A novel thermostable sulfite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli

A novel sulfite oxidase has been identified from Thermus thermophilus AT62. Despite this enzyme showing significant amino-acid sequence homology to several bacterial and eukaryal putative and identified sulfite oxidases, the kinetic analysis, performed following the oxidation of sulfite and with ferricyanide as the electron acceptor, already pointed out major differences from representatives of bacterial and eukaryal sources. Sulfite oxidase from T. thermophilus, purified to homogeneity, is a monomeric enzyme with an apparent molecular mass of 39.1 kDa and is almost exclusively located in the periplasm fraction. The enzyme showed sulfite oxidase activity only when ferricyanide was used as electron acceptor, which is different from most of sulfite-oxidizing enzymes from several sources that use cytochrome c as co-substrate. Spectroscopic studies demonstrated that the purified sulfite oxidase has no cytochrome like domain, normally present in homologous enzymes from eukaryotic and prokaryotic sources, and for this particular feature it is similar to homologous enzyme from Arabidopsis thaliana. The identified gene was PCR amplified on T. thermophilus AT62 genome, expressed in Escherichia coli and the recombinant protein identified and characterized.     

Identification of a novel multiple environmental factor‐responsive 1‐aminocyclopropane‐1‐carboxylate synthase gene,NT‐ACS2, from tobacco

Many abiotic environmental factors elicit the production of stress‐ethylene in higher plants. To elucidate the molecular mechanisms underlying the regulation of stress‐ethylene production and the physiological roles played by stress‐ethylene in stress responses of plants, we studied the gene expression of ACC synthase in tobacco plants that had been subjected to environmental stresses. Four new tobacco ACC synthase cDNA fragments, NT‐ACS2, NT‐ACS3, NT‐ACS4 and NT‐ACS5, were identified and sequenced. It was found that NT‐ACS2 could be induced by wounding, cold temperature and, especially, sunlight. NT‐ACS4 was induced at a faster kinetics by wounding. The multiple environmental stress‐responsive (MESR) NT‐ACS2 gene was found to contain three introns and four exons and encode a polypeptide of 484 amino acids, 54·6 kDa and pI 6·87. Computer analysis of the 3·4 kb 5 ′ flanking region upstream of the ACS coding region revealed the existence of a group of putative cis‐acting regulatory elements potentially conferring wounding, chilling, and UV light inducibility. Phylogenetic analysis of ACC synthase genes of different plant origins indicated that the chill‐inducible NT‐ACS2 gene is closely related to a chilling‐inducible citrus ACS gene.     

Molybdoenzymes in Drosophila. IV. Further characterization of the cinnamon phenotype

Summary Mutations at the cin gene display drastically lowered levels of the molybdoenzymes, xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), and lack pyridoxal oxidase (PO) and sulfite oxidase (SO) activities. Certain mutations at cin also display varying degrees of female sterility, which is maternally affected. Here we characterize five new cin alleles with respect to the molybdoenzyme activities as well as the molybdenum cofactor, commonly required for molybdoenzyme activity. In complementing cin heterozygotes we find that, in addition to the previously reported unusually high levels of XDH and AO activities, there are unusually elevated levels of SO activity, as well as complementation for PO activity. The levels of immunologically crossreacting material in such heterozygotes indicate that the elevated levels of molybdoenzyme activities cannot be due to increases in the number of enzyme molecules. Measurements of the level of molybdenum cofactor activity normally present in XDH, AO, PO, and SO point to the possibility that a larger fraction of the enzyme molecules are active in these heterozygotes. The possible role of SO with respect to cinnamon's female sterility is also discussed.     

An E3 ubiquitin ligase from Nicotiana benthamiana targets the replicase of Bamboo mosaic virus and restricts its replication

One up-regulated host gene identified previously was found involved in the infection process of Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. The full length cDNA of this gene was cloned by 5′ and 3′-rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved really interesting new gene (RING) domain and a transmembrane domain. The gene might function as an ubiquitin E3 ligase. We designated this protein in Nicotiana benthamiana as ubiquitin E3 ligase containing RING domain 1 (NbUbE3R1). Further characterization by using Tobacco rattle virus-based virus-induced gene silencing (loss-of-function) revealed that increased BaMV accumulation was in both knockdown plants and protoplasts. The gene might have a defensive role in the replication step of BaMV infection. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed in N. benthamiana plants. The wild-type NbUbE3R1-orange fluorescent protein (NbUbE3R1-OFP), NbUbE3R1/△TM-OFP (removal of the transmembrane domain) and NbUbE3R1/mRING-OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting the accumulation of BaMV. The common feature of these constructs was the intact substrate-interacting domain. Yeast two-hybrid and co-immunoprecipitation experiments used to determine the possible viral-encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an up-regulated gene, NbUbE3R1 that plays a role in BaMV replication.     

Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

The Arabidopsis LecRK‐VI.2 associates with the pattern‐recognition receptor FLS2 and primes Nicotiana benthamiana pattern‐triggered immunity

Pattern‐triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad‐spectrum disease resistance. PTI is activated upon perception of microbe‐associated molecular patterns (MAMPs) by pattern‐recognition receptors (PRRs). We have recently demonstrated that the L‐type lectin receptor kinase‐VI.2 (LecRK‐VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull‐down, bimolecular fluorescence complementation and co‐immunoprecipitation analyses that LecRK‐VI.2 associates with the PRR FLS2. We also demonstrated that LecRK‐VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK‐VI.2 were indeed more resistant to virulent hemi‐biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK‐VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK‐VI.2 in N. benthamiana primed PTI‐mediated reactive oxygen species production, mitogen‐activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK‐VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.     

Molecular characterization of four genes involved in sulfur metabolism in <Emphasis Type="Italic">Porphyra purpurea</Emphasis> (Roth) C. Agardh

Sulfated polysaccharides (carrageenans and agars) are among the most important products of red algae that are used as food additives as well as in molecular biology research. The quality and value of the product is greatly dependent on the levels and sites of sulfation of the polysaccharides. Little information is currently available on the molecular details of sulfur metabolism in red algae. Considering the economic importance of sulfated polysaccharide, elucidating the molecular details of sulfur metabolism in these organisms could help in future endeavors to improve algal commercial value, e.g., through genetic engineering. A cDNA library from the red alga Porphyra purpurea (Roth) C. Agardh was used to isolate four cDNAs with homology to genes encoding known sulfur assimilation enzymes: sulfate adenyltransferase (ATP sulfurylase), adenosine 5′-phosphosulfate kinase (APSK), sulfite reductase, and cysteine synthase. These cDNAs were characterized with respect to their molecular properties and a cDNA with homology to APSK was used to functionally complement an Escherichia coli auxotroph APSK⁻ mutant. The other cDNAs are being similarly characterized with respect to their ability to produce functional enzymes. Elucidation of the regulation of expression of these genes will aid in future research to determine the biochemical and genetic details of the sulfate assimilation pathway as well as its genetic manipulation in red algae.     

The hypersensitive induced reaction 3 (HIR3) gene contributes to plant basal resistance via an EDS1 and salicylic acid‐dependent pathway

The hypersensitive‐induced reaction (HIR) gene family is associated with the hypersensitive response (HR) that is a part of the plant defense system against bacterial and fungal pathogens. The involvement of HIR genes in response to viral pathogens has not yet been studied. We now report that the HIR3 genes of Nicotiana benthamiana and Oryza sativa (rice) were upregulated following rice stripe virus (RSV) infection. Silencing of HIR3s in N. benthamiana resulted in an increased accumulation of RSV RNAs, whereas overexpression of HIR3s in N. benthamiana or rice reduced the expression of RSV RNAs and decreased symptom severity, while also conferring resistance to Turnip mosaic virus, Potato virus X, and the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. Silencing of HIR3 genes in N. benthamiana reduced the content of salicylic acid (SA) and was accompanied by the downregulated expression of genes in the SA pathway. Transient expression of the two HIR3 gene homologs from N. benthamiana or the rice HIR3 gene in N. benthamiana leaves caused cell death and an accumulation of SA, but did not do so in EDS1‐silenced plants or in plants expressing NahG. The results indicate that HIR3 contributes to plant basal resistance via an EDS1‐ and SA‐dependent pathway.     

Structure and induction pattern of a novel proteinase inhibitor class II gene of tobacco

A cDNA and a corresponding genomic clone encoding a protein with partial identity to type II proteinase inhibitors from potato, tomato and Nicotiana alata, were isolated from tobacco libraries. The protein of 197 amino acids contains a putative signal peptide of 24 residues and three homologous domains, each with a different reactive site. The tobacco PI-II gene is not expressed in leaves of healthy plants, but is locally induced in leaves subjected to different types of stress (TMV infection, wounding, UV irradiation) and upon ethephon treatment. As opposed to the analogous PI-II genes of potato and tomato, the tobacco gene is not systemically induced by wounding or pathogenic infection. A far-upstream region in the PI-II promoter, containing various direct and indirect repeats, shares considerable sequence similarity to a similar region in the stress-inducible Cu/Zn-superoxide dismutase gene of N. plumbaginifolia.     

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

A novel approach is developed for coordinated expression of multiple proteins from a single transgene in plants. An Ssp DnaE mini‐intein variant engineered for hyper‐N‐terminal autocleavage is covalently linked to the foot‐and‐mouth disease virus 2A (F2A) peptide with unique ribosome skipping property, via a peptide linker, to create an ‘IntF2A’ self‐excising fusion protein domain. This IntF2A domain acts, in cis, to direct highly effective release of its flanking proteins of interest (POIs) from a ‘polyprotein’ precursor in plants. This is successfully demonstrated in stably transformed cultured tobacco cells as well as in different organs of transgenic tobacco plants. Highly efficient polyprotein processing mediated by the IntF2A domain was also demonstrated in lettuce and Nicotiana benthamiana based on transient expression. Protein constituents released from the polyprotein precursor displayed proper function and accumulated at similar levels inside the cells. Importantly, no C‐terminal F2A extension remains on the released POIs. We demonstrated co‐expression of as many as three proteins in plants without compromising expression levels when compared with those using single‐protein vectors. Accurate differential cellular targeting of released POIs is also achieved. In addition, we succeeded in expressing a fully assembled and functional chimeric anti‐His Tag antibody in N. benthamiana leaves. The IntF2A‐based polyprotein transgene system overcomes key impediments of existing strategies for multiprotein co‐expression in plants, which is particularly important for gene/trait stacking.     

Tissue-specific regulation of rice molybdenum cofactor sulfurase gene in response to salt stress and ABA

Molybdenum-containing aldehyde oxidase is a key enzyme for catalyzing the final step of abscisic acid (ABA) biosynthesis in plants. Sulfuration of the molybdenum cofactor (MoCo) is an essential step for activating aldehyde oxidase. The molybdenum cofactor sulfurase (MCSU) that transfers the sulfur ligand to aldehyde oxidase-bound MoCo is thus considered an important factor in regulating the ABA levels in plant tissues. In this study, we identified the rice MCSU cDNA (OsMCSU), which is the first MCSU gene cloned in monocot species. According to the functional domain analysis of the predicted amino acid sequence, the OsMCSU protein contains a Nifs domain at its N-terminus and a MOSC domain at the C-terminus. Expression of the OsMCSU gene was up-regulated by salt stress in root tissues of rice seedlings, but this effect was not observed in leaf tissues. In roots, regulations of OsMCSU expressions could be mediated by both ABA-dependent and ABA-independent signaling pathways under salt stress condition.     

NbLRK1, a lectin-like receptor kinase protein of <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>, interacts with <Emphasis Type="Italic">Phytophthora infestans</Emphasis> INF1 elicitin and mediates INF1-induced cell death

Phytophthora infestans INF1 elicitin causes the hypersensitive response (HR) in Nicotiana benthamiana (Kamoun et al. in Plant Cell 10:1413–1425, 1998). To identify N. benthamiana proteins that interact with INF1, we carried out a yeast two-hybrid screen. This screen resulted in the isolation of a gene NbLRK1 coding for a novel lectin-like receptor kinase. NbLRK1 interacted with INF1 through its VIb kinase subdomain. Purified INF1 and NbLRK1 proteins also interacted in vitro. INF1 treatment of N. benthamiana leaves induced autophosphorylation of NbLRK1. Most importantly, virus-induced gene silencing (VIGS) of NbLRK1 delayed INF1-mediated HR in N. benthamiana. These data suggest that NbLRK1 is a component of the N. benthamiana protein complex that recognizes INF1 elicitor and transduces the HR signal.     

Elicitin‐like proteins Oli‐D1 and Oli‐D2 from Pythium oligandrum trigger hypersensitive response in Nicotiana benthamiana and induce resistance against Botrytis cinerea in tomato

The biocontrol agent Pythium oligandrum and its elicitin‐like proteins oligandrins have been shown to induce disease resistance in a range of plants. In the present study, the ability of two oligandrins, Oli‐D1 and Oli‐D2, to induce an immune response and the possible molecular mechanism regulating the defence responses in Nicotiana benthamiana and tomato were investigated. Infiltration of recombinant Oli‐D1 and Oli‐D2 proteins induced a typical immune response in N. benthamiana including the induction of a hypersensitive response (HR), accumulation of reactive oxygen species and production of autofluorescence. Agrobacterium‐mediated transient expression assays revealed that full‐length Oli‐D1 and Oli‐D2 were required for full HR‐inducing activity in N. benthamiana, and virus‐induced gene silencing‐mediated knockdown of some of the signalling regulatory genes demonstrated that NbSGT1 and NbNPR1 were required for Oli‐D1 and Oli‐D2 to induce HR in N. benthamiana. Subcellular localization analyses indicated that both Oli‐D1 and Oli‐D2 were targeted to the plasma membrane of N. benthamiana. When infiltrated or transiently expressed in leaves, Oli‐D1 and Oli‐D2 induced resistance against Botrytis cinerea in tomato and activated the expression of a set of genes involved in the jasmonic acid/ethylene (JA/ET)‐mediated signalling pathway. Our results demonstrate that Oli‐D1 and Oli‐D2 are effective elicitors capable of inducing immune responses in plants, probably through the JA/ET‐mediated signalling pathway, and that both Oli‐D1 and Oli‐D2 have potential for the development of bioactive formulae for crop disease control in practice.