Destabilization of potato spindle tuber viroid by mutations in the left terminal loop.

Infectivity studies with highly infectious RNA inocula generated by ribozyme cleavage were used to compare the biological properties of three apparently nonviable mutants of potato spindle tuber viroid (PSTVd). One of these mutants (PSTVd-P) contains three nucleotide substitutions in the left terminal loop, and mechanical inoculation of tomato seedlings with RNA transcripts at levels equivalent to 10(3)-10(5) times the ID50 for PSTVd-Intermediate failed to result in systemic infection. Viable progeny containing a spontaneous C-->G change at position 4 could, however, be recovered from transgenic Nicotiana benthamiana plants that constitutively expressed PSTVd-P RNA. The initial mutations in PSTVd-P led to an overall weakening of its native structure in vitro, and the precisely-full-length molecule released by ribozyme cleavage in vivo was also unstable. Even RT-PCR analysis failed to reveal detectable amounts of circularized PSTVd-P among the RNAs isolated from uninfected plants. Predicted stabilizing effects of a spontaneous mutation at position 4 suggest that the appearance of viable progeny was dependent on a combination of events: errors by host RNA polymerase II during transcription of the mutant transgene coupled with a strong selective pressure against alterations in the native structure of PSTVd.

Role of the variable domain in modulating potato spindle tuber viroid replication.

Potato spindle tuber viroid (PSTVd) is believed to undergo a series of specific structural transitions during replication. The variable domain of PSTVd is known to contain sequences that are important for replication/accumulation as well as one of three "premelting regions" which control breakdown of the native structure in vitro. We have examined the structural and biological effects of five single and two double nucleotide substitutions within premelting region 3 in an effort to isolate temperature-sensitive mutations affecting PSTVd replication or pathogenesis. None of these mutants replicated as rapidly as the wild type, and a variety of spontaneous sequence changes were detected in their progeny. Higher temperatures were able to partially overcome the inhibition of replication associated with a more stable secondary structure, but no well-defined temperature-sensitive PSTVd mutants were identified. Selective pressures arising from the interaction of assay temperature and structural stability in vivo appear capable of moving PSTVd populations between peaks of relatively high fitness. Depending on the exact nature and location of the mutation, selection may occur at the level of either the plus or the minus strand.

Molecular cloning and biochemical characterization of a receptor-like serine/threonine kinase from rice.

A receptor-like protein kinase, OsPK10, has been cloned from rice (Oryza sativa). The 2.8 kb cDNA contains an open reading frame capable of encoding a peptide sequence of 824 amino acids. The topological features of the predicted OsPK10 protein include an N-terminal signal peptide, a cysteine-rich extracellular ligand-binding domain, a membrane-spanning segment, and a cytoplasmic domain possessing all the hallmarks of catalytic domains of eukaryotic protein kinases. The cytoplasmic domain was selectively expressed in Escherichia coli and assayed for kinase activity. The results show the protein is capable of autophosphorylation using either ATP or GTP as the phosphate donor. Phosphoamino acid analysis reveals phosphorylation of threonines, consistent with the substrate specificity indicated by sequence motifs in the catalytic core. A single amino acid substitution of Glu for Lys-528 completely abolishes autophosphorylation activity. DNA gel blot analyses suggest that the haploid rice genome contains a single copy of the OsPK10 gene. OsPK10 transcripts appear to be more abundant in shoots than in roots of rice seedlings.

Cloning and characterization of a novel member of protein kinase family from soybean.

The cDNA coding for a novel protein kinase from soybean (Glycine max L.), named GmPK6, was sequenced. The primary sequence of GmPK6 consists of 462 amino acids with an N-terminal sequence similar to the central region of Xenopus U1 snRNP 70K protein, and a C-terminal kinase domain representing structural mosaicism with features diagnostic of both protein serine/threonine and tyrosine kinases in eukaryotic organisms. The GmPK6 gene is expressed as 2.5 kb transcript in a variety of tissues.

Molecular identification of a soybean protein kinase gene family by using PCR.

In this study we report identification of six members of a protein kinase gene family from soybean (Glycine max L.). Two fully degenerate oligonucleotide primers corresponding to two conserved motifs (DLK-PENV and GTHEYLAPE) in the catalytic domains of eukaryotic protein serine/threonine kinases were used in a polymerase chain reaction (PCR) to amplify soybean cDNA. Sequence analysis showed that 28 of the PCR sequences represented six different putative protein serine/threonine kinases. These results not only demonstrate that catalytic domains of protein kinases are highly conserved between plants and other eukaryotes but also suggest that there are multiple genes encoding protein kinases in plants.

Restoration of shooty morphology of a nontumorous mutant of Nicotiana glauca x N. langsdorffii by cytokinin and the isopentenyltransferase gene.

The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids.

Anther culture of wheat (Triticum aestivum L.) F1's and their reciprocal crosses.

Anthers from three sets of wheat (Triticum aestivum L. em. Thell) F1's and their reciprocal crosses, made between parental lines differing greatly in their ability to produce microspore derived callus, were cultured on the Chinese potato medium so that we could 1) more clearly define the role of nuclear or cytoplasmic factors within T. aestivum in transferring the ability to undergo in vitro androgenesis, and 2) to briefly review the gametic representation and disease screening potential of the resulting polyhaploid wheat plants. The microspore derived calli values from F1's were slightly less than the midparental value. Statistical analysis indicated that the ability of each F1 to produce callus either did not significantly differ from that of the respective parental line having the highest androgenic yield or it exceeded its respective parental line having the lowest yield. No differences were noted between the members of each pair of reciprocal crosses. The results indicate that the transfer of in vitro androgenic ability to F1 hybrids is not dependent upon the maternal cytoplasm source. Polyhaploid plants, carrying the Pm 3 a powdery mildew resistance gene, expressed resistance to culture 4 a of powdery mildew.