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Brome mosaic virus (BMV), a tripartite RNA plant virus, accumulates RNA3-derived defective RNAs (D-RNAs) in which 477–500 nucleotides (nt) are deleted in the central region of the 3a protein open reading frame (ORF), after prolonged infection in barley. In the present study, six artificial D-RNAs (AD-RNAs), having deletions of the same size as the naturally occurring D-RNA but at different positions in the 3a ORF, were constructed and tested for their amplification and encapsidation in barley protoplasts by coinoculation with BMV RNA1 and 2, or RNA1, 2, and 3. Northern blot analysis of RNA accumulation in total and virion fractions showed that deletions of 492 nt in the 3′-proximal and the 5′-proximal regions of the 3a ORF decreased encapsidation efficiency of the AD-RNAs compared with that of RNA3, whereas deletions in the central region enhanced encapsidation efficiency. The present results also show that deletion positions affect competition with RNA3 in the amplification and encapsidation of AD-RNAs.

A hybrid Cowpea chlorotic mottle virus (CCMV) [CCMV(B3a)] in which the CCMV 3a movement protein gene is replaced by the 3a (B3a) gene of Brome mosaic virus cannot infect cowpea systemically. Previously, analysis of RNA3 cDNA clones constructed from cowpea-adapted mutants derived from CCMV(B3a) revealed that a single codon change in the B3a gene allowed CCMV(B3a) to infect cowpea systemically. In this study, to extend the analysis of the CCMV(B3a) adaptation mechanism, we directly sequenced B3a gene RT-PCR products prepared from 28 cowpea plants in which cowpea-adapted mutants appeared, and found seven patterns of a codon change localized at five specific positions in the central region (Ser 118 , Glu 132 , Glu 138 , Gln 178 , and Ser 180 ). All of the patterns involved an amino acid change to Lys or Arg. Mutational analysis of the B3a gene demonstrated that a single codon change resulting in either Lys or Arg at any of the five positions was sufficient for the adaptation of CCMV(B3a) to cowpea. In contrast, CCMV(B3a) variants with a codon change resulting in Lys or Arg at three other positions (137, 155, and 161) in the B3a gene not only showed lack of systemic infection of cowpea but also showed weakened initial cell-to-cell movement in the inoculated leaves and diminished B3a accumulation in protoplasts. These results suggest that adaptive changes in the B3a gene are site-specifically selected in cowpea plants.

Cucumber mosaic cucumovirus (CMV) and brome mosaic bromovirus (BMV) have many similarities, including the three-dimensional structure of virions, genome organizations, and requirement of the coat protein (CP) for cell-to-cell movement. We have shown that a chimeric BMV with the CMV 3a movement protein (MP) gene instead of its own cannot move from cell to cell in Chenopodium quinoa, a common permissive host for both BMV and CMV. Another chimeric BMV was constructed by replacing both MP and CP genes of BMV with those of CMV (MP/CP-chimera) and tested for its infectivity in C. quinoa, to determine whether the CMV CP has some functions required for the CMV MP-mediated cell-to-cell movement and to exhibit functional difference between CPs of BMV and CMV. Cell-to-cell movement of the MP/CP-chimera occurred, and small local lesions were induced on the inoculated leaves. A frameshift mutation introduced in the CMV CP gene of the MP/CP-chimera resulted in a lack of cell-to-cell movement of the chimeric virus. These results indicate that the viral movement mediated by the CMV MP requires its cognate CP. Deletion of the amino-terminal region in CMV CP, which is not obligatory for CMV movement, also abolished cell-to-cell movement of the MP/CP-chimera. This may suggest some differences in cell-to-cell movement of the MP/CP-chimera and CMV. On the other hand, the sole replacement of BMV CP gene with that of CMV abolished viral cell-to-cell movement, suggesting a possibility that the viral movement mediated by the BMV MP may also require its cognate CP. Functional compatibility between MP and CP in viral cell-to-cell movement is discussed.

Biosynthesis of melanin by Colletotrichum lagenarium is essential for appressorial penetration of its host plants. Carpropamid is a novel melanin-inhibiting fungicide for control of rice blast caused by Magnaporthe grisea. In C. lagenarium, appressorial and colonial melanization were inhibited by carpropamid. Accumulation of melanin intermediates, scytalone, was also shown in the culture medium containing carpropamid. One of the enzymes involved in the melanin biosynthesis, scytalone dehydratase catalyzes the conversion of scytalone to 1,3,8-trihydroxynaphthalene. We previously cloned SCD1 gene coding for scytalone dehydratase of C. lagenarium. In this study, a recombinant SCD1 expression vector was constructed using pMAL system and the recombinant scytalone dehydratase was purified from transformed Escherichia coli. Recombinant SCD1 showed scytalone dehydratase activity in an in vitro reaction using scytalone as substrate. The activity was assayed by measuring the production of 1,3,8-trihydroxynaphthalene or disappearance of scytalone, which was detectable by reverse-phase high-pressure liquid chromatography. Dehydration of scytalone to 1,3,8-trihydroxynaphthalene was inhibited by carpropamid in the in vitro reaction.

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 1 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.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes, but distinct host ranges: BMV systemically infects the monocot barley, while CCMV systemically infects the dicot cowpea. We have previously shown that in approximately 10% of inoculated cowpea plants, a CCMV hybrid [CCMV(B3a)] with the 3a cell-to-cell movement protein gene replaced by that of cowpea-nonadapted BMV directs systemic infections, which are caused by secondary mutation(s) of the hybrid virus. Here, to further analyze the role of RNA3 in adaptation to a new host, RNA3 cDNA clones were constructed from total RNA recovered from the uninoculated upper leaves of systemically infected cowpea plants inoculated with CCMV(B3a). Sequence and mutational analysis of two such RNA3 clones revealed that a single codon change (A776 → C) in a conserved motif of the 3a movement protein gene conferred compatibility for systemic infection of a new host, cowpea, suggesting that this site in the 3a gene is directly or indirectly involved in crucial host interactions associated with host-range specificity. The adaptive hybrid viruses carrying this mutation induced exacerbated symptoms, while wt CCMV appeared nearly symptomless, showing that the bromovirus 3a movement protein gene can significantly contribute to regulating symptom development. However, introducing this cowpea-adaptive mutation into the BMV genome had little effect on the ability of BMV to systemically infect barley.

The effects of some chemical disinfectants, organic solvents, hydrogen ions, heat, ultraviolet (UV) irradiation and ozone on the inactivation of striped jack nervous necrosis virus (SJNNV) were investigated. SJNNV was inactivated by contact with a final concentration of 50 ppm of sodium hypochlorite, calcium hypochlorite, benzalkonium chloride and iodine for 10 min at 20 °C. Cresol concentrations of more than 10 000 ppm were required to inactivate SJNNV, and no inactivation of SJNNV by formalin was detected at any concentration tested. The effective concentrations of ethanol and methanol were 60% and 50%, respectively, but SJNNV was resistant to ether and chloroform. SJNNV was inactivated by high alkalinity, pH 12 for 10 min at 20 °C, and also inactivated by heat treatment at 60 °C for 30 min. Inactivation of SJNNV by UV irradiation was observed at an intensity of 410 μW cm−2 for 244 s. Ozone at 0.1 μg ml−1 as a total residual oxidant was required to inactivate SJNNV for 2.5 min. Washing fertilized eggs and the treatment of sea water with ozone decreased the rate of occurrence of VNN.

Many particles of a non-occluded baculovirus with a membrane 70 × 330 nm in size were observed in the nuclei of the mid-gut glands of larval Kuruma shrimp ( Penaeus japonicus ) with mid-gut necrosis using an electron microscope. Nucleotides of the baculovirus were isolated from the diseased larvae in the presence of Triton X-100. The nucleocapsid was a bacilliform virion, approximately 50 nm in width and 260 nm in length. From the purified preparations of nucleocapsids of the virus, two major proteins with molecular weights of 35 and 14 kDa were identified by SDS-polyacrylamide gel electrophoretic analysis. The viral genomic DNA extracted from the nucleocapsids was digested with Bam HI and Sau 3AI, but not with Eco RI, Pst I, Xho I and Sal I. The molecular weight of the DNA was estimated to be approximately 85.1 × 10 6 from the agarose gel electrophoretic pattern of the Bam HI fragments. A part of the Bam HI fragments was cloned, labeled with 32 P-CTP and used for Southern blot analysis as a probe. The fragment hybridized to DNA isolated from the diseased larvae, but not to DNA isolated from healthy larvae.

1,8-Dihydroxynaphthalene (1,8-DHN) was isolated from Cochliobolus heterostrophus pale green mutant M13BL19 ( Alb1 + Brn1 + Sal1 + Pgr1-1 ) as the active substance which restored melanization of HE1AS73 ( Alb1-8 Brn1-2 Sal1-1 Pgr1 + ). HE1AS73 oxidized 1,8-DHN, 1-naphthol, N,N -dimethyl- p -phenylenediamine, tetramethylbenzidine, syringaldazine, and 3-(3,4′-dihydroxyphenyl)alanine, but did not oxidize p -cresol and l -tyrosine. None of these compounds was oxidized by HE5R13 ( Alb1-8 Brn1-2 Sal1-1 Pgr1-1 ). The syringaldazine oxidation activity of HE1AS73 was inhibited by diethyldithiocarbamate, cetyltrimethylammonium bromide, and dithiopyrimidine. These results indicate that the Pgr1 + strain contained p -diphenol oxidase which was absent from the Pgr1 − strain. We consider from these results that p -diphenol oxidase, in which Pgr1 is involved, plays a role in melanin synthesis of C. heterostrophus and 1,8-DHN is a natural substrate of this enzyme.

Three mutants of Colletotrichum lagenarium , with appressoria that could not penetrate nitrocellulose membranes or cell walls of the host plant, were obtained by mutagenic treatment. Appressoria of these mutants were identical in shape and colour to those of the parent strain. Scanning electron microscopy revealed that one mutant (83182) formed a penetration peg at the basal part of the appressoria and that the other mutants (82335 and 83348) did not form a penetration peg. The latter mutants are considered to have a defect in morphogenesis of the penetration peg. Application of Czapek liquid medium to mutant 83182 after appressorium formation partially restored appressorial penetration without halo formation; this mutant synthesized a protein, P-95, which was closely related to cellulose degrading enzymes. These results suggest that mutant 83182 is deficient in a secretion or translocation system for these enzymes.

A melanin biosynthetic enzyme, scytalone dehydratase, which converts scytalone to 1,3,8-trihydroxynaphthalene was purified from mycelia of Cochliobolus miyabeanus. Enzymatic reactions were carried out under anaerobic conditions to prevent unfavorable oxidative reactions. This enzyme had an optimum pH near 8.2 and mol w 23,000 Da. The enzymatic reaction did not require NADPH as a cofactor and was not inhibited by the melanin biosynthesis inhibitors, tricyclazole, PP 389, pyroquilon, PCBA, and phthalide

Using the thymidine kinase gene-containing plasmid pTK-1 and the mouse fibroblast thymidine kinase-deficient LTK − cell line, we obtained results indicating that the plasmid molecules suspended in the external medium were introduced into the nuclei by pricking cells on the nuclei with a microneedle. Approx. 25% of the cells pricked in the presence of the plasmid pTK-1 showed TK gene expression, and 2% of the cells became stable TK + transformants. This ‘pricking’ method is applicable for introducing DNA into cell nuclei and is more efficient than the conventional calcium phosphate transformation method.

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.

Cerulenin [(2S) (3R)2,3-epoxy-4-oxo-7,10-dodecadienoylamide], a polyketide synthesis inhibitor, inhibited appressorial pigmentation of Colletotrichum lagenarium at concentrations higher than 10 μg/ml. The inhibitory concentrations of cerulenin inhibited neither spore germination nor appressorium formation but did inhibit penetration of nitrocellulose membranes by penetrating hyphae from appressoria. The colorless appressoria germinated laterally on nitrocellulose membranes and rarely penetrated them. In the presence of cerulenin, treatment with scytalone, a melanin precursor for this fungus, restored appressorial pigmentation and also penetration by appressoria of nitrocellulose membranes. In Czapek liquid medium, mutant 8015 which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene in the melanin biosynthetic pathway produced scytalone after 9 days of incubation when hyphal growth reached the maximum level. Application of 10 μg/ml of cerulenin at 9 days of incubation inhibited further production of scytalone by mutant 8015. From these results, it is concluded that appressorial melanin of C. lagenarium was synthesized via the polyketide pathway.

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 × 103 were observed in BMV-infected protoplasts from three plant species (a systemic host, a local lesion host, and a nonhost of BMV).

Regulation of melanin biosynthesis in relation to appressorium differentiation ofColletotrichum lagenarium was investigated. When spores of the parent strain 104-T were incubated at 24°C, appressorial pigmentation started at 6 h of incubation and was preceded by appressorim swelling; appressoria were darkly pigmented at 12 h of incubation. The same time course of appressorial pigmentation was observed in albino mutant 79215 when scytalone, a natural precursor of melanin biosynthesis, was applied before the swelling of appressoria. In accordance with this result, [14C]scytalone was not incorporated into germlings of albino mutant 79215 before the swelling of appressoria. Cycloheximide applied 1 h or more after incubation of spores of the parent strain 104-T, or of albino mutant 79215 treated with scytalone, inhibited neither appressorium formation nor appressorial pigmentation. These results indicate that enzymes involved in melanin biosynthesis subsequent to scytalone are preexisting enzymes or synthesized as inactive forms during 1 h of incubation, and that they are activated during appressorium differentiation. In addition, an early step(s) prior to scytalone in the melanin biosynthesis of appressoria was temperature sensitive; when colorless appressoria of the parent strain 104-T formed during 6 h of incubation at 24°C were postincubated at 32°C, the appressoria did not melanize, whereas application of scytalone to the postincubation at 32°C permitted melanization of the appressoria. Also, albino mutant 79215 formed melanized appressoria during postincubation at 32°C in the presence of scytalone. These results indicate that high temperatures inhibit melanin biosynthesis by inhibiting one or more steps prior to scytalone synthesis.

Melanin biosynthesis by appressoria was studied in relation to their penetrating ability using tricyclazole [5-methyl-1,2,4-triazolo(3,4-b)benzothiazole], pp 389 [4,5-dihydro-4-methyltetra-zolo(1,5-a)quinazolin-5-one], and pyroquilon [1,2,5,6-tetrahydropyrrolo(3,2,1-i,j)quinolin-4-one], and color mutants of Colletotrichum lagenarium. Tricyclazole at 100 μM inhibited melanin biosynthesis by appressoria of C. lagenairum 104-T, and caused accumulation of 3,4-dihydro-4,8-dihydroxy-1(2H)naphthalenone (DDN) in the culture medium. By contrast, DDN was not detected in culture media of tricyclazole-treated mutant 8015, which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene (1,3,8-THN). Vermelone restored melanization of appressoria of albino mutant 79215 and of the parent strain 104-T treated with tricyclazole, pp 389, and pyroquilon; however, scytalone restored melanization only in appressoria of albino mutant 79215. These results indicate that tricyclazole, pp 389, and pyroquilon inhibit the conversion of 1,3,8-THN to vermelone in the melanin biosynthetic pathway of appressoria of C. lagenarium. Colorless appressoria formed in the presence of the three melanin-inhibiting chemicals germinated laterally on nitrocellulose membranes and rarely penetrated the membranes. On the other hand, when pigmented appressoria were restored by application of vermelone in the presence of the three chemicals, lateral germination of the appressoria was largely suppressed, and the membranes were effectively penetrated. From these results, it is concluded that the major effect of tricyclazole, pp 389, and pyroquilon on appressoria of C. lagenarium, causing failure of penetration, is the inhibition of melanization. Effects of the chemicals on other metabolic functions can be precluded as significant factors affecting the penetration process.

Melanin biosynthesis of appressoria inColletotrichum lagenaru was studied using color mutants. Mutant 8015 ofC. lagenarium obtained byN-methyl-N′-nitro-N-nitroso-guanidine treatment formed a red-brown colony and secreted a substance which restored black coloration to colonies of albino mutants. This substance was identified as scytalone (3,4-dihydro-3,6,8-trihydroxy-1(2H)naphthalenone). Nineteen albino mutants formed colorless appressoria, but in the presence of 0.75 mM scytalone, the albino mutants formed darkly pigmented appressoria indistinguishable from those of the parent strain. Furthermore, the time course of appressorial pigmentation of albino mutants in the presence of scytalone was the same as that of the parent strain. On nitrocellulose membranes and host cucumber cotyledons, the colorless appressoria of albino mutants germinated laterally to form secondary appressoria, and consequently had little ability to form penetration hyphae. In the presence of 0.75 mM scytalone, however, the pigmented appressoria penetrated nitrocellulose membranes and host cucumber cell walls similarly to those of the parent strain. From these results, we conclude that scytalone is a normal precusor of melanin in appressoria, and that appressorial pigmentation is essential for the formation of penetration hyphae inC. lagenarium.

Color mutants of Cochliobolus miyabeanus defective in melanin biosynthesis were isolated. Although the wild-type strain KU-13 formed dark green colonies, color mutants formed white, brown, and gray colonies or white colonies with red pigment secretion. From the white mutant which secreted red pigment, designated scy , a melanin precursor which restored melanization of albino mutants alm-1 was isolated and identified as scytalone. This indicated that scy mutant was defective in the conversion of scytalone to 1,3,8-trihydroxynaphthalene and that melanin of this fungus is of pentaketide origin formed from oxidation of 1,8-dihydroxynaphthalene. Albino mutants alm-1 were considered to be defective in pentaketide cyclization and brown mutants brm were considered to be defective in the conversion of 1,3,8-trihydroxynaphthalene to vermelone. Albino mutants alm-2 whose coloration was not restored by application of scytalone were also isolated. The alm-2 gene was believed to be a gene transactively regulating the pentaketide cyclization and conversion of scytalone. From crossing experiments among the color mutants, it was indicated that alm-1, alm-2 , and brm were linked and that scy segregates independently of these three mutant loci. Crossing of a methionine requiring mutant with alm and scy indicated that the three loci segregate independently of each other.