Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization.

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.

Cloning and characterization of a melanin biosynthetic THR1 reductase gene essential for appressorial penetration of Colletotrichum lagenarium.

Melanin biosynthesis of Colletotrichum lagenarium is essential for appressorial penetration of the host plant. A melanin deficient mutant 9141 (Thr-) has a defect in the conversion of 1,3,8-trihydroxynaphthalene to vermelone in the melanin biosynthetic pathway. The mutant formed nonmelanized appressoria and had little infectivity on cucumber leaves. A cosmid clone pCR1 was selected from a cosmid library of wild-type C. lagenarium by means of a heterologous probe BRM2, one of the clustered genes involved in melanin biosynthesis of Alternaria alternata. pCR1 transformed the Thr- mutant 9141 to wild-type phenotype. A DNA fragment (THR1) homologous to BRM2 was subcloned from pCR1 and the nucleotide sequence determined. THR1 contains one open reading frame that encodes a protein of 282 amino acids. A transformant resulting from gene disruption showed a light brown phenotype different from the dark brown phenotype of the wild-type 104-T. The transformant formed nonmelanized appressoria and had little infectivity. The THR1 amino acid sequence contains a region highly similar to the Ver1 gene involved in the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis by Aspergillus parasiticus and to the T4HN reductase gene involved in the conversion of 1,3,6,8-tetrahydroxynaphthalene to scytalone and 1,3,8-trihydroxynaphthalene to vermelone in melanin biosynthesis by Magnaporthe grisea. Expression of the THR1 gene during spore germination of C. lagenarium was detected by RNA blotting. We propose that the C. lagenarium THR1 gene encodes a reductase involved in conversion of 1,3,8-trihydroxynaphthalene to vermelone.

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis.

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.

Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea.

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.

Nucleotide sequence analysis of cDNA encoding the coat protein of cucumber mosaic virus: genome organization and molecular features of the protein.

The cDNA sequence coding for the coat protein of cucumber mosaic virus (Japanese Y strain) was cloned, and its nucleotide sequence was determined. The sequence contains an open reading frame that encodes the coat protein composed of 218 amino acids. The nucleotide and deduced amino acid sequences of the coat protein of this strain were compared with those of the Q strain; the homologies of the sequences were 78% and 81%, respectively. Further study of the sequences gave an insight into the genome organization and the molecular features of the coat protein. The coding region can be divided into three characteristic regions. The N-terminal region has conserved features in the positively charged structure, the hydropathy pattern and the predicted secondary structure, although the amino acid sequence is varied mainly due to frameshift mutations. It is noteworthy that the positions of arginine residues in this region are highly conserved. Both the nucleotide and amino acid sequences of the central region are well conserved. The amino acid sequence of the C-terminal region is not conserved, because of frameshift mutations, however, the total number of amino acids is conserved. The nucleotide sequence of the 3'-noncoding region is divergent, but it could form a tRNA-like structure similar to those reported for other viruses. Detailed investigation suggests that the Y and Q strains are evolutionarily distant.

mRNA amplification system by viral replicase in transgenic plants.

We have constructed transgenic tobacco plants (M1x2-FCP2IFN plants) expressing viral RNA replication genes of brome mosaic virus (BMV) and BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN-gamma) gene. In M1x2-FCP2IFN plants the RNA3 derivative expressed from the integrated cDNA was replicated and subgenomic RNA (i.e. mRNA of IFN-gamma) was produced by BMV replicase. The accumulation level of the mRNA of IFN-gamma was approximately 5-fold higher than that by the cauliflower mosaic virus (CaMV) 35S RNA promoter. In addition IFN-gamma accumulated in M1x2-FCP2IFN plants.

The Alternaria alternata Melanin Biosynthesis Gene Restores Appressorial Melanization and Penetration of Cellulose Membranes in the Melanin-Deficient Albino Mutant of Colletotrichum lagenarium

Colletotrichum lagenarium and Alternaria alternata produce a dark pigment, melanin. The C. lagenarium PKS1 and A. alternata ALM genes are involved in polyketide synthesis in the melanin biosynthesis pathway. PKS1 encodes a type I polyketide synthase. For functional comparison of the ALM gene with the PKS1 gene, we examined whether the A. alternata ALM gene could restore melanin synthesis in C. lagenarium albino mutant (Pks1(-)). The ALM gene transformed the albino mutant (Pks1(-)) to melanin-producing phenotypes, designated CAL transformants. The pigment intensity of both melanized colonies and appressoria of CAL transformants was weaker than that of the wild type. Ultrastructural studies of the cell walls of appressoria demonstrated that CAL transformants formed an outer melanized layer, as did the wild type. On the other hand, the thin inner and middle layers were less electron-dense than those of the wild type. CAL transformants were able to penetrate cellulose membranes as effectively as the wild type. By contrast, the penetration frequency of CAL transformants on cucumber cotyledons was remarkably reduced compared to that of the wild type. During conidial germination, the PKS1 transcript accumulated de novo in both the wild-type and CAL transformants after the start of conidial incubation. On the other hand, ALM transcript accumulated in conidia of CAL transformants before the start of conidial incubation.

Cloning of the fish cell line SSN-1 for piscine nodaviruses.

Six cell clones were derived from the SSN-1 cell line, which is composed of a mixed cell population and persistently infected with a C-type retrovirus (SnRV). These clones were susceptible to 4 piscine nodavirus strains belonging to different genotypes (SJNNV, RGNNV, TPNNV and *BFNNV [striped jack, redspotted grouper, tiger puffer and barfin flounder nervous necrosis viruses]). Three clones, designated A-6, E-9, and E-11, were highly permissive to nodavirus infection and production. The virus-induced cytopathic effects appeared as cytoplasmic vacuoles and intensive disintegration at 3 to 5 d post-incubation. These observations were highly reproducible and formed the basis for a successful virus titration system. Quantitative analysis using the cloned E-11 cell line clearly revealed differences in the optimal growth temperatures among the 4 genotypic variants: 25 to 30 degrees C for strain SGWak97 (RGNNV), 20 to 25 degrees C for strain SJNag93 (SJNNV), 20 degrees C for strain TPKag93 (TPNNV), and 15 to 20 degrees C for strain JFIwa98 (BFNNV). Electron microscopy demonstrated SnRV retrovirus particles only in A-6 and E-9 cells, but PCR amplification for the pol gene and LTR region of the proviral DNA indicated the presence of the retrovirus in the other clones, including E-11. The cell clones obtained in the present study will be more useful for qualitative and quantitative analyses of piscine nodaviruses than the SSN-1 cell line.

RNA polymerase activity and protein synthesis in brome mosaic virus-infected protoplasts.

RNA-dependent RNA polymerase activity and protein synthesis in brome mosaic virus (BMV)-infected and uninfected protoplasts were investigated during the course of viral replication. In protoplast homogenates, the membrane-bound RNA polymerase activity resistant to actinomycin D was enhanced by BMV infection up to 30-fold that found in mock-inoculated protoplasts. The activity was first detected 7 to 8 hr postinfection. It reached a maximum at around 30 hr postinfection, then decreased gradually while virus and the viral antigen in the protoplasts, as measured by infectivity and fluorescent antibody staining, respectively, continued to increase. The bound enzyme activity was not enhanced by inoculation with BMV RNA 3 and/or RNA 4. Four proteins with molecular weights of 120, 110, 36, and 19.5 x 103 were observed in BMV-infected protoplasts from three plant species (a systemic host, a local lesion host, and a nonhost of BMV).

Effects of temperature and antibiotics on appressorium formation in spores of Colletotrichum lagenarium.

Effects of temperature (32 degrees C), cycloheximide, and blasticidin-S on spore germination and appressorium formation of Colletotrichum lagenarium were investigated. Temperature treatment at 32 degrees C, given just before the emergence of the germ tube 4 h after incubation at 24 degrees C, significantly inhibited appressorium formation. Cycloheximide (1 ppm) or blasticidin-S (7 ppm) appeared to have reversed the effect of 32 degrees C treatment by producing appressoria in 30% of the germinated spores.

Expression and characterization of the 3a movement protein of cowpea chlorotic mottle bromovirus.

Cowpea chlorotic mottle bromovirus (CCMV) 3a protein is required for cell-to-cell movement of the virus in host plants. The 3a protein was produced in Escherichia coli using an expression vector. Gel retardation analysis and UV cross-linking experiments demonstrated that the CCMV 3a protein (CC3a) bound single-stranded (ss) RNA cooperatively without sequence specificity. Binding competition analysis showed that CC3a bound ss-nucleic acids more strongly than double-stranded nucleic acids.

Resistant tobacco plants from protoplast-derived calluses selected for their resistance to Pseudomonas and Alternaria toxins.

Protoplast-derived calluses of tobacco (Nicotiana tabacum cv. 'Samsun') were selected for their resistance to toxins from Pseudomonas syringae pv. tabaci, which causes wildfire disease, and from Alternaria alternata pathotype tobacco, which causes brown spot. A number of plants were regenerated from each of the toxin-selected protoplast-derived calluses. A large percentage of the plants obtained from the second selection cycle calluses were resistant to infection by these pathogens. Resistance to wildfire disease, however, seems to be unrelated to resistance to brown spot disease. Variations in the morphological characteristics of the regenerated plants were found. Results of an assay of the R1 generation indicate that the resistance shown by R0 plants against both disease is heritable.

Genotypic and phenotypic analysis of bromovirus adaptive mutants derived from a single plant.

Eight adaptive mutant clones have been made from the total RNA extracted from uninoculated upper leaves of a single cowpea plant exhibiting systemic infection after inoculation with a hybrid cowpea chlorotic mottle bromovirus (CCMV) with the 3a movement protein gene of CCMV replaced by that of cowpea-nonadapted brome mosaic bromovirus (BMV). Sequence and mutational analyses of these clones showed genotypic and phenotypic diversity of the cloned virus population, but all examined clones had the adaptive mutation, A to C at position 776 within the BMV 3a gene, required for the systemic infection of cowpea. The data support the quasispecies model for RNA virus population, and suggest that the maintenance of the adaptive mutation may be due to powerful selection pressure in an infection process.

Temporal Transcriptional Pattern of Three Melanin Biosynthesis Genes, PKS1, SCD1, and THR1, in Appressorium-Differentiating and Nondifferentiating Conidia of Colletotrichum lagenarium.

A phytopathogenic fungus, Colletotrichum lagenarium, produces melanized appressoria that display temperature-sensitive differentiation. Conidia incubated in water at 24(deg)C germinated, and germ tubes differentiated into melanized appressoria. On the other hand, conidia incubated in water at 32(deg)C germinated and produced germ tubes that elongated without appressorium differentiation. Conidia in 0.1% yeast extract solution at 32(deg)C germinated and developed into vegetative hyphae. In this study, we investigated the temporal transcriptional pattern of cloned melanin biosynthesis genes, PKS1, SCD1, and THR1, in these differentiating and nondifferentiating conidia. During appressorium differentiation, de novo transcripts of the three melanin biosynthesis genes accumulated 1 to 2 h after the start of conidial incubation at 24(deg)C, and the amount of transcripts began to decrease at 6 h. In conidia germinating in water at 32(deg)C, the transcriptional pattern of the PKS1, SCD1, and THR1 genes was similar to that of these genes in appressorium-forming conidia, although no appressoria were formed. However, in conidia in 0.1% yeast extract solution at 32(deg)C, transcripts of the three melanin biosynthesis genes hardly accumulated.

Expression of brome mosaic virus-encoded replicase genes in transgenic tobacco plants.

We introduced replicase genes of brome mosaic virus (BMV) to Nicotiana tabacum cv. Petit Habana (SR1) using two different types of transformation vectors containing cDNAs of BMV RNA 1 and RNA 2. One type (V type) contains cDNA from which complete viral RNAs are transcribed. These RNAs can function as templates for viral replicase. The other type (M type) contains cDNA from which viral RNAs without their 3' non-coding regions are transcribed; these RNAs can only function as mRNA. Viral replicase expressed from the integrated cDNAs in both V and M type transgenic plants can complement an infection by BMV RNA 3.

Deletion of the C-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell.

The movement protein (MP) gene of brome mosaic virus (BMV) was precisely replaced with that of cucumber mosaic virus (CMV). Infectivity tests of the chimeric BMV on Chenopodium quinoa, a permissive host for cell-to-cell movement of both BMV and CMV, showed that the chimeric BMV failed to move from cell to cell even though it replicated in protoplasts. A spontaneous mutant of the chimeric BMV that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the experiments. A cloned cDNA representing the genomic RNA encoding the MP of the chimeric BMV mutant was analyzed and found to contain a mutation in the CMV MP gene resulting in deletion of the C-terminal 33 amino acids of the MP. Directed mutagenesis of the CMV MP gene showed that the C-terminal deletion was responsible for the movement capability of the mutant. When the mutation was introduced into CMV, the CMV mutant moved from cell to cell in C. quinoa, though the movement was less efficient than that of the wild-type CMV. These results indicate that the CMV MP, except the C-terminal 33 amino acids, potentiates cell-to-cell movement of both BMV and CMV in C. quinoa. In addition, since C. quinoa is a common host for both BMV and CMV, these results suggest that the CMV MP has specificity for the viral genomes during cell-to-cell movement of the virus and that the C-terminal 33 amino acids of the CMV MP are involved in that specificity.

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection.

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.

Restoration of pathogenicity of a penetration-deficient mutant of Collectotrichum lagenarium by DNA complementation.

Infection by Colletotrichum lagenarium requires formation of an appressorium and of a penetration peg. A mutant, 83,348, defective in morphogenesis of the penetration peg was unable to penetrate into cellulose membranes or infect cucumber leaves. DNA transformation using a wild-type genomic library constructed in pKVB resulted in two transformants, Ppr1 and Ppr2, with restored penetration peg formation, from 2,000 benomyl-resistant transformants. However, penetration into cellulose membranes by these transformants ranged from 30 to 40% compared to greater than 90% by wild-type. Southern-blot hybridization showed that a single copy of a cosmid clone had integrated into the genome of the transformants. A 12.0-kbp fragment of the cosmid vector with the flanking region of wild-type genomic DNA was recovered by plasmid rescue from Ppr1. Using the flanking DNA sequences as a probe for colony blot hybridization, a genomic clone was identified and designated pRP46. Transformants obtained following transformation with pRP46 were able to penetrate cellulose membranes. The penetration frequency of pRP46 transformants ranged from 25 to 65%. Transformants were also pathogenic on cucumber.