Barley stripe mosaic virus-encoded proteins triple-gene block 2 and gammab localize to chloroplasts in virus-infected monocot and dicot plants, revealing hitherto-unknown roles in virus replication.

Replication of Barley stripe mosaic virus (BSMV), genus Hordeivirus, is thought to be associated with vesicles in proplastids and chloroplasts, but the molecular details of the process and identity of virus proteins involved in establishing the virus replication complexes are unknown. In addition, BSMV encodes a triple-gene block of movement proteins (TGBs) that putatively share functional roles with their counterparts in other hordei-, pomo- and pecluviruses, but detailed information on the intracellular locations of the individual TGBs is lacking. Here, the subcellular localizations of BSMV-encoded proteins TGB2 and gammab fused to green or red fluorescent proteins were examined in epidermal cells of Nicotiana benthamiana and barley (Hordeum vulgare 'Black Hulless'). The fusion proteins were expressed from a BSMV vector or under the control of the cauliflower mosaic virus 35S promoter. The subcellular localizations were studied by confocal laser-scanning microscopy (CLSM). CLSM studies showed that both proteins were recruited to chloroplasts in the presence of viral RNA and that virus RNA, coat protein and gammab protein were detected in plastid preparations from infected leaves. Electron microscope images of thin sections of virus-infected leaves revealed abnormal chloroplasts with cytoplasmic inclusions containing virus-like particles. In addition, cellular localizations of BSMV TGB2 suggest subtle differences in function between the hordei-like TGB2 proteins. The results indicate that TGB2 and gammab proteins play a previously unknown functional role at the site of virus replication.

Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria.

Cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) catalyses the conversion of cinnamoyl-CoAs into their corresponding cinnamaldehydes, i.e. the first step of the phenylpropanoid pathway specifically dedicated to the monolignol biosynthetic branch. In previous work, we described the isolation and characterisation of the first cDNA encoding CCR in Eucalyptus (Lacombe, E., Hawkins, S., Van Dorsselaere, J., Piquemal, J., Goffner, D., Poeydomenge, O., Boudet, A.M., Grima-Pettenati, J., 1997. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant Journal 11, 429--441) and shown the role of this enzyme in controlling the carbon flux into lignins (Piquemal, J., Lapierre, C., Myton, K., O'Connell, A., Schuch, W., Grima-Pettenati, J., Boudet, A.M., 1998. Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant Journal 13, 71--83). Here, we report the characterisation of two functionally and structurally distinct cDNA clones, AtCCR1 and AtCCR2 (81.6% protein sequence identity) in Arabidopsis thaliana. The two recombinant proteins expressed in Escherichia coli are able to use the three cinnamoyl-CoAs tested but with different levels of efficiency. AtCCR1 is five times more efficient with feruloyl-CoA and sinapoyl-CoA than AtCCR2. In addition, the two genes are differentially expressed during development and in response to infection. AtCCR1 is preferentially expressed in tissues undergoing lignification. In contrast, AtCCR2, which is poorly expressed during development, is strongly and transiently induced during the incompatible interaction with Xanthomonas campestris pv. campestris leading to a hypersensitive response. Altogether, these data suggest that AtCCR1 is involved in constitutive lignification whereas AtCCR2 is involved in the biosynthesis of phenolics whose accumulation may lead to resistance.

A novel myb oncogene homologue in Arabidopsis thaliana related to hypersensitive cell death.

A novel myb oncogene homologue (AtMYB30) has been isolated by differential screening of a cDNA library prepared from Xanthomonas campestris pv. campestris (X. campestris)-inoculated Arabidopsis thaliana cells cultured in the presence of cycloheximide. AtMYB30 is a single-copy gene, and the encoded protein contains a MYB domain highly homologous to other plant and animal MYB proteins. Analyses of transcript levels in A. thaliana plants, or in cultured A. thaliana cells infected with either virulent or avirulent strains of the pathogens X. campestris and Pseudomonas syringae pv. tomato, showed that maximal levels of transcription of this gene occurred during the hypersensitive response. Furthermore, in A. thaliana mutants affected in the control of cell death initiation (lsd3, lsd4 and lsd5), constitutive expression or expression in lesion-positive plants was observed, while in suppressors of the mutations lsd5 and lsd4, AtMYB30 transcripts did not accumulate. However, AtMYB30 expression could not be detected in the lsd1 mutant, which was hyper-responsive to cell death initiators and unable to limit the extent of cell death, whatever the environmental conditions. The results presented here suggest a strong correlation between AtMYB30 and genetically controlled cell death, with a role in the initiation of cell death rather than in the limitation of its extent. Our results further indicate that the lsd mutants constitute an appropriate genetic model for studying the role of this gene in hypersensitive cell death, and their relation to different steps of the pathway(s) leading to cell death.

Identification of new early markers of the hypersensitive response in Arabidopsis thaliana(1).

New molecular markers of the hypersensitive response (HR) of Arabidopsis thaliana to the bacterial pathogen Xanthomonas campestris pv. campestris (X.c.c.) have been identified by differential screening of a cDNA library constructed from suspension cells inoculated by an HR-inducing strain in the presence of cycloheximide. Seven families of genes (called Athsr) have been isolated, show similarities to voltage-dependent anion channels (VDAC) and alternative oxidases, or are novel proteins. Athsr genes have shown to be specifically or preferentially expressed during the HR. These data suggest that Athsr genes might be involved in early events conditioning the establishment of the HR.

Bax-induced cell death in tobacco is similar to the hypersensitive response.

Bax, a death-promoting member of the Bcl-2 family of proteins, triggered cell death when expressed in plants from a tobacco mosaic virus vector. Analysis of Bax deletion mutants demonstrated a requirement for the BH1 and BH3 domains in promoting rapid cell death, whereas deletion of the carboxyl-terminal transmembrane domain completely abolished the lethality of Bax in plants. The phenotype of cell death induced by Bax closely resembled the hypersensitive response induced by wild-type tobacco mosaic virus in tobacco plants carrying the N gene. The cell death-promoting function of Bax in plants correlated with accumulation of the defense-related protein PR1, suggesting Bax activated an endogenous cell-death program in plants. In support of this view, both N gene- and Bax-mediated cell death was blocked by okadaic acid, an inhibitor of protein phosphatase activity. The ability of Bax to induce cell death and a defense reaction in plants suggests that some features of animal and plant cell death processes may be shared.

Molecular cloning of a sulfotransferase in Arabidopsis thaliana and regulation during development and in response to infection with pathogenic bacteria.

A cDNA clone (RaRO47) encoding a sulfotransferase (ST) has been isolated from Arabidopsis cell suspensions. The deduced polypeptide of 302 amino acids is highly related to plant flavonol sulfotransferases (FSTs), characterized for the first time in Flaveria, and also to STs from animal tissue. The expression of the Arabidopsis ST gene(s) corresponding to RaR047 was examined during different developmental stages. It was found that, at the level of steady-state mRNA, expression of gene(s) encoding this ST was rapidly induced in the aerial parts of young seedlings, and during growth of Arabidopsis cell cultures. No expression could be detected in roots. Treatment of Arabidopsis seedlings with hormonal or stress-related compounds, showed that RaR047 mRNA accumulation was more particularly induced in response to salicylic acid and methyl jasmonate. Furthermore, in the leaves of mature plants or in cell suspensions, accumulation of RaR047 mRNA was observed upon infection with bacterial pathogens. This expression was observed preferentially in response to avirulent pathogens causing an hypersensitive reaction, as compared to virulent pathogens, which lead to disease.

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.