Colorado potato beetle larvae on potato plants expressing a locust proteinase inhibitor.

The natural defence system of plants often involves inhibitors of digestive enzymes of their pests. Modem and environmental-friendly methods try to increase this plant resistance by expressing heterologous protease inhibitors in crops. Here we report the effects of expressing a gene from desert locust (Schistocerca gregaria) encoding two serine protease inhibitors in potato on Colorado potato beetle (Leptinotarsa decemlineata) larvae. The gene encoding both peptides on a single chain was used for Agrobacterium-mediated transformation of potato plants. The presence of the active inhibitor protein in the leaves was verified. The feeding bioassays in the laboratory showed that despite the low level of the peptide in leaves, CPB larvae on transgenic plants have grown slightly but significantly more slowly than those on control potato plants. The results support the notion that expression of multifunctional proteinase inhibitors of insect origin in plants might be a good strategy to improve insect resistance.

Tissue-specific signal(s) activate the promoter of a metallocarboxypeptidase inhibitor gene family in potato tuber and berry.

The molecular basis of the differential expression of the GM7-type metallocarboxypeptidase inhibitor (MCPI) genes in tuberizing (StMCPI) and non-tuberizing Solanum species (SbMCPI) was investigated. It was shown that the StMCPI is encoded by a gene family in Solanum tuberosum (potato), but SbMCPI might be a single-copy gene in the non-tuberizing species Solanum brevidens. The StMCPI promoter shows evolutionary relatedness to the S. brevidens-derived SbMCPI and to the fruit-specific tomato promoter 2A11. Both StMCPI and SbMCPI promoter regions were able to confer tuber- and berry-specific expression for the beta-glucuronidase reporter gene in potato suggesting that the difference in MCPI gene expression is in trans regulatory factors between the tuberizing and the non-tuberizing Solanum species. The MCPI promoters did not respond to metabolic, environmental or hormonal signals in leaves. Thus, the MCPI genes are regulated in a different way than the other known tuber-specific genes and potentially are suitable for biotechnological application in potato to provide specific transgene expression in tuber and berry.

Potato StubSNF1 interacts with StubGAL83: a plant protein kinase complex with yeast and mammalian counterparts.

StubSNF1 is a potato cDNA that encodes a protein kinase similar to the yeast SNF1 gene involved in transcriptional regulation of glucose-repressible genes. The yeast SNF1 functions in a complex with GAL83/SIP1/SIP2 and SNF4 proteins. We have used StubSNF1 as bait in a yeast two-hybrid system to screen for potato cDNAs encoding proteins that bind to StubSNF1. Three overlapping cDNAs, two different in size, were isolated. DNA sequence analysis revealed that they were orthologues of the yeast GAL83/SIP1/SIP2 genes and their mammalian counterparts, AMPK beta-subunits. The direct interaction between the potato proteins StubGAL83 and StubSNF1 was shown by an in vitro binding assay. Southern and Northern hybridisations revealed that StubGAL83 exists in a low copy number in the potato genome and is highly (but organ-specifically) expressed in potato. In contrast, StubSNF1 possesses low transcript levels in each organ, except in flowers where high amounts of StubSNF1 mRNA could be detected. We demonstrate here that StubGAL83 can also interact with yeast SNF4 in a yeast two-hybrid system suggesting that plant SNF1 kinases may function in complexes similar to those detected in yeast and mammals.

Potato protein kinase StCPK1: a putative evolutionary link between CDPKs and CRKs.

Calcium-dependent protein kinases (CDPKs) in plants are characterized by a four-domain structure including conserved sequences in the catalytic domain, and in the C-terminal calmodulin-like domain. Based on this conservation we have PCR-amplified and isolated a potato cDNA clone (StCPK1) from a library representing an early stage of tuber development. DNA sequence analysis revealed that in the catalytic domain, StCPK1 shares more homology with CDPK-related kinases than with CDPKs; however, like CDPKs, it possesses canonical EF-hands at the calmodulin-like 3' end. StCPK1 exists in a few copies in the potato genome and is abundantly expressed in the sepals of mature flowers. Floral expression of genes homologous to StCPK1 appears to be widespread in the family Solanaceae.

Isolation and sequence analysis of a cDNA and a related gene for cytochrome P450 proteins from Solanum chacoense.

Inosine-containing degenerate PCR primers corresponding to the heme-binding domain of cytochrome P450 proteins have been synthesized and used for cloning cDNAs by the RT-PCR technique from Solanum chacoense. One clone in which the primer was immediately followed by sequences corresponding to the remaining part of the conserved domain was obtained. A leaf cDNA and a genomic library were constructed from S. chacoense. Clones homologous to the PCR fragment were isolated by plaque hybridization from both libraries (CYPs.ch-1 and CYPs.ch-2, respectively). Based on DNA sequence analysis, the selected clones are 87.6% identical and belong to the CYP71 family. The CYPs.ch genes are present in multiple copies in the S. chacoense as well as in the S. tuberosum genome with some polymorphisms. The CYPs.ch transcripts are slightly induced by methyl jasmonate and abscisic acid in S. chacoense foliage.

Comparative studies on potato tuber development using an in vitro tuber induction system.

A method for synchronized in vitro tuber induction in a Hungarian cultivar of Solanum tuberosum designated "Keszthelyi 855" has been developed. It was shown that in this system tuberization and stolon elongation primarily depend on the level of sucrose in the medium. The cytokinin, 6-bensylaminopurine (BAP), also enhances the efficiency of tuber formation, however, only at sucrose concentration above 4% (w/v). The synchronized plant culture provided starting material for isolation of genes specifically expressed in tuberizing Solanum species during the early stage of tuber development. In comparison with the non-tuberizing Solanum brevidens, three types of specific transcripts have been obtained by differential screening. Based on DNA sequence analysis the genes isolated code for the major tuber proteins, patatin and proteinase inhibitors.

Starch synthesis, and tuber storage protein genes are differently expressed in Solanum tuberosum and in Solanum brevidens.

Studying in vitro stem cuttings of Solanum tuberosum induced for tuberization and those of a non-tuberizing Solanum species, differences both in morphology and in gene expression were detected. Stolon formation essentially depended on light while tuberization was triggered by the elevated level of sucrose in the medium. Genes involved in starch synthesis were induced by sucrose in both species, however, starch granules were detected only in potato. A new tuber specific cDNA clone, GM7, encoding a putative metallocarboxypeptidase inhibitor and the cDNA of a proline rich cell wall protein with S. brevidens specific expression were isolated by differential screening. Sucrose mediated transcription of the tuber storage proteins like patatin and proteinase inhibitors (Kunitz-type, winI, GM7) failed in S. brevidens.

The cycHJKL genes of Rhizobium meliloti involved in cytochrome c biogenesis are required for "respiratory" nitrate reduction ex planta and for nitrogen fixation during symbiosis.

We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix-) and "respiratory" nitrate reduction (Rnr-). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr- and Fix- phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Cc11 and Cc12 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multisubunit cytochrome c haem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.

Isolation and characterization of a water-stress-inducible cDNA clone from Solanum chacoense.

A rich source of valuable genes are wild species. Solanum chacoense Bitter with its extreme resistance to viruses, insects and drought, is a good example. In the present study, a stress gene, designated DS2, has been isolated from S. chacoense. We have shown that the expression of the gene is organ-specific being detected in leaf, stem and stolon, but not in root, tuber or flower. Treatment of detached leaves with abscisic acid (ABA), salicylic acid or methyl jasmonate resulted in only very moderate accumulation of DS2 mRNA. Thus, DS2 represents a very rare type of the water-stress-inducible genes whose signalling pathway is not primarily related to ABA. Based on DNA sequence analysis, DS2 encodes a putative protein starting with 20 amino acids homologous to the ABA- and water-stress-inducible, ripening-related (ASR) proteins of tomato continued by an insert of 155 amino acids structurally similar to certain LEAs (late embryogenesis-abundant proteins) and ending in 88 amino acids homologous again to the ASR sequences and to an unpublished partial cDNA fragment isolated from the root of rice. The N-terminal region of the DS2 protein is hydrophilic with ten 13-mer amino acid motifs and random coil structure. In contrast, the C-terminus predicts an alpha-helix and possesses a bipartite nuclear targeting sequence motif. These data suggest that the function of the DS2 may be the protection of the nuclear DNA from desiccation.

Solanum brevidens possesses a non-sucrose-inducible patatin gene.

The patatin gene is the best known "tuber-specific" gene of potato (Solanum tuberosum). Patatin is encoded by a multigene family that can be divided into two classes. Class I genes are highly expressed in tubers and are sucrose inducible, while class II genes are under developmental control and are expressed mainly in root tips. Here we report the isolation and characterization of cDNA clones corresponding to a patatin gene of the non-tuberizing Solanum species S. brevidens. We show that the gene is 94-100% homologous to the class I type patatin genes of S. tuberosum; the homology includes the sequences in the 5' and the 3' untranslated regions. However, the patatin gene of S. brevidens is regulated like class II type patatin genes and cannot be transcriptionally activated by elevated levels of sucrose. This result further supports the idea that the components required for tuberization may be present in non-tuberizing solanaceous plants, but are regulated differently.

nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum.

The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.

Inhibition of tumor induction in tobacco by Agrobacterium tumefaciens and nodulation induced by Rhizobium meliloti in the presence of phenothiazines and structurally related compounds.

Plasmids of Agrobacterium tumefaciens and Rhizobium meliloti carrying Kanamycin resistance genes were eliminated from 1.4 to 0.2% of the growing bacterial cultures by promethazine and imipramine. As a result of plasmid elimination, the A. tumefaciens plasmidless isolate was not able to induce crown gall tumor on tobacco plants. The plasmidless R. meliloti strain failed to induce nodule formation on alfalfa plants. The efficiency of nodulation was decreased when the bacteria were grown in the presence of the drugs. The antiplasmid effects of the drugs were not prevented by opines, (nopaline and octopine) in Escherichia coli F'lac cells.

Six nodulation genes of nod box locus 4 in Rhizobium meliloti are involved in nodulation signal production: nodM codes for D-glucosamine synthetase.

The nucleotide sequence of the nod box locus n4 in Rhizobium meliloti was determined and revealed six genes organized in a single transcriptional unit, which are induced in response to a plant signal such as luteolin. Mutations in these genes influence the early steps of nodule development on Medicago, but have no detectable effect on Melilotus, another host for R. meliloti. Based on sequence homology, the first open reading frame (ORF) corresponds to the nodM gene and the last to the nodN gene of Rhizobium leguminosarum. The others do not exhibit similarity to any genes sequenced so far, so we designated them as nolF, nolG, nolH and nolI, respectively. We found that the n4 locus, and especially the nodM and nodN genes, are involved in the production of the root hair deformation (Had) factor. NodM exhibits homology to amidotransferases, primarily to the D-glucosamine synthetase encoded by the glmS gene of Escherichia coli. We demonstrated that in E. coli the regulatory gene nodD together with luteolin can activate nod genes. On this basis we showed that nodM complemented an E. coli glmS- mutation, indicating that nodM can be considered as a glmS gene under plant signal control. Moreover, exogenously supplied D-glucosamine restored nodulation of Medicago by nodM mutants. Our data suggest that in addition to the housekeeping glmS gene of R. melioti, nodM as a second glmS copy provides glucosamine in sufficient amounts for the synthesis of the Had factor.

Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction.

A fix region of Rhizobium meliloti 41 involved both in symbiotic nodule development and in the adsorption of bacteriophage 16-3 was delimited by directed Tn5 mutagenesis. Mutations in this DNA region were assigned to four complementation units and were mapped close to the pyr-2 and pyr-29 chromosomal markers. Phage inactivation studies with bacterial cell envelope preparations and crude lipopolysaccharides (LPS) as well as preliminary characterization of LPS in the mutants indicated that these genes are involved in the synthesis of a strain-specific LPS. Mutations in this DNA region resulted in a Fix- phenotype in AK631, an exopolysaccharide (EPS)-deficient derivative of R. meliloti 41; however, they did not influence the symbiotic efficiency of the parent strain. An exo region able to restore the EPS production of AK631 was isolated and shown to be homologous to the exoB region of R. meliloti SU47. By generating double mutants, we demonstrated that exo and lps genes determine similar functions in the course of nodule development, suggesting that EPS and LPS may provide equivalent information for the host plant.

Production of root hair deformation factors by Rhizobium meliloti nodulation genes in Escherichia coli: HsnD (NodH) is involved in the plant host-specific modification of the NodABC factor.

The role of the hsnD (nodH) gene in the determination of the host-specific nodulation ability of Rhizobium meliloti was studied by expressing the common nodulation genes (nodABC) with or without the hsnD gene in Escherichia coli and testing for biological activity on various leguminous plants. In this way, four categories of plants were established. Upon infection with E. coli carrying the nodABC construct, root hair deformation (Had) was detected on clovers while the hsnD gene was additionally needed for the elicitation of the same response on alfalfa and sweet clover. A weak root hair deformation was seen on siratro by inoculation with E. coli harbouring the nodABC genes and was highly increased when hsnD was also introduced. Cowpea and Desmodium did not respond to any of the E. coli strains constructed. Exudates or cytosolic fractions of the respective E. coli derivatives elicited the same root hair deformation as the intact bacteria. These data indicate that not only the nodABC gene products but also the hsnD product are involved in the synthesis of Had factors. Subclones expressing only the nodA, nodB, or nodC genes or the same genes in pairs (nodAB, nodBC, nodAC) did not provide a compound with activity comparable to the NodABC factor, suggesting that all three genes are required for the production of the Had factor which is active on clover. Coinoculation of alfalfa plants with two strains of E. coli, one carrying the nodABC genes and the other expressing only hsnD, or combining exudates or cytosolic fractions from these strains did not result in root hair deformation on alfalfa. These data indicate that the HsnD protein itself or its product is not an additional alfalfa-specific extracellular signal but more likely is enzymatically involved in the modification of the basic compound determined by the nodABC genes.

Regulation of nod gene expression in Bradyrhizobium japonicum.

The best inducers of nod::lacZ translational fusions in Bradyrhizobium japonicum are isoflavones, primarily genistein and daidzein. Upstream of the nodABC genes in B. japonicum is a novel gene, nodY, which is coregulated with nodABC. Measurements of the activity of lacZ fusions to the nodD gene of B. japonicum show that this gene is inducible by soybean seed extract and selected flavonoid chemicals. The induction of the nodY ABC and nodD operons appears to require a functional nodD gene, indicating that the nodD gene product controls its own synthesis as well as other nod genes.

A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).

Rhizobium meliloti insertion element ISRm2 and its use for identification of the fixX gene.

Two of the three plasmids of the wild-type Rhizobium meliloti 41 (pRme41a and pRme41c) carry a copy of ISRm2, a 2.7-kilobase-long transposable element. ISRm2 is terminated by 22-base-pair (bp) inverted repeat sequences, exhibiting some homology to the inverted repeats of elements generating 9-bp target sequence duplication. Transposition of ISRm2 results in a duplication of 8 bp in length, rather rare among transposable elements. DNA sequences homologous to an internal fragment of ISRm2 were found in several Rhizobium species. Transposition of ISRm2 into fixation and nodulation genes located on the symbiotic plasmid pRme41b was detected at a high frequency. Exact locations of two copies of ISRm2 which transposed into the nod-nif region on the megaplasmid were determined. In one case, integration into the protein-coding region of the hsnD gene that determines a host specificity function of nodulation occurred. In the other mutant, ISRm2 was localized upstream of nifA, where a short open reading frame coding for a new fix gene (fixX) was identified. The product of fixX is a ferredoxin carrying a characteristic cluster of cysteine residues. On the basis of the observation that the arrangement of the ISRm2 copies is identical in the free-living wild-type cells and in nitrogen-fixing nodules, we concluded that the involvement of ISRm2 transposition in the development of nitrogen-fixing symbiosis is unlikely.

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation.

Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod-) or nitrogen fixation (Fix-). Seven Nod- mutants were isolated from strain USDA110 and from strain 61 A101 C, 4 Nod- mutants and 14 Fix- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His- Nod- mutants of USDA110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three non-auxotrophic Nod- mutants and one His-Nod- mutant of USDA110. Homogenotization of the cloned fragments into wild-type strain USDA110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.

Genes for the catabolism and synthesis of an opine-like compound in Rhizobium meliloti are closely linked and on the Sym plasmid.

In alfalfa nodules induced by Rhizobium meliloti strain L5-30 the compound L-3-O-methyl-scyllo-inosamine (3-O-MSI) is synthesized. This compound is also catabolized specifically by this strain. Its biological properties are therefore similar to the Agrobacterium opines. To answer the question whether opine-like compounds ("Rhizopines") play a role in a plant symbiotic interaction, we isolated the genes for the catabolism of 3-O-MSI (moc genes) and for the induction of its synthesis in the nodule [mos gene(s)]. moc and mos genes were shown to be closely linked and located on the Sym plasmid of L5-30, suggesting that they have co-evolved and may be important in symbiosis. These genes have been cloned into a broad host-range vector that can be mobilized into other R. meliloti strains where they are expressed. The location of the mos genes in the bacteria extends the opine concept, initially developed for a plant pathological interaction, to a symbiotic one.