E. Tapio Palva

The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh.

We have isolated a rab-related (responsive to ABA) gene, rab18 from Arabidopsis thaliana. The gene encodes a hydrophilic, glycine-rich protein (18.5 kDa), which contains the conserved serine- and lysine-rich domains characteristic of similar RAB proteins in other plant species. The rab18 mRNA accumulates in plants exposed to low temperature, water stress or exogenous ABA but not in plants subjected to heat shock. This stress-related accumulation of the rab18 mRNA is markedly decreased in the ABA-synthesis mutant aba-1, the ABA-response mutant abi-1 or in wild-type plants treated with the carotenoid synthesis inhibitor, fluridone. Exogenous ABA treatment can induce the rab18 mRNA in the aba-1 mutant but not in the abi-1 mutant. These results provide direct genetic evidence for the ABA-dependent regulation of the rab18 gene in A. thaliana.

Differential expression of two related, low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh

Plant cold acclimation is correlated to expression of low-temperature-induced (lti) genes. By using a previously characterized lti cDNA clone as a probe we isolated a genomic fragment that carried two closely located lti genes of Arabidopsis thaliana. The genes were structurally related with the coding regions interrupted by three similarly located short introns and were transcribed in the same direction. The nucleotide sequences of the two genes, lti78 and lti65, predict novel hydrophilic polypeptides with molecular weights of 77856 and 64510, respectively, lti78 corresponding to the cDNA probe. Of the 710 amino acids of LTI78 and 600 amino acids of LTI65, 346 amino acids were identical between the polypeptides, which suggests that the genes may have a common origin.Both lti78 and lti65 were induced by low temperature, exogenous abscisic acid (ABA) and drought, but the responsiveness of the genes to these stimuli was markedly different. Both the levels and the temporal pattern of expression differed between the genes. Expression of lti78 was mainly responsive to low temperature, that of lti65 to drought and ABA. In contrast to the induction of lti78, which follows separate signal pathways during low-temperature, ABA and drought treatment, the drought induction of lti65 is ABA-dependent and the low-temperature induction appears to be coupled to the ABA biosynthetic pathway. This differential expression of two related genes may indicate that they have some-what different roles in the stress response.

Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh.

A cDNA clone corresponding to a novel low-temperature-induced Arabidopsis thaliana gene, named lti140, was employed for studies of the environmental signals and the signal pathways involved in cold-induced gene expression. The single-copy lti140 gene encodes a 140 kDa cold acclimation-related polypeptide. The lti140 mRNA accumulates rapidly in both leaves and roots when plants are subject to low temperature or water stress or are treated with the plant hormone abscisic acid (ABA), but not by heat-shock treatment. The low-temperature induction of lti140 is not mediated by ABA, as shown by normal induction of the lti140 mRNA in both ABA-deficient and ABA-insensitive mutants and after treatment with the ABA biosynthesis inhibitor fluridone. The effects of low temperature and exogenously added ABA are not cumulative suggesting that these two pathways converge. The induction by ABA is abolished in the ABA-insensitive mutant abi-1 indicating that the abi-1 mutation defines a component in the ABA response pathway. Accumulation of the lti140 mRNA in plants exposed to water stress was somewhat reduced by treatment with fluridone and in the ABA-insensitive mutant abi-1 suggesting that the water stress induction of lti140 could be partly mediated by ABA. It is concluded that three separate but converging signal pathways regulate the expression of the lti140 gene.

Bacteriophage T4 resistant mutants of the plant pathogen Erwinia carotovora

Bacterial lipopolysaccharide (LPS) has been implied in a variety of pathogenic and symbiotic plant-bacterium interactions. In order to study the role of LPS in pathogenicity of Erwinia carotovora, a broad host range phytopathogen, we isolated LPS defective mutants of two subspecies of Erwinia carotovora, subsp. carotovora (Ecc) and subsp. astroseptica (Eca). This was accomplished by using the Escherichia coli phage T4 as a selective agent. Screening of Erwinia isolates revealed that some of them were sensitive to T4 and thus T4 could be employed in mutant isolation. Fully T4 resistant mutants were all shown to be defective in their LPS structure. Preliminary pathogenicity tests on tobacco did not, however, reveal any decrease in the virulence of the LPS defective strains.

Structure and organization of two closely related low-temperature-induced dhn/lea/rab-like genes in Arabidopsis thaliana L. Heynh

We have isolated a 7 kb EcoRI genomic fragment from Arabidopsis thaliana which contains, in a tandem arrangement, two closely related dhn/lea/rab-like genes, lti29 (formerly named lti45) and cor47, corresponding to previously isolated cDNA clones. Both transcripts have been shown to accumulate in response to low temperature (LT), abscisic acid (ABA) and dehydration. Alignment of the amino acid sequences of the deduced polypeptides showed that they are 67% identical. The calculated molecular masses of the two polypeptides were 29 kDa for LTI29 and 30 kDa for COR47. Both polypeptides contain one conserved serine-stretch and three lysine-rich repeats characteristic of DHN/LEA/RAB-like proteins. In addition, both LTI29 and COR47 harbour and N-terminal acidic repeat only found in a few members amongst the DHN/LEA/RAB proteins. The close distance between the two genes (separated by 2.7 kb) and their tandem organization in the A. thaliana genome as well as the overall homology at the nucleotide sequence level of the coding region suggest that the two genes have evolved through a duplication event. This seems to be a common feature among A. thaliana LT-reponsive genes.

Cold induced gene expression in Arabidopsis thaliana L.

Exposure of Arabidopsis thaliana L. to an acclimation temperature (+4°C) results in a rapid increase of frost tolerance from −3°C to −7°C. This increase could be correlated to changes in soluble protein pattern. Analysis of in vitro translation products from isolated mRNA suggests that induction acts at the transcriptional level.

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.

Expression of pehA-bla gene fusions in Erwinia carotovora subsp, carotovora and isolation of regulatory mutants affecting polygalacturonase production

SummaryIn vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75–2, 75–5 and 75–6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 as PehA protein, respectively, fused to the mature part of the periplasmic β-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75–5 and 75–6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp− mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR−), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR− mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR−1 as well as PehA−1 mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.

The ompS gene of Vibrio cholerae encodes a growth-phase-dependent maltoporin.

The outer membrane of Vibrio cholerae contains a maltose‐inducible major protein, OmpS (43 kDa), that is common to different isolates. Nucleotide sequence analysis of the corresponding structural gene, ompS, revealed an open reading frame encoding a 412‐amino‐acid polypeptide. The amino acid sequence of OmpS is similar to that of LamB, the Escherichia coli maltoporin, and to ScrY of Klebsilla pneumoniae, although the antigenic determinants of these proteins are different. The cloned ompS gene complemented an ompS mutation of V. cholerae and the corresponding polypeptide could function as a maltoporin in a LamB‐ mutant of E. coli. The promoter region of ompS is highly homologous to the malK‐lamB promoter of E. coli and the ompS gene is controlled by MalT in E. coli. This indicates that the same kind of regulatory mechanism is used to activate the ompS expression in V. cholerae and malK‐lamB expression in E. coli. An ompS‐lacZ transcriptional fusion was used to demonstrate a dual control in ompS expression; the ompS gene is responsive to the inducers maltose and trehalose but in their absence it is also expressed in response to growth‐phase. These different modes of induction might be of importance during different stages of V. cholerae infection.

Occurrence of bacteriophage T4 receptor in Erwinia carotovora

SummaryWe have tested for the presence of the receptor for the Escherichia coli phage T4 in different isolates of the plant pathogenic enterobacteria Erwinia carotora subsp. carotovora and subsp. atroseptica. Several of the isolates appeared to contain a functional T4 receptor as shown by phage adsorption and phage-induced lysis of the bacteria. Two of the isolates could even sustain lytic growth of T4. In addition, we show that the transducing derivative of T4, T4GT7, can be employed to transfer plasmids from E. coli to E. carotovora thus opening up new possibilities for genetic analysis of Erwinia.