Efficient regeneration systems for two closely related Moricandia species possessing a C3 or C3-C4 intermediate photosynthetic character.

The C(3)-C(4) intermediate species Moricandia arvensis ( Brassicaceae) and its closest C(3) relative, Moricandia moricandioides, represent model species for studying the C(3)-C(4) photosynthetic character relative to the C(3) phenotype. In order to enable transgenic analyses in these two species, optimal regeneration systems based on leaf and/or stem internode segments were developed, and genotypes suitable for in vitro tissue culture were identified. Evaluation of the regeneration capability of 30 M. arvensis genotypes and 12 M. moricandioides genotypes revealed that all could form callus. However, shoots were only produced by 40% of the M. arvensis genotypes and 8% of the M. moricandioides genotypes. The two Moricandia species showed significant genotypic differences with respect to callus formation and shoot regeneration. For the 12 regenerative M. arvensis genotypes, 29-100% of the explants developed shoots, while 71% of the explants from the single regenerable M. moricandioides genotype formed shoots. The genotype used, the choice of stem or leaf explants and the composition of the medium (i.e. concentrations of different hormones and salts) significantly affected plant regeneration (chi-square analyses, P<0.05). Whole plants could be obtained in the greenhouse after 3-3.5 months for M. arvensis genotypes and after 4-4.5 months for M. moricandioides.

Rice tungro spherical virus polyprotein processing: identification of a virus-encoded protease and mutational analysis of putative cleavage sites.

Rice tungro spherical virus encodes a large polyprotein containing motifs with sequence similarity to viral serine-like proteases and RNA polymerases. Polyclonal antisera raised against domains of the putative protease and polymerase in fusion with glutathione S-transferase detected a protein of about 35 kDa and, in very low amounts, a protein of about 70 kDa, respectively, in extracts from infected plants. In in vitro transcription/translation systems and in Escherichia coli we demonstrated a proteolytic activity in the C-terminal region of the polyprotein. This protease rapidly cleaved its polyprotein precursors in vitro. Mutating a potential cleavage site located N-terminal to the protease domain, Gln2526-Asp2527, diminished processing. The transversion mutation at the putative C-terminal cleavage site of the protease, at Gln2852-Ala2853, led to a delayed and partial processing.

Amino acids of alfalfa mosaic virus coat protein that direct formation of unusually long virus particles.

In contrast to most alfalfa mosaic virus (AMV) strains (YSMV, S, M and 425), AMV strains VRU and 1 5/64 can form abnormally long virus particles, an ability which has been linked to the coat protein (CP). In order to study this phenomenon, the CP-encoding RNAs 3 of AMV strains VRU and 1 5/64 were cloned and fully sequenced. Comparative sequence analyses of AMV RNA 3 sequences derived from different strains revealed two non-conservative amino acid substitutions, Ser65 and Leu175, which occur exclusively in the closely related VRU- and 15/64-CPs. When these amino acid alterations were introduced into the CP of AMV strain 425 unusually long virus particles were assembled. This confirms that amino acids Ser66 and Leu175 of the CPs of AMV strains VRU and 15/64 are involved in the formation of tubular virus particles.

Rice tungro spherical virus: nucleotide sequence of the 3' genomic half and studies on the two small 3' open reading frames.

Rice tungro spherical virus (RTSV) consists of a single-stranded RNA genome of about 12 kilobases that contains one large open reading frame, ORF 1 and two small ORFs 2 and 3 at its 3' end (Shen et al., 1993, Virology 193:621-630); it was suggested that ORF 2 was expressed via a frameshift. To study the genomic information of RTSV and the variation between different RTSV isolates, the 3' half of a Philippine isolate and parts of a Thai and an Indian isolate were cloned and sequenced. Significant sequence differences were found in ORF 2 and in the 3' non-translated region. Additional stop codons have been revealed in the previously described ORF 2 in several independent clones from the three different virus isolates, the most conserved stop codon in the middle of ORF 2 being confirmed by direct RNA sequencing. These results suggest that ORF 2 could only express a peptide of about 5 kDa instead of 12 kDa as proposed earlier. Polyclonal antisera were raised against ORF 2 and 3 proteins as fusions with glutathione-S-transferase. Using these antisera we failed to detect any virus-specific peptides in extracts from infected rice plants and in virus preparations. The nucleotide sequence of the 3' end of our RTSV isolates contains several small ORFs and does not contain a repeat of 256 nucleotides found in the published sequence. These results indicate that RTSV could contain an unusually long 3' non-coding region of 1240 nucleotides in length.

Complete nucleotide sequence of the RNA 3 from subgroup II of cucumber mosaic virus (CMV) strain: Trk7.

The complete nucleotide sequence (2209 nucleotides) of the RNA 3 of the cucumber mosaic virus strain Trk7 was determined. Trk7-CMV possesses two open reading frames which encode the 3a protein (279 amino acids) and the coat protein (218 amino acids). Sequence analysis proved the earlier biological and serological classification of the RNA 3 molecule of Trk7-CMV in subgroup II. A high degree of homology was found in the strains Q and Kin of CMV, while the average similarity between the two different subgroups is much lower.

Cloning and sequencing of potato virus Y (Hungarian isolate) genomic RNA.

A sequence of 9703 nucleotides (nt) is reported for the genomic RNA of potato virus Y (Hungarian isolate, PVY-H), which causes necrotic rings around the buds on the tubers and mottling of leaves. The sequence contains one large open reading frame of 3061 amino acids (aa), a noncoding region of 189 nt at the 5' end and a 330-nt 3' nontranslated region. The nt sequence and the predicted aa sequence of the polyprotein of PVY-H were analysed pairwise with the only available complete sequence of PVY strain N (PVYn) and with the partial sequences of different PVY strains, as well as with other potyviruses and potyvirus-related plant viruses. The overall relationship between PVY-H and PVYn shows a nt sequence identity of 88.5% and an aa sequence identity of 94.2%. The lowest degree of homology was detected at the 5' terminus of the genome, including the 5' noncoding region (70.3%) and the 275-aa P1 protein (78%). A fivefold sequence repeat block of 5'-UUUCA was found in the 5' noncoding region of PVY-H, which seems to be characteristic of PVY strains.

Efficient pathogen-derived resistance induced by integrated potato virus Y coat protein gene in tobacco.

The coat protein (CP) gene from potato virus Y (Hungarian isolate, PVY-H) was engineered into Agrobacterium tumefaciens binary vector for expression in different tobacco lines. Three different Nicotiana tabacum breeding lines were transformed and the integration of the CP gene was confirmed by PCR technique using genomic DNA preparations. The transcription and expression of the integrated CP gene was detected by Northern and Western blots. Pathogen-derived resistance was demonstrated by inoculation of the R1 progeny of the transformed lines with purified PVY-H. The efficiency of protection varied between different transgenic plants ranging from almost complete to no protection. Five CP expressing tobacco lines were resistant to challenge infection with PVY-H as indicated by attenuation or absence of symptom development associated with reduction or lack of detectable virus accumulation. Data from Western blots showed that there is no correlation between the level of the expressed CP and the extent of protection. This suggests that the mechanism of the observed resistance is independent of the level of CP accumulation in the transgenic tobacco plants.