Paxillus involutus Forms an Ectomycorrhizal Symbiosis and Enhances Survival of PtCOMT-modified Betula pendula in vitro

Abstract The ability of the PtCOMT (caffeate/5-hydroxyferulate O-methyltransferase from Populus tremuloides L.) - modified Betula pendula Roth. lines to form symbiosis with an ectomycorrhizal (ECM) fungus Paxillus involutus Batsch Fr. was studied in vitro. Lignin precursor gene PtCOMT was introduced into two B. pendula clones under the control of the cauliflower mosaic virus 35S promoter or the promoter of the sunflower polyubiquitin gene UbB1. Of the four transgenic lines, one 35SPtCOMT line (23) had a decreased syringyl/guaiacyl (S/G) ratio of root lignin, and two UbB1-PtCOMT lines (110 and 130) retarded root growth compared to the control clone. Both control clones and all transgenic lines were able to form ECMs with P. involutus, but the transgenic lines differed from the controls in the characteristics of the ECMs. The number of lateral roots covered with fungal hyphae and/or development of a Hartig net (HN) were reduced in line 23 with a decreased S/G ratio, and in lines 110 and 130 with slower root formation and changed root morphology, respectively. However, line 23 benefited more from the inoculation in lateral root formation than the control, and in lines 110 and 130 the percentage of viable plants increased most due to inoculation. The results show that B. pendula plants genetically transformed with the lignin gene PtCOMT could form mycorrhizal symbiosis regardless of changes in either the root S/G ratio or development. The benefits of the symbiosis were variable even in the closed in vitro system, and dependent on the clone or transgenic line and the ECM fungal symbiont.

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Constitutive Expression of Pea Defense Gene DRR206 Confers Resistance to Blackleg (Leptosphaeria maculans) Disease in Transgenic Canola (Brassica napus)

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.5.410 ◽

1999 ◽

Vol 12 (5) ◽

pp. 410-418 ◽

Cited By ~ 92

Author(s):

Yaping Wang ◽

Goska Nowak ◽

David Culley ◽

Lee A. Hadwiger ◽

Brian Fristensky

Keyword(s):

Brassica Napus ◽

Transgenic Plants ◽

Leptosphaeria Maculans ◽

Constitutive Expression ◽

Single Copy ◽

Cauliflower Mosaic Virus ◽

Adult Plant ◽

35S Promoter ◽

Transgenic Lines

To identify genes effective against the blackleg fungus Leptosphaeria maculans (Phoma lingam), we have transformed canola (Brassica napus) with four pea (Pisum sativum) genes under constitutive control by the cauliflower mosaic virus 35S promoter: PR10.1, chitinase, DRR206, and defensin. Transgenic lines containing single-copy T-DNA insertions for each gene were screened for both seedling (cotyledonary) and adult plant resistance. Lines for which pea DRR206 mRNA was expressed showed decreased disease scores, compared with non-expressing transgenic lines. Transgenic plants expressing pea defensin showed a slight enhancement of resistance, while for PR10 and chitinase transgenics there was little or no enhancement of resistance. Resistance to L. maculans cosegregated with DRR206 transgenes. Extracts from DRR206 and defensin transgenic plants inhibited fungal germination in vitro. DRR206 transgenic plants also demonstrated decreased hyphal growth at inoculation sites. While the precise function of DRR206 remains to be determined, these results suggest that it does play an important role in defense against fungi.

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Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3 - 9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana

Functional Plant Biology ◽

10.1071/fp12051 ◽

2012 ◽

Vol 39 (9) ◽

pp. 764 ◽

Cited By ~ 6

Author(s):

Gi-Ho Lee ◽

Seong-Han Sohn ◽

Eun-Young Park ◽

Young-Doo Park

Keyword(s):

Gene Silencing ◽

Nicotiana Benthamiana ◽

Fluorescent Protein ◽

Cauliflower Mosaic Virus ◽

Camv 35S Promoter ◽

35S Promoter ◽

Camv 35S ◽

Histone Deacetylase 1 ◽

Transgenic Lines ◽

Green Fluorescent

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host’s defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3–9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

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Expression of thaumatin, a new type of alternative sweetener in rice

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha48311676 ◽

2020 ◽

Vol 48 (3) ◽

pp. 1276-1291

Author(s):

Shahina AKTER ◽

Md. Amdadul HUQ ◽

Yu-Jin JUNG ◽

Kwon-Kyoo KANG

Keyword(s):

Transgenic Rice ◽

Oryza Sativa L ◽

Plasmid Vector ◽

Single Copy ◽

Cauliflower Mosaic Virus ◽

Pcr Analysis ◽

35S Promoter ◽

Alternative Sweetener ◽

Sweet Proteins ◽

Transgenic Lines

Sweet proteins are the natural alternative to the artificial sweeteners as well as flavor enhancers. Among other sweet protein, thaumatin protein was isolated from Thaumatococcus daniellii Benth plant fruit. In this study, pinII Ti plasmid vector was constructed with thaumatin gene, where thaumatin was placed under the control of the duel cauliflower mosaic virus 35S promoter into rice (Oryza sativa L. var. japonica cv. ‘Dongjinbyeo’) by Agrobacterium-mediated transformation to generate transgenic plants. Thirteen plant lines were regenerated and the transgenic rice lines were confirmed by different molecular analysis. The genomic PCR result revealed that all of the plant lines were transgenic. The single copy and intergenic plant lines were selected by Taqman PCR analysis and FST analysis, respectively. Expression of thaumatin gene in transgenic rice resulted in the accumulation of thaumatin protein in the leave. Thaumatin protein was also accumulated in leave of T1 generation. Sensory analysis result suggested that the thaumatin protein expressing transgenic lines exerted sweet tasting activity. These results demonstrated that thaumatin was expressed in transgenic rice plants.

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Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta1

Biochemical Journal ◽

10.1042/bj20021453 ◽

2003 ◽

Vol 373 (3) ◽

pp. 987-992 ◽

Cited By ~ 54

Author(s):

Eng-Kiat LIM ◽

Gillian S. HIGGINS ◽

Yi LI ◽

Dianna J. BOWLES

Keyword(s):

Enzyme Activity ◽

Caffeic Acid ◽

Northern Blot Analysis ◽

Transgenic Arabidopsis ◽

Structural Features ◽

Cauliflower Mosaic Virus ◽

Leaf Extracts ◽

Transgenic Lines ◽

First Time

Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms of caffeic acid are known to exist: the 3-O- and 4-O-glucosides and the glucose ester. This study describes for the first time a glucosyltransferase [UDP-glucose:glucosyltransferase (UGT)] that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid. The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro. The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs. The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter. Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides. The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3-O-glucoside. The data are discussed in the context of the utility of UGTs for natural product biotransformations.

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Improved Cold-resistant Performance in Transgenic Grape (Vitis vinifera L.) Overexpressing Cold-inducible Transcription Factors AtDREB1b

HortScience ◽

10.21273/hortsci.44.1.35 ◽

2009 ◽

Vol 44 (1) ◽

pp. 35-39 ◽

Cited By ~ 23

Author(s):

Wanmei Jin ◽

Jing Dong ◽

Yuanlei Hu ◽

Zhongping Lin ◽

Xuefeng Xu ◽

...

Keyword(s):

Transgenic Plants ◽

Vitis Vinifera ◽

Blot Analysis ◽

Freezing Point ◽

Cold Treatment ◽

Cauliflower Mosaic Virus ◽

Vitis Vinifera L ◽

35S Promoter ◽

Transgenic Lines ◽

H 26

Dehydration response element binding (DREB)1b is a cold-inducible transcription factor in Arabidopsis thaliana. DREB1b driven by cauliflower mosaic virus 35S promoter was genetically introduced into grape Vitis vinifera L. cv. Centennial Seedless through Agrobacterium-mediated transformation for improving its cold resistance and exploring new genetic breeding approaches to obtain cold-resistant cultivars. In this study, Southern blot analysis showed the DREB1b gene was integrated into the transgenic grapevines with one to two copies. Northern blot analysis showed the presence of DREB1b transcripts in the independent transgenic lines 3, 5, 6, and 7. Further characterization of transgenic grapevines confirmed that both electrolyte leakage conductivity and the freezing point of the transgenic plants were lower than those of wild-type plants. After the cold treatment at –4 °C for 12 h, 26% of transgenic plants wilted among which 95% plants recovered once being placed under the condition of temperature 22 to 25 °C. However, subjected to the same treatment, 98% of nontransgenic plants wilted and only 2% recovered. Our results lead to the conclusion that activity of DREB1b in the transgenic grape could significantly improve its resistance to cold stress.

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Functional analysis of a class I patatin gene SK24-1 in microtuber formation of transgenic potatoes

Canadian Journal of Plant Science ◽

10.4141/cjps07031 ◽

2008 ◽

Vol 88 (4) ◽

pp. 593-598 ◽

Cited By ~ 1

Author(s):

Huaijun Si ◽

Jun Liu ◽

Jian Huang ◽

Conghua Xie

Keyword(s):

Escherichia Coli ◽

Cdna Clone ◽

Antisense Rna ◽

Cauliflower Mosaic Virus ◽

Class I ◽

35S Promoter ◽

Transformed Plants ◽

Transgenic Potatoes ◽

Class I Patatin

Expression of a class I patatin cDNA clone, SK24-1, in Escherichia coli revealed that the cDNA clone possessed lipid acyl hydrolase (LAH) activity. Transformed potato plants were obtained via Agrobacterium-mediated transformation using the chimeric constructs containing the sense and antisense cDNA under the control cauliflower mosaic virus 35S (CaMV 35S) promoter. In some sense transformed plants, both sense patatin RNA and LAH activity were increased and further resulted in a significant increase of percentage of plantlets that formed microtubers and numbers of microtubers per plantlet in vitro. All antisense plants displayed a reduction in LAH activity. Both sense and antisense RNA could be detected in antisense plants, but transcripts of antisense RNA resulted in a reduction of endogenous sense RNA. Moreover, expression of antisense cDNA in some antisense transformed plants led to a significant decrease in the number of microtubers formed. These results suggest that SK24-1 was involved in regulating microtuber formation. Key words: Patatin, potato, Escherichia coli, sense RNA, antisense RNA

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Transcriptional silencing of RNAi constructs against nematode genes in Arabidopsis

Nematology ◽

10.1163/15685411-00002698 ◽

2013 ◽

Vol 15 (5) ◽

pp. 519-528 ◽

Cited By ~ 11

Author(s):

Tina Kyndt ◽

Hongli Ji ◽

Bartel Vanholme ◽

Godelieve Gheysen

Keyword(s):

Transcriptional Silencing ◽

Housekeeping Genes ◽

Cauliflower Mosaic Virus ◽

Heterodera Schachtii ◽

Camv 35S Promoter ◽

35S Promoter ◽

Expression Levels ◽

Camv 35S ◽

General Decline ◽

Transgenic Lines

In this research, Arabidopsis thaliana plants were transformed with hairpin constructs targeting cyst nematode (Heterodera schachtii) genes, driven by the cauliflower mosaic virus (CaMV) 35S promoter: two housekeeping genes (the splicing factor Hs-U2AF and the vacuolar Hs-H+ATPase) and one candidate effector gene (the ubiquitin extension protein Hs-ubi). Expression of the dsRNA appeared to be extremely variable between and within homozygous T3 lines and even between tissues. Infection experiments showed up to 50% reduction in nematode infection for some transgenic lines. The results varied not only between lines containing the same construct but also between independent repetitions of the experiment. Further focusing on the Hs-U2AF-RNAi lines revealed large variations and a general decline of construct expression levels over the generations. Bisulphite sequencing of a 197 bp part of the CaMV 35S promoter revealed substantial methylation in this region and a negative correlation between the methylation level and expression of the hairpin construct. Taken together, our results show that host-generated RNAi can suffer from high levels of transcriptional silencing of the construct, leading to varying expression levels within and between transgenic lines.

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Research note: The 35S promoter is not constitutively expressed in the transgenic tropical actinorhizal tree Casuarina glauca

Functional Plant Biology ◽

10.1071/pp01121 ◽

2002 ◽

Vol 29 (5) ◽

pp. 649 ◽

Cited By ~ 31

Author(s):

Aziz Smouni ◽

Laurent Laplaze ◽

Didier Bogusz ◽

Fathia Guermache ◽

Florence Auguy ◽

...

Keyword(s):

Vascular Tissue ◽

Gus Expression ◽

Cauliflower Mosaic Virus ◽

Camv 35S Promoter ◽

35S Promoter ◽

Casuarina Glauca ◽

Camv 35S ◽

Highly Active ◽

One Year

The tropical nitrogen-fixing tree, Casuarina glauca Sieb. ex Spreng. was genetically transformed using Agrobacterium tumefaciens C58C1(pGV2260; pBIN19GUSINT). We report on the expression pattern conferred by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic C. glauca plants grown in vitro, and for one year in a greenhouse. Histochemical assays in shoots from in vitro plants revealed β-glucuronidase (GUS) staining in apical and axillary buds, and in nearly all tissues near the base of the stem. In roots, the CaMV 35S drove strong GUS expression in the apex and vascular tissue. In 1-year old plants grown in a greenhouse, the CaMV 35S promoter was highly active, except in peripheral suberized tissues. Transgenic C. glauca plants were nodulated by the actinomycete Frankia. Histochemical assays on vibratome sections of transgenic nodules demonstrated intense GUS activity in the vascular bundle, the phellogen, and in strands of uninfected cells filled with polyphenols. GUS expression was undetectable in Frankia-infected cells.

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EFFECT OF METAL NANOPARTICLES ON THE GROWTH OF NGOC LINH GINSENG (PANAX VIETNAMENSIS) LATERAL ROOTS CULTURED IN VITRO

HUE UNIVERSITY JOURNAL OF SCIENCE NATURAL SCIENCE ◽

10.26459/hueuni-jns.v126i1d.4524 ◽

2017 ◽

Vol 126 (1C) ◽

Author(s):

Duong Tan Nhut ◽

Nguyen Thi Nhat Linh ◽

Nguyen Hoang Loc ◽

Hoang Thanh Tung ◽

Vu Thi Hien ◽

...

Keyword(s):

Root Growth ◽

Metal Nanoparticles ◽

Lateral Root ◽

Root Formation ◽

Lateral Roots ◽

Metal Nanoparticle ◽

Ginseng Root ◽

Lateral Root Formation ◽

Triterpenoid Saponins

Panax vietnamensis (Ngoc Linh ginseng) plays critical roles in pharmaceutical industry because triterpenoid saponins from its roots produce medicine for improving health and treating many diseases. Metal nanoparticles reveal completely new or improved properties based on specific characteristics such as size, distribution and morphology compare to metal ion or salt; and their potential for in vitro plant cultures. Present study investigated the effects of metal nanoparticles including nZnO (0.5-2.5 mg/l), nAg (1-3 mg/l), and nCu (1-3 mg/l) supplemented in free-hormone-MS medium to in vitro Panax vietnamensis lateral root growth. Our results showed that metal nanoparticles have the positive effect on the growth of in vitro P. vietnamensis lateral roots with nAg, nCu, and nZnO. At different concentrations, in vitro P. vietnamensis lateral root growth also has various effects on the growth of lateral roots. In supplemented metal nanoparticle treatments, nCu is the most optimum for in vitro P. vietnamensis lateral root growth; the highest increase was obtained at 1.5 mg/l nCu treatment (99.3% lateral root formation and all root growth indexes are the highest). Besides, 2.5 mg/l nAg is also significantly noticed in ginseng root growth. However, the negative impact on the growth of the in vitro P. vietnamensis lateral roots showed when culture medium contained the highest concentration; such as the root growing inhibition of nCu and nAg above 2.5 mg/l. Especially, this decrease was higher with the application of nZnO0.5-2.5 mg/l (decrease the lateral root number) and 2.5 mg/l (decrease percent of lateral root formation).

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Cellular and molecular events in a newly organizing lateral root meristem

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1995.0135 ◽

1995 ◽

Vol 350 (1331) ◽

pp. 39-43 ◽

Cited By ~ 17

Keyword(s):

Lateral Root ◽

Root Formation ◽

Developmental Process ◽

Lateral Roots ◽

Root Apical Meristem ◽

Cdna Libraries ◽

Lateral Root Formation ◽

Ribosomal Protein Genes ◽

Efficient System

Spontaneous or auxin-induced lateral root formation in radish and Arabidopsis provides an efficient system in which to examine molecular and cellular events associated with the initiation of a new meristem. Subtracted cDNA libraries made at different times in lateral root initiation were used as a source of genes that are expressed differentially during this developmental process, and expression studies on a small gene family of ribosomal protein genes were conducted. From analysis of cell division patterns in pericycle cells the number of founder cells for lateral roots was established. By the use of in vitro growth assays lateral root formation was determined to be a two-stage process. First a primordium is formed, and subsequently a subset of primordial cells begins to function as the lateral root apical meristem. This mode of root development has implications for pattern formation in newly organizing organs.

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