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B i o t e c h n o l o g y news and views Stanton B. Gelvin

Biotechnology News and Views will appear quarterly. Its purpose will be to summarize interesting publications in the recent plant molecular biology literature.

Gene induction in plant-microbial interactions The genetic, biochemical, and physiological responses of plants to microorganisms has been an area of active research for decades. More recently, the molecular responses of specific genes, both in the host plant and in the microbial invader, have been analyzed. Because the precise functions of bacterial genes which are activated during the interaction are often unknown or difficult to assay, the use of 'reporter genes' whose activity is easy to assay has received increasing attention. These reporter genes, whose activities include antibiotic resistance, bioluminescence and /3-galactosidase, have been inserted as transcriptional and/or translational fusions into the bacterial chromosome or plasmids and the relevant activity measured before and after induction by plants or plant exudates. Such a system for delivering a /3-galactosidase gene relatively randomly into the genomes of several plant pathogenic bacteria has recently been described by Stachel et al. (EMBO J. 4, 891-898, 1985). The system includes a vector, p H o H o l , consisting of the transposon Tn 3 defective in transposase activity and which contains a promoterless lac operon positioned just inside one of the inverted repeats. The expression of the /3-galactosidase gene is dependent upon read-through transcription or translation of the gene into which p H o H o l has inserted. A second plasmid, pSShe, provides the transposase activity to effect transposition of the Tn3-1ac transposon. The authors have used this system to identify genes in the octopine catabolism (occ) region of an Agrobacterium tumefaciens Tiplasmid which are induced by octopine, and genes in the virulence (vir) region of the Ti-plasmid which are activated in response to cocultivation of the bacteria with plant cells. These vir-lac fusions strains of Agrobacterium have subsequently been

used by several laboratories to identify specific plant compounds which activate the vir genes. A similar approach has been used by Olson et al. (Biotechnology Feb., 143-149, 1985). These authors have employed a promoterless lac operon contained on the transposon mini mu-dI (Kan, lac) in the narrow host range suicide plasmid pGS6 to mutageneize a fast growing strain of Rhizobium japonicum, USDA 201. The relative randomness of mu insertion in this bacterium was demonstrated by the percentage (0.3%) of the recipients which were auxotrophic for a variety of amino acids or nucleosides, and by the percentage (4%) which were defective in nodulation or nitrogen fixation. Three of the 1000 mutants which were tested showed increased levels of/3-galactosidase activity after incubation of the bacteria with root extracts or exudates of soybean or kidney bean, but not of the non-hosts pea, clover, alfalfa, or corn. The induction therefore appears to be specific and such a system should be useful in identifying and characterizing bacterial genes activated during the early stages of nodulation. Such an analysis of genes necessary for the nodulation of alfalfa by Rhizobium meliloti has recently been initiated by Mulligan and Long (Proc. Natl. Acad. Sci. USA 82, 6609-6613, 1985). The nodulation (nod) genes nod A, B, and C form an operon and are located on a large megaplasmid. These genes are not highly expressed in free living bacteria grown in broth. Using lac translational fusions, the authors were able to show that the nod C gene is activated by plant exudates and that this activation is dependent upon an active copy of the nod D gene, which forms a different transcriptional unit on the R. meliloti megaplasmid. On the other side of things, plant-microbe interactions have been known for some time to alter gene activity in the plant host. One of the best stud-

368 ied such systems is the nodulation interaction between leguminous plants and Rhizobium species. Plant encoded proteins induced by the bacteria, such as the leghemoglobins, have been termed nodulins and several recent publications have investigated the biosynthesis and functions of these proteins. Govers et al. (EMBO J. 4, 861-867, 1985) have isolated mRNA from root nodules of pea various times after infection by Rhizobium leguminosarum and translated this RNA in vitro. The resultant proteins were subsequently analyzed by two-dimensional PAGE. Eight plant encoded genes, including four leghemoglobins and nodulins N-21, N-40, N-40', and N-68, were strongly induced during the nodulation process, but not necessarily with the same developmental kinetics. N-40' is expressed very early in nodulation, and both it and N-68 are expressed earlier than the leghemoglobin genes. N-21 mRNA is only seen late in nodule development, and is the major nodulin missing in the nodules of plants infected by nod + fix- bacteria. These results suggest that, except for N-21, the expression of many nodulins is not dependent upon bacteroid development, heme excretion, or nitrogen fixation. Two publications from D.P.S. Verma's laboratory have described the primary structure of two soybean nodulin genes. Nodulin-24 (Katinakis and Verma, Proc. Natl. Acad. Sci. USA 82, 4157-4161, 1985) is a polypeptide which is incorporated into the peri-bacteroid membrane synthesized during nodulation of soybean roots. It has an apparent molecular weight of 24000, but sequencing of the gene shows that it really is 15 100 daltons. The three central exons (of five) encode hydrophobic domains, apparently accounting for the aberrant mobility of the protein on SDS-polyacylamide gels. Each of these three exons is bounded by a 12-base pair inverted repeat, and thus each of these protein domains appears to be encoded by gene duplications resembling insertion sequences. Nodulin-35 is a subunit of a nodule-specific uricase, and cDNA and genomic clones for the soybean gene have recently been characterized (Nguyen et al., Proc. Natl. Acad. Sci. USA 82, 5040=5044, 1985). The gene is almost 5 kilobase pairs in length, containing eight exons and seven introns, several of which are quite large. The protein apparently is a nodule-specific uricase, in that its mRNA is not found in young soybean roots and

leaves which contain another uricase activity. The mRNA is translated on free cytoplasmic polyribosomes, and the protein, which does not contain a cleaved signal sequence, is located in peroxisomes. This is therefore the first report of the isolation of a gene for a peroxisomal polypeptide. Recent studies have also concentrated on the molecular biology of plant-fungal interactions. The production of the mRNA for hydroxyproline-rich glycoproteins (HRGPs), a n important constituent of plant cell walls, following infection of bean hypocotyls by the filamentous Ascomycete Colletotrichum lindenmuthianum has recently been described by Showalter et al. (Proc. Natl. Acad. Sci. USA 82, 6551-6555, 1985). This pathogen causes the disease anthracnose in beans. Using a tomato HRGP gene probe, the accumulation of this mRNA was demonstrated in elicitor treated bean suspension culture cells. In bean hypocotyls, using a race of the fungus which elicited an incompatible reaction, the increase in HRGP mRNA was rapid, corresponding well with the elicitation of the hypersensitive response by the plant. Using a pathogenic race of fungus to infect a susceptible cultivar of plant (compatible reaction), the accumulation of HRGP mRNA was considerably delayed. Interestingly, in both types of interactions cells far from the infection site accumulated HRGP mRNA.

Agrobacterium tumefaciens chromosomal genes affecting virulence Numerous molecular and genetic studies on Agrobacterium tumefaciens have concentrated on elucidating the function of bacterial genes involved with tumorigenesis. Most of these experiments have involved the analysis of the auxin and cytokinin biosynthetic genes in the T-DNA and the virulence (vir) genes on the Ti-plasmid. It has long been known, however, that genes on the bacterial chromosome can influence virulence (Garfinkel and Nester, J. Bacteriol. 144, 732-743, 1980). Some of these genes are involved with attachment of the bacteria to the plant cell wall (Douglas et al., J. Bacteriol. 152, 1265-1275, 1982). More recently, these authors have further characterized these chromosomal genes involved with bacterial attachment (Douglas et al., J. Bacteriol. 161, 850-860, 1985)

369 Cosmid clones containing large segments of the Agrobacterium chromosome were isolated from a number of Tn5 mutagenized strains which were avirulent and did not attach well to plant cells, and the mutations mapped. All mutations were localized to an 11 kilobase pair region of DNA, and marker exchange experiments with the mutagenized DNA and DNA from wildtype cells indicated that the mutant phenotype was associated with the presence of Tn5. The lac fusion transposon Tn3::HoHol was used to map more specifically these genes responsible for plant attachment. Two operons, designated chvA and B (for chromosomal virulence) were localized and shown to be transcribed in opposite orientations. ~galactosidase activity of these lac=chv fusions was not stimulated by cocultivation of these bacteria with plant cells indicating that, unlike certain vir genes, these chv genes are not inducible by plant products. Close et al. (J. Bacteriol. 164, 774-781, 1985) have recently isolated an Agrobacterium tumefaciens chromosomal mutant which can regulate the expression of certain Ti-plasmid vir genes. The mutation, called ros (rough surface) is pleiotropic, and bacteria harboring it show altered colony morphology and cold-sensitivity for growth. The mutation was originally isolated by selecting Agrobacterium colonies, containing a promoterless CAT gene inserted into a Ti-plasmid vir gene, which could grow on chloramphenicol. The vir gene into which the CAT gene was inserted is normally inactive except when the bacteria are cocultivated with plant cells, normally resulting in chloramphenicol sensitive bacteria. Plasmid transfer experiments indicated that the mutation was on the Agrobacterium chromosome and that the chloramphenicol resistance demonstrated by the ros strains did not merely result from mutations in vir gene sequences upstream of the fused CAT gene. The authors concluded that the ros gene product may function as a negative regulator of vir gene activity, and may therefore be involved in expressing Ti-plasmid functions essential for virulence.

Recombinant DNA technology and plant virology Increasingly, recombinant DNA technology has been applied to the analysis of DNA and RNA plant viruses and to viroids. In some instances, as

with cauliflower mosaic virus and reverse transcripts of viroids, the cloned DNA is directly infectious. In other cases, as with some RNA viruses (Ahlquist et al., Proc. Natl. Acad. Sci. USA 81, 7066-7070, 1984), the cloned sequences must be transcribed in vitro and the resulting RNA used for infection. Recently, in vitro mutagenesis of cloned cDNA fragments of the 3'-terminal region of brome mosaic virus (BMV) RNA 3 was carried out (Bujarski et al., Proc. Natl. Acad. Sci. USA 82, 5636-5640, 1985). Deletion mutations were made in the arms of the tRNA-like structure using restriction endonucleases and S1 nuclease, and the resulting DNA fragments placed in SP6 transcription vectors. Following in vitro transcription, the mutagenized RNA fragments were assayed in vitro for replication and aminoacylation activities. Specific deletions had various affects on these activities: Arm C contains a tyrosyl anticodon, and mutations in this arm inhibited replicase but not aminoacylation activity. Some mutations in arm B were detrimental to replicase activity, but most inhibited aminoacylation. The complete removal of arm D did not affect aminoacylation, and sometimes stimulated replicase activity. These results reiterated the importance of previous experiments in showing that the anti-codon arm of BMV is important for replicase template recognition. Previously, Tabler and Sanger (EMBO J. 3, 3055-4062, 1984) had shown that cloned single and double stranded copies of potato spindle tuber viroid (PSTV) are infectious. These same authors have followed up on these results and further investigated the infectivity of cloned PSTV multimers (Tablet and Sanger, EMBO J. 4, 2191-2199, 1985). From one to six PSTV cDNA copies were placed head-to-tail in SP6 transcription vectors and the RNA resulting from in vitro transcription (of either the + or - polarity) used to infect tomato plants. Except for the monomer + strand polarity construct, all + strand oligomers were highly infectious. Minus strand constructs were either avirulent or extremely attenuated i.n their ability to produce symptoms. When these - strand RNAs were capped or mixed with non-infectious + strand fragments, they became infectious. The reason for the lack of infectivity of the - strand RNA oligomers is curious in that both + and - strand PSTV DNA oligomers are infectious. The authors

370 suggest five possible reasons why the - strand RNA oligomers are not infectious, including problems involving the transport, processing, replication, and stability of these species. Protection of the - strand oligomers by capping or complexing with + strand fragments may alleviate some of these potential problems. In viroid infected plants, longer than unit length viroid plus strands have been detected. These have been postulated to be replication intermediates, produced perhaps by a rolling circle mechanism, which are cleaved by an endonuclease and ligated to form viroid monomers. Both double and single stranded viroid cDNA clones are infectious, but only in a multimer form. The suggestion has been made that the lack of infectivity of the monomer form of the viroid cDNA clones derives from the lack of two endonuclease processing sites. It should therefore be possible to construct greater than monomer (but less than dimer) length viroid cDNAs which, should they contain the postulated processing site, be infectious. This has recently been shown by Meshi et al. (Mol. Gen. Genet. 200, 199-206, 1985) using hop stunt viroid cDNA clones. Only clones containing two copies of a 64bp region of the viroid, one viroid unit length apart and in the correct orientation, were infectious. This region can form an intramolecular structure which is similar to those formed by most other viroids except avocado sunblotch viroid. The authors propose that, upon infection, the cDNA

clones are first transcribed in the host cells, cleaved to unit length monomers at the two processing sites, and then replicate as usual. Because the replication cycle of cauliflower mosaic virus (CaMV) involves the generation of a full length viral cRNA molecule, it may be expected that if this RNA (the 355 CaMV RNA) contains introns, these should be spliced out. That this may be true has recently been demonstrated by Hirochika et al. (EMBO J. 4, 1673-1680, 1985). These authors noted that an 856 bp site specific deletion often occurred during the replication of the CaMVS strain. The DNA sequence around the deletion site suggested that RNA donor and acceptor splicing consensus sequences may be used to remove an intron from the ORF I and II regions of the CaMV 355 RNA. This supposition is most likely correct, and was shown by the analysis of point mutations generated in the putative donor splicing sequence (a GT to GG or GA transversion was generated by in vitro site-directed mutagenesis). These new sequences were no longer used as donor splice sites; rather, new cryptic donor sites were utilized along with, in most instances, the same splice acceptor site. These results are consistent with the reversetranscription model of CaMV DNA replication. The authors suggested that, because the rate of splicing activity may be detrimental to maintaining an intact CaMV genome, the S strain may contain a mutation in the regulatory activity.

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