to CMV containing the chimeric RNA3 between CMV(Y) and a virulent strain of CMV indicated that the coat protein gene of CMV(Y) determined the localization of the virus in the inoculated leaves of C24. The RCY1 locus was mapped between two CAPS markers, DFR and T43968, which were located in the region containing genetically defined disease resistance genes and their homologues. These results indicate that the resistance response to CMV(Y) in C24 is determined by the combination of the coat protein gene and RCY1 on chromosome 5.

Replication of Tomato spotted wilt virus After Ingestion by Adult Thrips setosus is Restricted to Midgut Epithelial Cells.

ABSTRACT If acquisition access feeding (AAF) is first given after adult eclosion, none of the nine thrips species able to serve as tospovirus vectors can become infective. The previous cellular investigations of this phenomenon, carried out only in Frankliniella occidentalis, suggested that infectivity was prevented because the type member of the tospoviruses, Tomato spotted wilt virus (TSWV), was unable to enter the midgut of adult thrips. The present study extends a cellular view of tospovirus-thrips interactions to a species other than the western flower thrips, F. occidentalis. Our findings show that TSWV enters and replicates within the midgut of adult Thrips setosus, but does not infect cells beyond the midgut epithelia. After AAF as adult, TSWV replicated in T. setosus midgut cells as indicated by significant increases in nucleocapsid (N) protein detected by double-antibody sandwich enzyme-linked immunosorbent assay, and the presence of inclusions containing the S RNA-encoded nonstructural and N proteins revealed by microscopic observations. Electron microscopic observations of adult insects showed that no infection occurred in cells beyond the midgut epithelia, and insects subsampled from the same cohorts could not transmit TSWV. In contrast, electron microscopy observations of larval T. setosus revealed that TSWV infected the midgut and muscle cells, and adult insects developing from these cohorts had infected salivary glands and were able to transmit TSWV. Mature virions were observed only in the salivary glands of adults developing from infected larvae. Our findings suggest that the barrier to infectivity in T. setosus adults differs from that shown for F. occidentalis adults.

Novel mannose-binding rice lectin composed of some isolectins and its relation to a stress-inducible salT gene.

The novel mannose-binding rice lectin (MRL) purified by Sephadex G-50 or maltamyl Sepharose 4B affinity chromatography was not homogeneous, but the components were separated clearly by two dimensional polyacrylamide gel electrophoresis (1st; isoelectric focusing with Immobiline, 2nd; SDS-PAGE). The major spots were located at pI 4.85 and 4.74, and minor spots at pI 4.66, 4.56, and 4.44; all spots were distributed at about MW 45,000. Other faint spots were sometimes detected just below the major spots. In the western blot analysis, all the spots reacted with the monoclonal antibodies specific to MRL, which bound to MRL and inhibited the lectin activity to agglutinate rabbit erythrocytes. The proteins of the spots at pI 4.85, 4.77, 4.66, and 4.56 had lectin activity. The major proteins at pI 4.85 and 4.77 also had the common amino acid sequence at N-terminus, TLVKIGPWGGNGGSAQDISV, which is almost identical to salt and drought stress-inducible salT gene products in rice plants. High homology was also conserved in both the cDNA and the genomic clones encoding the MRL component at pI 4.85, which were selected with MRL-specific antibodies and an oligonucleotide designed from the partial amino acid sequence. All results suggest that MRL is composed of several isolectins, if not, related proteins having a common epitope and may belong to a family of stress-inducible proteins.

In vivo and in vitro phosphorylation of rice dwarf phytoreovirus Pns12 cytoplasmic nonstructural protein.

In vivo and in vitro phosphorylation and intracellular location of rice dwarf phytoreovirus Pns12, which is encoded by one of the twelve dsRNA genome segments, S12, and comprises 312 amino acids, was investigated. When [32P]phosphoric acid was incorporated into RDV-infected leafhopper cultured cells, labelled Pns12 was immunoprecipitated from the cells by a monospecific anti-Pns12 polyclonal antibody. Recombinant Pns12 was purified from Spodoptera frugiperda cells infected with AcRS12, a baculovirus recombinant carrying a full-length cDNA of RDV S12. Purified Pns12 was also demonstrated to be phosphorylated in vitro by a kinase activity present in host (rice, barley, wheat, leafhopper) cells and non-host (tobacco, spinach, white clover, S. frugiperda, mosquito, mammals) cells as well. Immunocytochemical studies showed Pns12 accumulated in the cytoplasm of infected cells, and frequently localized in a slightly electron-dense patch. These results demonstrated that RDV Pns12 was a cytoplasmic nonstructural phosphoprotein.

Phosphorylation and/or presence of serine 37 in the movement protein of tomato mosaic tobamovirus is essential for intracellular localization and stability in vivo.

The P30 movement protein (MP) of tomato mosaic tobamovirus (ToMV) is synthesized in the early stages of infection and is phosphorylated in vivo. Here, we determined that serine 37 and serine 238 in the ToMV MP are sites of phosphorylation. MP mutants in which serine was replaced by alanine at positions 37 and 238 (LQ37A238A) or at position 37 only (LQ37A) were not phosphorylated, and mutant viruses did not infect tobacco or tomato plants. By contrast, mutation of serine 238 to alanine did not affect the infectivity of the virus (LQ238A). To investigate the subcellular localization of mutant MPs, we constructed viruses that expressed each mutant MP fused with the green fluorescent protein (GFP) of Aequorea victoria. Wild-type and mutant LQ238A MP fusion proteins showed distinct temporally regulated patterns of MP-GFP localization in protoplasts and formation of fluorescent ring-shaped infection sites on Nicotiana benthamiana. However mutant virus LQ37A MP-GFP did not show a distinct pattern of localization or formation of fluorescent rings. Pulse-chase experiments revealed that MP produced by mutant virus LQ37A was less stable than wild-type and LQ238A MPs. MP which contained threonine at position 37 was phosphorylated, but the stability of the MP in vivo was very low. These studies suggest that the presence of serine at position 37 or phosphorylation of serine 37 is essential for intracellular localization and stability of the MP, which is necessary for the protein to function.

Role of arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression.

In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) and requires protein biosynthesis for ABA-dependent gene expression. Previous experiments established that a 67-bp DNA fragment of the rd22 promoter is sufficient for dehydration- and ABA-induced gene expression and that this DNA fragment contains two closely located putative recognition sites for the basic helix-loop-helix protein MYC and one putative recognition site for MYB. We have carefully analyzed the 67-bp region of the rd22 promoter in transgenic tobacco plants and found that both the first MYC site and the MYB recognition site function as cis-acting elements in the dehydration-induced expression of the rd22 gene. A cDNA encoding a MYC-related DNA binding protein was isolated by DNA-ligand binding screening, using the 67-bp region as a probe, and designated rd22BP1. The rd22BP1 cDNA encodes a 68-kD protein that has a typical DNA binding domain of a basic region helix-loop-helix leucine zipper motif in MYC-related transcription factors. The rd22BP1 protein binds specifically to the first MYC recognition site in the 67-bp fragment. RNA gel blot analysis revealed that transcription of the rd22BP1 gene is induced by dehydration stress and ABA treatment, and its induction precedes that of rd22. We have reported a drought- and ABA-inducible gene that encodes the MYB-related protein ATMYB2. In a transient transactivation experiment using Arabidopsis leaf protoplasts, we demonstrated that both the rd22BP1 and ATMYB2 proteins activate transcription of the rd22 promoter fused to the beta-glucuronidase reporter gene. These results indicate that both the rd22BP1 (MYC) and ATMYB2 (MYB) proteins function as transcriptional activators in the dehydration- and ABA-inducible expression of the rd22 gene.

Addition of nucleotides similar to deleted CAA repeats in the 5' non-coding region of tomato mosaic virus RNA following propagation.

Previously we made a series of deletion mutants in the 5' non-coding region of tomato mosaic tobamovirus (ToMV) RNA and checked their ability to replicate in tobacco protoplasts. Long deletions in this region caused the virus to lose the ability to replicate. Several mutants with deletions of about 10 nucleotides (short deletion mutants; SDM) retained the ability to replicate. In this study, we inoculated SDMs onto systemic host plants and observed their symptoms. One mutant (19/32) caused severe mosaic symptoms on some tobacco plants (Nicotiana tabacum cv. Samsun) but no symptoms on others. Virus accumulation in 19/32-inoculated plants paralleled the severity of symptoms. Four CAA repeat sequences were deleted in 19/32. Progeny 19/32 from plants showing severe systemic mosaic symptoms had acquired additional nucleotides in this region. We conclude that the CAA repeat sequence is related to the fitness of the virus population to replicate in whole plants rather than to translation of ToMV replicase genes.

Sequence-specific binding of protein factors to two independent promoter regions of the acidic tobacco pathogenesis-related-1 protein gene (PR-1).

Gel shift mobility analysis, using the proximal 0.3 kb fragment of the tobacco pathogenesis-related protein 1a gene (PR-1a) and nuclear extracts from healthy Samsun NN tobacco leaves, which do not produce PR-1 proteins, showed a broad shifted signal with low mobility. This signal was not detected with nuclear proteins from the interspecific hybrid of Nicotiana glutinosa x Nicotiana debneyi, which constitutively produces the PR-1a protein. Similar shifted signals were detected with both proximal and distal regions of the 0.3 kb fragment using nuclear proteins from healthy Samsun NN tobacco, but not with proteins from the interspecific hybrid. Further experiments, performed using 5' or 3' truncated fragments of the 0.3 kb fragment, identified two independent binding sites: a distal site between -179 and -168 bp from the transcription start site, and a proximal site between -61 and -37 bp. Footprint analysis revealed two protected sequences, a distal region between -184 and -172 bp, and a proximal region between -68 and -51 bp. These results indicate the presence of regulatory factor(s) for expression of the acidic PR-1a gene. The possibility of negative regulation of the gene is discussed.

Reinvestigation of intracellular localization of the 30K protein in tobacco protoplasts infected with tobacco mosaic virus RNA.

It has been shown that the 30K protein of tobacco mosaic virus (TMV) is responsible for the cell-to-cell movement function of the virus. It is still obscure how the protein is involved in this function at the molecular level. We formerly found that the 30K protein is localized to the plasmodesmata of TMV-infected plants. We also reported that the 30K protein was detected in a nuclei-rich fraction of TMV-infected protoplasts after biochemical fractionation. To clarify the inconsistency, the 30K protein was immunocytologically localized in TMV-infected protoplasts using a newly prepared antibody against the 30K protein. On some sections, the 30K protein was found near the nucleus but not in or on the nucleus. At later stages of infection a novel electron-transparent structure was detected in the cytoplasm where the 30K proteins were localized. This structure might reflect an intermediate form between its synthesis in the cytoplasm and its targeting to the plasmodesmata in whole plants.

Immunocytochemical localization of the 130K and 180K proteins (putative replicase components) of tobacco mosaic virus.

We have revealed the cellular localization of the putative replicase components of tobacco mosaic virus (TMV), 130K and 180K proteins, in TMV-infected tobacco leaves by the immunogold technique with antisera which specifically react with these two proteins. When sections of TMV-infected tobacco leaves were treated with anti-130K protein antiserum and then with protein A-gold complex, most of the gold label was strongly localized on granular inclusion bodies which were found specifically in the cytoplasm of TMV-infected cells. Very small amounts of label present in other regions, including the nuclei, chloroplasts, and mitochondria, seemed to be nonspecific. Gold-labeled 180K protein was also dispersed over the granular inclusion bodies. The granular inclusion bodies appeared to be oval-shaped structures with various diameters ranging from 0.2 to 2.8 microm. TMV particles were usually observed near the granular inclusion bodies as aggregates but not inside them. Considering the involvement of the 130K and 180K proteins in replication, the granular inclusion bodies may be the site for replication of TMV RNA.