Efficiency of the alpha-hairpinin SmAMP-X gene promoter from Stellaria media plant depends on selection of transgenic approach.

The antimicrobial activity of the alpha-HAIRPININ ANTIMICROBIAL PEPTIDE X (SmAMP-X gene, GenBank acc. No. HG423454.1) from Stellaria media plant has been shown in vitro. Here, we isolated the SmAMP-X gene promoter and found two genomic sequences for the promoter (designated pro-SmAMP-X and pro-SmAMP-X-Ψ2) with 83% identity in their core and proximal regions. We found that the abilities of these promoters to express the uidA reporter and the nptII selectable marker differ according to the structural organization of T-DNA in the binary vector used for plant transformation. Analysis of Agrobacterium-infiltrated Nicotiana benthamiana leaves, transgenic Arabidopsis thaliana lines, and transgenic Solanum tuberosum plants revealed that both promoters in the pCambia1381Z and pCambia2301 binary vectors generate 42-100% of the ß-glucuronidase (GUS) activity generated by the CaMV35S promoter. According to 5'-RACE (rapid amplification of cDNA ends) analysis, both plant promoters are influenced by the CaMV35S enhancer used to express selectable markers in the T-DNA region of pCambia1381Z and pCambia2301. The exclusion of CaMV35S enhancer from the T-DNA region significantly reduces the efficiency of pro-SmAMP-X-Ψ2 promoter for GUS production. Both promoters in the pCambia2300 vector without CaMV35S enhancer in the T-DNA region weakly express the nptII selectable marker in different tissues of transgenic N. tabacum plants and enable selection of transgenic cells in media with a high concentration of kanamycin. Overall, promoter sequences must be functionally validated in binary vectors lacking CaMV35S enhancer.

The complete genome sequence of Stellaria aquatica virus A, a new member of the genus Alphacarmovirus, family Tombusviridae.

A new member of the genus Alphacarmovirus was detected in Stellaria aquatica using high-throughput RNA sequencing analysis. The complete genome sequence of this new virus isolate, tentatively named "Stellaria aquatica virus A" (StAV-A), comprises 4,017 nucleotides with five predicted open reading frames (ORFs) and has a typical alphacarmovirus genome organization. Pairwise comparison of StAV-A with selected members of family Tombusviridae showed 44-58%, 32-64%, and 19-49% sequence identity for the overall nucleotide sequence, polymerase, and coat protein, respectively. Phylogenetic analysis of polymerase sequences places StAV-A alongside other members of the genus Alphacarmovirus in the family Tombusviridae.

Complete genome sequence of Stellaria aquatica virus B, a novel polerovirus that infects Stellaria aquatica.

The complete genome sequence of Stellaria aquatica virus B (StAVB), a new member of the genus Polerovirus that infects Stellaria aquatica, was determined using high-throughput RNA sequencing with confirmation by Sanger sequencing. The complete StAVB genome (GenBank accession no. OP389993) is 5,900 nucleotide (nt) long with seven open reading frames (ORF0-5 and ORF3a) that encode putative proteins (P0-P5 and P3a) in a similar configuration to that of other typical poleroviruses. Pairwise sequence comparisons with other poleroviruses showed 38-50% nt sequence identity in the complete genome and 13-24%, 36-45%, 7-68%, and 6-50% amino acid sequence identity in (aa), for the P0, P1-2, P3, and P4 protein, respectively. These data, together with the results of phylogenetic analysis, indicate that StAVB should be classified as a new member of the genus Polerovirus, family Solemoviridae.

A Synthetic Strong and Constitutive Promoter Derived from the Stellaria media pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants.

Synthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from Stellaria media ANTIMICROBIAL PEPTIDE1 (AMP1) and ANTIMICROBIAL PEPTIDE2 (AMP2). These promoters are more effective than the well-known Cauliflower mosaic virus 35S promoter. Although these promoters share about 94% identity, the pro-SmAMP1 promoter demonstrated stronger transient expression of a reporter gene in Agrobacterium infiltration of Nicotiana benthamiana leaves, while the pro-SmAMP2 promoter was more effective for the selection of transgenic tobacco (Nicotiana tabacum) cells when driving a selectable marker. Using the cap analysis of gene expression method, we detected no differences in the structure of the transcription start sites for either promoter in transgenic plants. For both promoters, we used fine-scale deletion analysis to identify 160 bp-long sequences that retain the unique properties of each promoter. With the use of chimeric promoters and directed mutagenesis, we demonstrated that the superiority of the pro-SmAMP1 promoter for Agrobacterium-mediated infiltration is caused by the proline-inducible ACTCAT cis-element strictly positioned relative to the TATA box in the core promoter. Surprisingly, the ACTCAT cis-element not only activated but also suppressed the efficiency of the pro-SmAMP1 promoter under proline stress. The absence of the ACTCAT cis-element and CAANNNNATC motif (negative regulator) in the pro-SmAMP2 promoter provided a more constitutive gene expression profile and better selection of transgenic cells on selective medium. We created a new synthetic promoter that enjoys high effectiveness both in transient expression and in selection of transgenic cells. Intact promoters with differing properties and high degrees of sequence identity may thus be used as a basis for the creation of new synthetic promoters for precise and coordinated gene expression.

Novel strong promoter of antimicrobial peptides gene pro-SmAMP2 from chickweed (Stellaria media).

BACKGROUND: In a previous study we found that in chickweed the expression level of the pro-SmAMP2 gene was comparable or even higher to that of the ß-actin gene. This high level of the gene expression has attracted our attention as an opportunity for the identification of novel strong promoters of plant origin, which could find its application in plant biotechnology. Therefore, in the present study we focused on the nucleotide sequence identification and the functional characteristics of the pro-SmAMP2 promoter in transgenic plants. RESULTS: In chickweed (Stellaria media), a 2120 bp promoter region of the pro-SmAMP2 gene encoding antifungal peptides was sequenced. Six 5'-deletion variants -2120, -1504, -1149, -822, -455, and -290 bp of pro-SmAMP2 gene promoter were fused with the coding region of the reporter gene gusA in the plant expression vector pCambia1381Z. Independent transgenic plants of tobacco Nicotiana tabacum were obtained with each genetic structure. GUS protein activity assay in extracts from transgenic plants showed that all deletion variants of the promoter, except -290 bp, expressed the gusA gene. In most transgenic plants, the GUS activity level was comparable or higher than in plants with the viral promoter CaMV 35S. GUS activity remains high in progenies and its level correlates positively with the amount of gusA gene mRNA in T3 homozygous plants. The activity of the рro-SmAMP2 promoter was detected in all organs of the transgenic plants studied, during meiosis and in pollen as well. CONCLUSION: Our results show that the рro-SmAMP2 promoter can be used for target genes expression control in transgenic plants.

[Expression of plant antimicrobial peptide pro-SmAMP2 gene increases resistance of transgenic potato plants to Alternaria and Fusarium pathogens].

The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.

Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.).

Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I50 value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene.

Transformation of tobacco and Arabidopsis plants with Stellaria media genes encoding novel hevein-like peptides increases their resistance to fungal pathogens.

Two novel antifungal hevein-like peptides, SmAMP1.1a and SmAMP2.2a, were previously isolated from seeds of Stellaria media. It has been established that these peptides accumulate in this weed as a result of proteolysis of two propeptides, pro-SmAMP1 and pro-SmAMP2. The primary structure of these propeptides is unique; in addition to having a signal peptide and negatively charged C-terminus, each of these structures consists of two hevein-like peptides of different length separated by a space rather than a single peptide. In this work, we demonstrated that the expression of the pro-SmAMP1 and pro-SmAMP2 genes was tissue-specific and increased substantially under exposure to fungal infection. To elucidate whether S. media has any advantages in defending against phytopathogens due to its unusual structure of pro-SmAMP1 and pro-SmAMP2, on the basis of the pro-SmAMP1 gene, we created three genetic constructs. Arabidopsis and tobacco plants were subsequently transformed with these constructs. Transgenic plants bearing the full-length pro-SmAMP1 gene exhibited the best resistance to the phytopathogens Bipolaris sorokiniana and Thielaviopsis basicola. The resistance of S. media plants to phytopathogenic fungi was likely due to the fungal-inducible expression of pro-SmAMP1 and pro-SmAMP2 genes, and due to the specific features of the primary structure of the corresponding propeptides. As a result of the processing of these propeptides, two different antimicrobial peptides were released simultaneously. Based on our results, we conclude that the genes for antimicrobial peptides from S. media may be promising genetic tools for the improvement of plant resistance to fungal diseases.

Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds.

Two novel highly homologous defensins, Sm-AMP-D1 and Sm-AMP-D2, were isolated from seeds of common chickweed Stellaria media L. (family Cariophyllaceae). They show sequence homology to defensins of the Brassicaceae plants and display strong inhibitory activity against phytopathogenic fungi and oomycetes in the micromolar range (IC(50)≤1µM). The cDNA sequences coding for Sm-AMP-D1 and Sm-AMP-D2 were obtained. They code for highly homologous precursor proteins, consisting of a signal peptide of 32 amino acid residues and the mature peptide domain of 50 amino acid residues. The Sm-AMP-D1 and Sm-AMP-D2 precursors differ by two amino acids: one in the signal peptide region, and the other, in the mature peptide domain. Two Sm-D1-encoding genes were identified in S. media genome by PCR amplification from the genomic DNA using Sm-D1-specific primers. They contain a single 599-bp intron in the signal peptide domain and differ from each other by nucleotide substitutions in the intron and 3'-untranslated regions, while the coding sequences are well conserved. One of the genes matched perfectly the sm-D1 cDNA sequence. The sm-D genes show promise for engineering pathogen resistance in crops and expand our knowledge on weed genomics.

Light irradiance differentially regulates endogenous levels of cytokinins and auxin in alpine and prairie genotypes of Stellaria longipes.

The growth patterns of plants from alpine (sun) and prairie (shade) ecotypes of Stellaria longipes in response to change in light irradiance was investigated and involvement of cytokinins (CKs), auxin (IAA) and abscisic acid (ABA) was studied to examine the mechanism behind phenotypic plasticity of these plants in response to light signalling. Low light irradiance induced shoot growth in plants of both ecotypes, but IAA levels were higher in plants from alpine, but not prairie ecotype. Dynamics of CK profiles in response to changing photosynthetically active radiation were quite different between ecotypes and changes were more pronounced in the plants of alpine ecotype, where opposite patterns in CK accumulation between low and normal light irradiances were observed. The plants of both ecotypes showed similar trends in ABA levels under low light irradiance. Thus, the highly plastic plants of prairie ecotype may have evolved mechanisms to control the growth in response to reduced light irradiance without major alterations in the levels of CKs or IAA. These results demonstrate that within species, plants from open habitats show less growth response to reduced light irradiance than plants from shaded habitats.

Isolation and characterization of PHYC gene from Stellaria longipes: differential expression regulated by different red/far-red light ratios and photoperiods.

We have cloned and characterized the phytochrome C ( PHYC) gene from Stellaria longipes. The PHYC gene is composed of a 110-bp 5'-untranslated leader sequence, a 3,342-bp coding region, and a 351-bp 3'-untranslated sequence. The Stellaria PHYC contains three long introns within the coding region at conserved locations as in most angiosperm PHY genes. DNA blot analysis indicates that the Stellaria genome contains a single copy of PHYC. Stellaria PHYC shares 60%, 58%, and 57% deduced amino acid identities with rice, Sorghum, and Arabidopsis PHYC, respectively. Phylogenetic analysis indicates that Stellaria PHYC is located in the dicot branch, but is divergent from Arabidopsis PHYC. The Stellaria PHYC is constitutively expressed in different plant organs, though the level of PHYC gene transcript in roots is slightly higher than in flowers, leaves, and stems. When 2-week old seedlings grown in the dark were exposed to constant white light, PHYC mRNA quickly accumulates within 1-12 h. When plants grown in darkness for 7 days were exposed to different red/far-red light (R/FR) ratios, the levels of PHYC mRNA at R/FR = 0.7 are much lower than under R/FR = 3.5. The levels of PHYC mRNA under short-day (SD) photoperiod are higher than under long-day (LD) photoperiod. Plants under SD conditions do not elongate, and are only about 1.7 cm tall at 19 days. In contrast, plants under LD conditions elongate with an average height of 21.2 cm at 19 days. The plants do not flower under SD conditions, but do so at 18-19 days under LD conditions. These results indicate that under SD conditions the high level of PHYC mRNA may inhibit stem elongation and flower initiation. In contrast, under LD conditions the high level of PHYC mRNA may promote stem elongation and flowering.