Overexpression of the A4-rolB gene from the pRiA4 of Rhizobium rhizogenes modulates hormones homeostasis and leads to an increase of flavonoid accumulation and drought tolerance in Arabidopsis thaliana transgenic plants.

MAIN CONCLUSION: Increased flavonol accumulation and enhanced drought tolerance in A4-rolB-overexpressing plants can be explained by the cooperative action of the SA and ROS signalling pathways. Clarification of function of the A4-rolB plast gene from pRiA4 of Rhizobium rhizogenes will allow a better understanding of the biological principles of the natural transformation process and its use as a tool for plant bioengineering. In the present study, we investigated whether the overexpression of A4-rolB gene could regulate two important processes, flavonoid biosynthesis and drought tolerance. In addition, we investigated some aspects of the possible machinery of the A4-rolB-induced changes in plant physiology, such as crosstalk of the major signalling systems. Based on the data obtained in this work, it can be presumed that constitutive overexpression of A4-rolB leads to the activation of the salicylic acid signalling system. An increase in flavonol accumulation and enhanced drought tolerance can be explained by the cooperative action of SA and ROS pathways.

Development of host strains and vector system for an efficient genetic transformation of filamentous fungi.

An ability to synthesize extracellular enzymes degrading a wide spectrum of plant and algae polymeric substrates makes many fungi relevant for biotechnology. The terrestrial thermophilic and marine fungal isolates capable of plant and algae degradation have been tested for antibiotic resistance for their possible use in a new genetic transformation system. Plasmids encoding the hygromycin B phosphotransferase (hph) under the control of the cauliflower mosaic virus 35S promoter, the trpC gene promoter of Aspergillus nidulans, and the Aureobasidium pullulans TEF gene promoter were delivered into the fungal cells by electroporation. The effectiveness of different promoters was compared by transformation and growth of Thermothelomyces thermophila (formerly Myceliophthora thermophila) on the selective medium and by real-time PCR analysis. A highly efficient transformation was observed at an electric-pulse of 8.5 kV/cm by using 10 µg of DNA per 1 × 105 conidia. Although all promoters were capable of hph expression in the Th. thermophila cells, the trpC promoter provided the highest level of hygromycin resistance. We further successfully applied plant binary vector pPZP for co-transformation of hph gene and enhanced green fluorescent protein gene that confirmed this transformation system could be used as an appropriate tool for gene function studies and the expression of heterologous proteins in micromycetes.

The rolB plant oncogene affects multiple signaling protein modules related to hormone signaling and plant defense.

The rolB plant oncogene of Agrobacterium rhizogenes perturbs many biochemical processes in transformed plant cells, thereby causing their neoplastic reprogramming. The oncogene renders the cells more tolerant to environmental stresses and herbicides and inhibits ROS elevation and programmed cell death. In the present work, we performed a proteomic analysis of Arabidopsis thaliana rolB-expressing callus line AtB-2, which represents a line with moderate expression of the oncogene. Our results show that under these conditions rolB greatly perturbs the expression of some chaperone-type proteins such as heat-shock proteins and cyclophilins. Heat-shock proteins of the DnaK subfamily were overexpressed in rolB-transformed calli, whereas the abundance of cyclophilins, members of the closely related single-domain cyclophilin family was decreased. Real-time PCR analysis of corresponding genes confirmed the reliability of proteomics data because gene expression correlated well with the expression of proteins. Bioinformatics analysis indicates that rolB can potentially affect several levels of signaling protein modules, including effector-triggered immunity (via the RPM1-RPS2 signaling module), the miRNA processing machinery, auxin and cytokinin signaling, the calcium signaling system and secondary metabolism.

Green synthesis of silver nanoparticles using transgenic Nicotiana tabacum callus culture expressing silicatein gene from marine sponge Latrunculia oparinae.

In the present investigation, transgenic tobacco callus cultures and plants overexpressing the silicatein gene LoSilA1 from marine sponge Latrunculia oparinae were obtained and their bioreduction behaviour for the synthesis of silver nanoparticles (AgNPs) was studied. Synthesized nanoparticles were characterized using UV-visible spectroscopy, Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic flame electron microscopy (AFM) and nanoparticle tracking analysis (NTA). Our measurements showed that the reduction of silver nitrate produced spherical AgNPs with diameters in the range of 12-80 nm. The results of XRD analysis proved the crystal nature of the obtained AgNPs. FTIR analysis indicated that particles are reduced and stabilized in solution by the capping agent, which is likely to be proteins present in the callus extract. Interestingly, the reduction potential of LoSiLA1-transgenic callus line was increased three-fold compared with the empty vector-transformed calli. The synthesized AgNPs were found to exhibit strong antibacterial activity against Escherichia coli and Agrobacterium rhizogenes. The present study reports the first evidence for using genetic engineering for activation of the reduction potential of plant cells for synthesis of biocidal AgNPs.

The rolB gene activates secondary metabolism in Arabidopsis calli via selective activation of genes encoding MYB and bHLH transcription factors.

It is known that the rolB gene of Agrobacterium rhizogenes increases the production of secondary metabolites in transformed plant cells, but its mechanism of action remains unclear. In this report, we demonstrate that rolB expression in Arabidopsis thaliana calli led to the activation of most genes encoding secondary metabolism-specific MYB and bHLH transcription factors (TFs), such as MYB11, MYB12, MYB28, MYB76, MYB34, MYB51, MYB122, TT2 and TT8. Accordingly, a higher transcript abundance of main biosynthetic genes related to these factors was detected. The rolB-transformed calli produced 3-fold higher levels of indolic glucosinolates (GSs) compared with normal calli but did not produce secondary metabolites from other groups. Enhanced accumulation of indolic GSs was caused by activation of MYB34, MYB51 and MYB122, and the absence of aliphatic GSs in transformed calli was caused by the inability of rolB to induce MYB29. The inability of rolB-calli to produce flavonoids was caused by the lack of MYB111 expression, induced by the rolB-mediated conversion of MYB expression from cotyledon-specific to root-specific patterns. The high specificity of rolB on secondary metabolism-specific TFs was demonstrated for the first time.

The rolB gene activates the expression of genes encoding microRNA processing machinery.

The rolB gene of Agrobacterium rhizogenes renders cells more tolerant of environmental stresses and increases their defense potential. However, these effects, coupled with the developmental abnormalities caused by rolB, have not yet been explained. In rolB-transformed Arabidopsis thaliana cells, we detected a 2.2 to 7-fold increase in the expression of genes encoding core and accessory proteins (DCL1, SE, HYL1, AGO1, TGH, DDL, HEN1, AGO4 and RDR2) of the microRNA processing machinery. However, the rolB gene did not affect the expression of DCL2, DCL3 and HST. The diverse and complex effects of rolB on transformed plant cells may be attributable to changes caused by this gene in particular RNA silencing pathways.

Recent advances in the understanding of Agrobacterium rhizogenes-derived genes and their effects on stress resistance and plant metabolism.

It is commonly accepted that the plant pathogens Agrobacterium rhizogenes and Agrobacterium tumefaciens, acting via their T-DNA oncogenes, disturb hormone metabolism or hormone perception pathways in plants, thereby attaining their aim of successful pathogenesis. In this work, we summarize recent data on the A. rhizogenes rolC and rolB oncogenes in comparison to the A. tumefaciens 6b oncogene with respect to their effects on the physiology of transformed cells. The newly discovered functions of the rol genes include the modulation of secondary metabolism, the modulation of levels of intracellular ROS and stress resistance of transformed cells, changed sucrose metabolism, and the inhibition of programmed cell death. We show that the rol genes do not have suppressive effects on plant innate immunity; rather, these genes activate plant defense reactions. The existence of not only the hormone-related mechanism of pathogenicity but also the defense-related mechanism of pathogenicity during plant-Agrobacterium interactions is suggested.

Can plant oncogenes inhibit programmed cell death? The rolB oncogene reduces apoptosis-like symptoms in transformed plant cells.

The rolB oncogene was previously identified as an important player in ROS metabolism in transformed plant cells. Numerous reports indicate a crucial role for animal oncogenes in apoptotic cell death. Whether plant oncogenes such as rolB can induce programmed cell death (PCD) in transformed plant cells is of particular importance. In this investigation, we used a single-cell assay based on confocal microscopy and fluorescent dyes capable of discriminating between apoptotic and necrotic cells. Our results indicate that the expression of rolB in plant cells was sufficient to decrease the proportion of apoptotic cells in steady-state conditions and diminish the rate of apoptotic cells during induced PCD. These data suggest that plant oncogenes, like animal oncogenes, may be involved in the processes mediating PCD.

The rolB gene suppresses reactive oxygen species in transformed plant cells through the sustained activation of antioxidant defense.

The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.

CDPK-driven changes in the intracellular ROS level and plant secondary metabolism.

Heterologous expression of a constitutively active calcium-dependent protein kinase (CDPK) gene was previously shown to increase secondary metabolite production in cultured cells of Rubia cordifolia, but the critical question of how CDPK activates secondary metabolism remains to be answered. In this article, we report that the expression of the Arabidopsis CDPK gene, AtCPK1, in R. cordifolia cells caused moderate and stable elevation of intracellular reactive oxygen species (ROS) levels. In contrast, the non-active, mutated AtCPK1 gene did not cause such an effect. The active AtCPK1 also increased cell size, likely by restricting cell division. These results are consistent with the model in which constitutive expression of AtCPK1 mimics the effects of elicitors, acting on secondary metabolism via the activation of ROS production.

Occurrence of a silicatein gene in glass sponges (Hexactinellida: Porifera).

Silicatein genes are involved in spicule formation in demosponges (Demospongiae: Porifera). However, numerous attempts to isolate silicatein genes from glass sponges (Hexactinellida: Porifera) resulted in a limited success. In the present investigation, we performed analysis of potential silicatein/cathepsin transcripts in three different species of glass sponges (Pheronema raphanus, Aulosaccus schulzei, and Bathydorus levis). In total, 472 clones of such transcripts have been analyzed. Most of them represent cathepsin transcripts and only three clones have been found to represent transcripts, which can be related to silicateins. Silicatein transcripts were identified in A. schulzei (Hexactinellida; Lyssacinosida; Rosselidae), and the corresponding gene was called AuSil-Hexa. Expression of AuSil-Hexa in A. schulzei was confirmed by real-time PCR. Comparative sequence analysis indicates high sequence identity of the A. schulzei silicatein with demosponge silicateins described previously. A phylogenetic analysis indicates that the AuSil-Hexa protein belongs to silicateins. However, the AuSil-Hexa protein contains a catalytic cysteine instead of the conventional serine.

Engineering high yields of secondary metabolites in Rubia cell cultures through transformation with rol genes.

Among the different methods currently used to improve yields of secondary metabolites in cultured plant cells, the method involving transformation by rol genes represents an example of relatively new technology. These genes, isolated from plasmids of the plant pathogen Agrobacterium rhizogenes, are potential activators of secondary metabolism in transformed cells from the Solanaceae, Araliaceae, Rubiaceae, Vitaceae, and Rosaceae families. In some cases, the activator effect of individual rol genes was sufficient to overcome the inability of cultured plant cells to produce large amounts of secondary metabolites. Stimulation of production characteristics of cultured plant cells mediated by the rol genes was shown to be remarkably stable over long-term cultivation. In this chapter, we describe transformation of Rubia cordifolia L. cells with the rol genes as an example of metabolic engineering of secondary metabolites.

Silicatein genes in spicule-forming and nonspicule-forming Pacific demosponges.

Silicatein genes are known to be involved in siliceous spicule formation in marine sponges. Proteins encoded by these genes, silicateins, were recently proposed for nanobiotechnological applications. We studied silicatein genes of marine sponges Latrunculia oparinae collected in the west Pacific region, shelf of Kuril Islands. Five silicatein genes, LoSilA1, LoSilA1a, LoSilA2, and LoSilA3 (silicatein-alpha group), LoSilB (silicatein-beta group), and one cathepsin gene, LoCath, were isolated from the sponge L. oparinae for the first time. The deduced amino acid sequence of L. oparinae silicateins showed high-sequence identity with silicateins described previously. LoCath contains the catalytic triad of amino acid residues Cys-His-Asn characteristic for cathepsins as well as motifs typical for silicateins. A phylogenetic analysis places LoCath between sponge silicateins-beta and L-cathepsins suggesting that the LoCath gene represents an intermediate form between silicatein and cathepsin genes. Additionally, we identified, for the first time, silicatein genes (AcSilA and AcSilB) in nonspicule-forming marine sponge, Acsmall a, Cyrillicnthodendrilla sp. The results suggest that silicateins could participate also in the function(s) unrelated to spiculogenesis.

Suppression of reactive oxygen species and enhanced stress tolerance in Rubia cordifolia cells expressing the rolC oncogene.

It is known that expression of the Agrobacterium rhizogenes rolC gene in transformed plant cells causes defense-like reactions, such as increased phytoalexin production and expression of pathogenesis-related proteins. In the present study, we examined whether this phenomenon is associated with increased production of reactive oxygen species (ROS). Single-cell assays based on confocal microscopy and fluorogenic dyes (2,7-dichlorofluorescein diacetate and dihydrorhodamine 123) showed reduced steady-state levels of ROS in rolC-expressing Rubia cordifolia cells as compared with normal cells. Paraquat, a ROS inducer, caused significant ROS elevation in normal cells but had little effect on rolC-transformed cells. Likewise, ROS elevation triggered by a light stress was suppressed in transformed cells. Our results indicate that the rolC gene acts as a ROS suppressor in unstressed cells and its expression prevents stress-induced ROS elevations. We detected a two- to threefold increase in tolerance of rolC-transformed cells to salt, heat, and cold treatments. Simultaneously, rolC-transformed cells maintained permanently active defensive status, as found by measuring isochorismate synthase gene expression and anthraquinone production. Thus, the oncogene provoked multiple effects in which ROS production and phytoalexin production were clearly dissociated.

Individual and combined effects of the rolA, B, and C genes on anthraquinone production in Rubia cordifolia transformed calli.

It is known that the rolA, rolB, and rolC genes of Agrobacterium rhizogenes T-DNA affect processes of plant development and activate the synthesis of secondary metabolites in transformed plant cells. Although a synergistic activity of the rol genes on root formation is well-documented, little is known about their individual and combined action on secondary metabolism. In the present investigation, we provide evidence indicating that individual rolA, rolB, and rolC genes are capable of increasing biosynthesis of anthraquinones (AQs) in transformed calli of Rubia cordifolia. The stimulatory effect was due to the increased transcription of a key gene of AQ biosynthesis, the isochorismate synthase (ICS) gene. The strongest AQ-stimulating activity was shown for an R. cordifolia culture expressing rolB at high levels, where rolB ensured a 15-fold increase of AQ accumulation compared with the control, non-transformed calli. A tyrosine phosphatase inhibitor abolished the rolB-induced increase of AQ production, thus indicating the involvement of tyrosine (de)phosphorylation in the rolB-mediated AQ stimulation. The rolA- and rolC-expressing cultures produced 2.8- and 4.3-fold higher levels of AQs, respectively, when compared with the control calli. However, the effect of rolA, rolB, and rolC on AQ biosynthesis was not synergistic because rolA and rolC apparently attenuated the stimulatory effect of rolB on AQ biosynthesis. Therefore, the rol-gene-mediated signals that promote root formation and those which activate biosynthesis of secondary metabolites seem to have a point of divergence.