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The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gene function. This technique has so far been difficult to apply to plants because the frequency of gene disruption following transformation with constructs containing DNA homologous to genomic sequences is low (0.01 to 0.1%). It has recently been shown that high rates of gene disruption (up to 90%) can be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of gene disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isolated segments of 13 other closely related members of the Cab gene family. In the 199-bp stretch sequenced, the 13 new members of the Cab family show an average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 304 silent substitutions and 16 substitutions that lead to a change in the amino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab genes) into a vector containing a selectable hygromycin resistance marker, and used this construct to transform P. patens. In three of nine stable transformants tested, the construct had inserted in, and disrupted, the ZLAB1 gene. There was no discernible phenotype associated with the disruption. We have therefore shown that gene disruption is reproducible in P. patens and that the requirement for sequence homology appears to be stringent, therefore allowing the role of individual members of a gene family to be analysed in land plants for the first time.

Three nodulation-deficient (nod) mutants of Rhizobium leguminosarum were isolated following insertion of the transposon Tn5 into pRL1JI, the R. leguminosarum plasmid known to carry the nodulation genes. DNA adjacent to the nod: Tn5 alleles was subcloned and used to probe a cosmid clone bank containing DNA from a Rhizobium strain carrying pRL1JI. Two cosmid clones which showed homology with the probe contained about 10 kb of DNA in common. The R. leguminosarum host-range determinants were found to be present within this 10 kb common region since either of the cosmid clones could enable a cured R. phaseoli strain to nodulate peas instead of Phaseolus beans, its normal host. Electron microscopy of nodules induced by Rhizobium strains cured of their normal symbiotic plasmid but containing either of the two cosmid clones showed bacteroid-forms surrounded by a peri-bacteroid membrane, indicating that normal infection had occurred. Thus it is clear that this 10 kb region of nodDNA carries the genes that determine host range and that relatively few bacterial genes may be involved in nodule and bacteroid development.

Mutant strains of Rhizobium leguminosarum have been isolated in which the transposon Tn5 was inserted into the cloned nodulation genes. The mutant strains were examined for their ability to induce nodules and to induce normal root hair curling on peas. Five distinct classes of nod mutations were identified on the basis of the phenotypes of the various mutant strains, and of the mapped locations of the nod:: Tn5 alleles. Three classes of mutants (I, II and III) were defective in nodulation and two classes (IV and V) were delayed in nodule appearance. Mutations of classes I, II and III types affected root hair curling and a subcloned 6.6 kb fragment of DNA containing this region conferred the ability to induce root hair curling upon a Rhizobium strain deleted of its nod genes. Analysis of protein products formed after in vitro transcription and translation of the plasmid DNA corresponding to the class I, II, III and IV regions indicated that proteins of molecular weights 48,000, 45,000, 36,000 and 34,000 were formed. A comparison of protein products formed by equivalent plasmids carrying nod:: Tn5 alleles indicated that the class I mutations blocked the synthesis of the 45,000 molecular weight peptide and that the two products of molecular weights 36,000 and 34,000 were absent from the class II mutants. It is concluded that at least five nod genes are involved in nodulation and the polypeptide products of some of these genes have been identified.