Activation of hsr203, a plant gene expressed during incompatible plant-pathogen interactions, is correlated with programmed cell death.

hsr203J is a tobacco gene whose activation is rapid, highly localized, and specific for incompatible interactions between tobacco and the bacterial pathogen Ralstonia solanacearum. The effect of other hypersensitive response (HR)-inducing pathogens and elicitors has been tested with transgenic plants containing the hsr203J promoter-GUS reporter gene fusion, and confirms the generality of the preferential inducibility of the hsr203J gene promoter during incompatible interactions: bacterial and viral pathogens inducing an HR in tobacco were able to induce the promoter fusion, as were inducers of HR-like responses such as harpin, elicitins, and PopA1 proteins. A tomato hsr203 homologous cDNA was isolated (Lehsr203) and used to examine the effect of avr gene products on the expression of such genes. Lehsr203 was shown to be rapidly and transiently induced in leaves of the tomato Cf-9 line, following Avr9 product infiltration, but not in those of the Cf-0 line. Among potential effectors of HR or resistance such as H2O2, salicylic acid, methyl jasmonate, and 2,6-dichloro-isonicotinic acid (INA), none is able to induce a significant increase in promoter activation. In contrast, heavy metals that cause leaf necrosis can trigger such an activation. In addition, hsr203-GUS fusion expression is detected in transgenic tobacco lines expressing the bO gene and exhibiting spontaneous HR-like lesions. Taken together, these results demonstrate a strong correlation between hsr203 and genetically controlled cell death in tobacco and tomato. The expression of this gene should be a useful marker for programmed cell death occurring in response not only to diverse pathogens, but also to diverse death-triggering extracellular agents.

Transformation of Azotobacter vinelandii with plasmids RP4 (IncP-1 group) and RSF1010 (IncQ group).

Multicopy plasmid RSF1010 and four of its in vitro-constructed derivatives were mobilized by the self-transmissible RP4 plasmid into Azotobacter vinelandii UW. Modifications of the Escherichia coli transformation procedure of Cohen et al. (Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114, 1972) allowed transformation of A. vinelandii strains UW and ATCC 12837 with purified RP4 or RSF1010 deoxyribonucleic acid.

HSR203 antisense suppression in tobacco accelerates development of hypersensitive cell death.

Activation of the tobacco gene hsr203 is rapid, highly localized, specific for incompatible plant-pathogen interactions, and strongly correlated with programmed cell death occurring in response to diverse pathogens. Functional characterization of hsr203 gene product has shown that HSR203 is a serine hydrolase that displays esterase activity. We show here that transgenic tobacco plants deficient in HSR203 protein exhibit an accelerated hypersensitive response when inoculated with an avirulent strain of Ralstonia solanacearum. This response was accompanied by a maximal level of cell death and a drastic inhibition of in planta bacterial growth. Transgenic plants deficient in HSR203 were also found to show increased resistance in a dosage-dependent manner to Pseudomonas syringae pv. pisi, another avirulent bacterial pathogen, and to virulent and avirulent races of Phytophthora parasitica, a fungal pathogen of tobacco, but not to different virulent bacteria. Surprisingly, expression of another hsr gene, hsr515, and that of the defence genes PR1-a and PR5, was strongly reduced in the transgenic lines. Our results suggest that hsr203 antisense suppression in tobacco can have pleiotropic effects on HR cell death and defence mechanisms, and induces increased resistance to different pathogens.

Developmental and pathogen-induced activation of an msr gene, str 246C, from tobacco involves multiple regulatory elements.

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5' flanking DNA sequence from the str246C gene fused to the beta-glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5' deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 bp was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.

[Abdominal aortic aneurysm and Bourneville's tuberous sclerosis]. / Anévrysme de l'aorte abdominale et sclérose tubéreuse de Bourneville.

The authors report on a case of tuberous sclerosis diagnosed in the neonatal period on the basis of intracardiac tumor, rib anomalies and cerebral calcifications. At 4.5 months of age the infant presented acute abdominal pains which led to the discovery (ultrasound identification confirmed by CT scan) of a giant ectasia of the whole abdominal aorta. The infant died 2 days later from the rupture of this aortic aneurysm.

Identification of a novel pathogen-responsive element in the promoter of the tobacco gene HSR203J, a molecular marker of the hypersensitive response.

The tobacco gene, HSR203J, which is specifically activated during the early steps of incompatible plant/pathogen interactions has been shown to be a molecular marker of the hypersensitive response (HR). It constitutes an ideal model for the identification of HR-responsive cis-regulatory elements. As a first step in the promoter dissection, deletion mutants of the 5' flanking sequence of HSR203J fused to the GUS reporter gene were analyzed. Then, the construction and study of chimeric constructs containing HSR203J promoter fragments fused to a minimal promoter enabled us to identify a 28-bp regulatory element located between -106 and -79 upstream of the transcription initiation site. This element has been shown to be necessary and sufficient for transcriptional activation in response to pathogen. It contains a 10-bp palindrome followed by its imperfect repeat. The mutagenesis of these two sequence elements led to the identification of a 12-bp motif termed HSRE (HSR203 responsive element) responsible for the marked induction of the HSR203J gene during the HR. Since this DNA region did not show any homology with known regulatory sequences, this 12 bp motif corresponds to a novel cis-regulatory element.

Effects of antisense oligodeoxynucleotide hybridization on in vitro translation of potato virus Y RNA.

Potato virus Y (PVY), a potyvirus, has an RNA genome containing 9704 nucleotides of which 185 belong to the 5' nontranslated region (NTR). Contrary to most eukaryotic mRNAs that have a cap structure, the potyvirus RNA has a genome-linked protein (VPg). In order to understand the mechanisms of PVY RNA translation initiation, hybrid-arrest translation was used to localize sequences involved in binding of proteins and/or ribosomes. The 5' NTR was fused to the beta-glucuronidase (GUS) reporter gene. Six antisense oligodeoxynucleotides were used for hybridization, and the efficiency of the in vitro translation of the hybridized mRNA was modified to different levels depending upon the position of the oligodeoxynucleotide used. The highest inhibition was obtained with an oligodeoxynucleotide hybridized to the 5' end. In addition, translation of GUS mRNA containing the PVY 5' NTR was greatly enhanced when this mRNA was capped. These results differ from those obtained with the tobacco etch virus (TEV) and three picornaviruses, but are similar to those obtained with capped mRNA.

Nucleotide sequence of potato virus Y (N Strain) genomic RNA.

The complete nucleotide sequence of the genomic RNA of the potyvirus potato virus Y strain N (PVYn) was obtained from cloned cDNAs. This sequence is 9704 nucleotides long and can encode a polyprotein of 3063 amino acids. The positions of the cleavage sites at the N terminus of the capsid and cytoplasmic inclusion proteins have been determined. Other putative protein cleavage sites have been deduced by searching for consensus sequences and by analogy with the polyprotein of the tobacco vein mottling virus and of the tobacco etch virus. Comparison of the PVY polyprotein sequence with that of other potyvirus polyproteins shows similarities in genome organization and a high level of identity along most of the polyprotein, except for the putative proteins flanking the helper component. A search for specific protein motifs has revealed the existence of a potential metal-binding site at the putative N terminus of the helper component in potyviruses. The possible functions of this structure are discussed.