Stable Plastid Transformation in Nicotiana benthamiana

Seyed Javad Davarpanah,정서희,Yaw Joo Kim,박연일,민성란,유장렬,정원중 한국식물학회 2009 Journal of Plant Biology Vol.52 No.3

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.

Effects of CO2 enrichment, LED inter-lighting, and high plant density on growth of Nicotiana benthamiana used as a host to express influenza virus hemagglutinin H1

Lingling Shang,Linda Gaudreau,Michèle Martel,Dominique Michaud,Steeve Pepin,André Gosselin 한국원예학회 2018 Horticulture, Environment, and Biotechnology Vol.59 No.5

Plants are being recognized as promising hosts for molecular farming and several molecular tools have been developed over the last two decades to optimize recombinant protein yields. However, the effects of basic growth factors on protein yield have been much less studied. Here, we investigated the effects of supplemental light emitting diode (LED) inter-lighting, CO2enrichment, and plant density on growth and recombinant protein yield of Nicotiana benthamiana used as a host to express the vaccine antigen influenza virus hemagglutinin H1. LED inter-lighting improved plant growth and recombinant protein yield on a per-plant basis. CO2 enrichment also enhanced plant growth, but its effect on recombinant protein yield was not significant. By comparison, high plant density decreased recombinant protein production per plant, mainly because of its negative impact on protein accumulation on a per-plant basis. On a whole-crop area basis, supplemental lighting, CO2 enrichment, and high plant density improved plant growth, while only LED inter-lighting and high plant density positively impacted recombinant protein yield. We suggest that LED inter-lighting and an elevated plant density should be used to maximize H1 antigen yield in large-scale protein production systems using N. benthamiana.

Rpi-blb2-mediated late blight resistance in Nicotiana benthamiana requires SGT1 and salicylic acid-mediated signaling but not RAR1 or HSP90

Oh, S.K.,Kim, H.,Choi, D. North-Holland Pub ; Elsevier Science Ltd 2014 FEBS letters Vol.588 No.7

The Rpi-blb2 recognizes the presence of the Phytophthora infestans AVRblb2 and initiates effector-triggered immunity (ETI). We performed gain-of-function and loss-of-function studies in Nicotiana benthamiana to elucidate Rpi-blb2-mediated resistance to P. infestans. Rpi-blb2 triggered a hypersensitive response through SGT1-mediated, but not RAR-mediated or HSP90-mediated, pathways. NbSGT1 was also required for basal and ETI-mediated by Rpi-blb2 in N. benthamiana. Moreover, salicylic acid (SA) affected basal defense and Rpi-blb2-mediated resistance against P. infestans. The increased susceptibility of Rpi-blb2-transgenic plants in the NahG-background correlated with reduced levels of SA. These findings provide evidence for the roles of SGT1- and SA-signaling in Rpi-blb2-mediated resistance against P. infestans.

오이모자이크바이러스 2b 유전자 발현 담배의 형태 및 전사체 분석

손성한,김윤희,안율균,김도선,원소윤,김정선,최홍수 한국식물병리학회 2015 식물병연구 Vol.21 No.3

오이모자이크바이러스 2b 유전자는 전사후유전자침묵(PTGS)을 억제하는 기능을 가진 억제인자이다. 식물체내 2b 유전자 기능을 분석하기 위해 Nicotiana benthamiana에 형질전환하였고 형태변화와 유전자 발현변화를 분석하였다. 8계통의2b 유전자 형질전환체 중에서 1개의 유전자가 T0 개체에 삽입된 계통은 3개였다. 2b 유전자 형질전환체는 일반적으로 종자확보가 어려웠지만 다행히 일부 배수화되지 않은 계통(hemizygote) 에서는 소량의 종자가 확보되어 계통유지가 가능하였다. 고정계통의 전사체를 해독하여 대조와 비교분석한 바, 2b 유전자는 특정유전자의 발현을 선택적으로 증대시키는 것이 아닌다수의 유전자를 비선택적으로 발현을 증대시키는 것으로 판단되었다. 이러한 결과는 2b 유전자가 세포질에 존재하는 다양한 RNA의 대사중 분해를 억제하여 세포질내 RNA가 축적되고이로 인해 단백질 합성도 증대되어 정상적 생장발달이 저해되고 기형적인 형태의 식물체가 되는 요인으로 판단된다. Cucumber mosaic virus possesses 2b gene known as a suppressor of post-transcriptional gene silencing (PTGS). To investigate its function and effect in plant, transgenic Nicotiana benethamiana expressing 2b gene was developed and analyzed in phenotypic characteristics and differential gene expression (DEG) comparing with wild-type. Eight lines of transgenic plants (T0) were obtained with difficulty and showed severe deformed phenotypes in leaves, flowers, petioles and etc. Moreover, transgenic plants were hardly able to set seeds, but small amounts of seeds were barely produced in some of transgene-hemizygous plants. DEG analysis showed that transgenic plant ectopically accumulated diverse RNA transcripts at higher levels than wild-type probably due to the disturbance in RNA metabolism, especially of RNA decay, caused by 2b-mediated inhibition of PTGS. These ectopic accumulations of RNAs disrupt protein and RNA homeostasis and then subsequently lead to abnormal phenotypes of transgenic plants.

Encapsidation of genomic but not subgenomic Turnip yellow mosaic virus RNA by coat protein provided in trans

Cho, Tae-Ju,Dreher, Theo W. Elsevier 2006 Virology Vol.356 No.1

<P><B>Abstract</B></P><P>We have studied the encapsidation requirements of <I>Turnip yellow mosaic virus</I> (TYMV) genomic and subgenomic RNA using an “agroinfiltration” procedure involving transient expression of RNAs and coat protein (CP) in <I>Nicotiana benthamiana</I> leaves. Although <I>N. benthamiana</I> is a nonhost, expression of TYMV RNA in its leaves by agroinfiltration resulted in efficient local infection and production of the expected virions containing genomic and subgenomic RNAs together with empty capsids. A nonreplicating genomic RNA with a mutation in the polymerase domain was efficiently encapsidated by CP provided in trans, even though RNA levels were a thousand-fold lower than in normal infections. In contrast, encapsidation of CP mRNA was not observed under these conditions, even when the CP mRNA had authentic 5′- and 3′-termini. Deletion of the 3′-tRNA-like structure from the genomic RNA did not alter the encapsidation behavior, suggesting that this feature does not play a role in the encapsidation of TYMV RNA. Our results indicate differences in the encapsidation process between genomic and subgenomic RNAs, and suggest an interaction between RNA replication and the packaging of subgenomic RNA.</P>

DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants

Jeon, Young,Ahn, Chang Sook,Jung, Hyun Ju,Kang, Hunseung,Park, Guen Tae,Choi, Yeonhee,Hwang, Jihwan,Pai, Hyun-Sook Oxford University Press 2014 Journal of experimental botany Vol.65 No.1

<P>This study investigated protein characteristics and physiological functions of DER (Double Era-like GTPase) of higher plants. <I>Nicotiana benthamiana</I> DER (NbDER) contained two tandemly repeated GTP-binding domains (GD) and a C-terminal domain (CTD) that was similar to the K-homology domain involved in RNA binding. Both GDs possessed GTPase activity and contributed to the maximum GTPase activity of NbDER. NbDER fused to green fluorescent protein was localized primarily to chloroplast nucleoids. <I>Arabidopsis der</I> null mutants exhibited an embryonic lethal phenotype, indicating an essential function of DER during plant embryogenesis. Virus-induced gene silencing of NbDER resulted in a leaf-yellowing phenotype caused by disrupted chloroplast biogenesis. NbDER was associated primarily with the chloroplast 50S ribosomal subunit <I>in vivo</I>, and both the CTD and the two GD contributed to the association. Recombinant proteins of NbDER and its CTD could bind to 23S and 16S ribosomal RNAs <I>in vitro</I>. Depletion of NbDER impaired processing of plastid-encoded ribosomal RNAs, resulting in accumulation of the precursor rRNAs in the chloroplasts. NbDER-deficient chloroplasts contained significantly reduced levels of mature 23S and 16S rRNAs and diverse mRNAs in the polysomal fractions, suggesting decreased translation in chloroplasts. These results suggest that DER is involved in chloroplast rRNA processing and ribosome biogenesis in higher plants.</P>

A Super-Absorbent Polymer Combination Promotes Bacterial Aggressiveness Uncoupled from the Epiphytic Population

Lee, Bo-Young,Kim, Dal-Soo,Ryu, Choong-Min The Korean Society of Plant Pathology 2008 Plant Pathology Journal Vol.24 No.3

Plant leaf surface is an important niche for diverse epiphytic microbes, including bacteria and fungi. Plant leaf surface plays a critical frontline defense against pathogen infections. The objective of our study was to evaluate the effectiveness of a starch-based super-absorbent polymer(SAP) combination, which enhances water potential and nutrient availability to plant leaves. We evaluated the effect of SAP on the maintenance of bacterial populations. In order to monitor bacterial populations in situ, a SAP mixture containing Pseudomonas syringae pv. tabaci that expressed recombinant green fluorescent protein(GFPuv) was spray-challenged onto whole leaves of Nicotiana benthamiana. The SAP combination treatment enhanced bacterial robustness, as indicated by disease severity and incidence. Unexpectedly, bacterial numbers were not significantly different between leaves treated with the SAP combination and those treated with water alone. Furthermore, young leaves treated with the SAP combination had more severe symptoms and a greater number of bacterial spots caused by primary and secondary infections compared to young leaves treated with the water control. In contrast, bacterial cell numbers did not statistically differ between the two groups, which indicated that measurement of viable GFP-based bacterial spots may provide a more sensitive methodology for assessing virulence of bacterial pathogens than methods that require dilution plating following maceration of bacterial-inoculated leaf tissue. Our study suggests that the SAP combination successfully increased bacterial aggressiveness, which could either be used to promote the ability of biological agents to control weedy plants or increase the robustness of saprophytic epiphytes against competition from potentially harmful microbes.

A Super-Absorbent Polymer Combination Promotes Bacterial Aggressiveness Uncoupled from the Epiphytic Population

이보영,김달수,류충민 한국식물병리학회 2008 Plant Pathology Journal Vol.24 No.3

Plant leaf surface is an important niche for diverse epiphytic microbes, including bacteria and fungi. Plant leaf surface plays a critical frontline defense against pathogen infections. The objective of our study was to evaluate the effectiveness of a starch-based super-absorbent polymer (SAP) combination, which enhances water potential and nutrient availability to plant leaves. We evaluated the effect of SAP on the maintenance of bacterial populations. In order to monitor bacterial populations in situ, a SAP mixture containing Pseudomonas syringae pv. tabaci that expressed recombinant green fluorescent protein (GFPuv) was spray-challenged onto whole leaves of Nicotiana benthamiana. The SAP combination treatment enhanced bacterial robustness, as indicated by disease severity and incidence. Unexpectedly, bacterial numbers were not significantly different between leaves treated with the SAP combination and those treated with water alone. Furthermore, young leaves treated with the SAP combination had more severe symptoms and a greater number of bacterial spots caused by primary and secondary infections compared to young leaves treated with the water control. In contrast, bacterial cell numbers did not statistically differ between the two groups, which indicated that measurement of viable GFP-based bacterial spots may provide a more sensitive methodology for assessing virulence of bacterial pathogens than methods that require dilution plating following maceration of bacterialinoculated leaf tissue. Our study suggests that the SAP combination successfully increased bacterial aggressiveness, which could either be used to promote the ability of biological agents to control weedy plants or increase the robustness of saprophytic epiphytes against competition from potentially harmful microbes.

Complete genome sequence of a novel soybean mosaic virus isolated from soybean seeds in Korea

신유섭,박태선,박지수,민동주,김예영,박상민,이수헌,김상민,이봉춘,조원경,홍진성 한국미생물학회 2023 미생물학회지 Vol.59 No.3

Soybean mosaic virus (SMV), a member of the genus Potyvirus, primarily damages soybeans. Although most SMV strains do not infect Solanaceae plants, an SMV isolate obtained from soybean seeds (SMV-Gm) was confirmed to induce systemic infection in Nicotiana benthamiana. Its complete genome sequence was determined using reverse transcription (RT)- PCR and cloning, showing that it comprises 9588 nucleotides, with a GC content of 41.65% and a single large polyprotein, consistent with the typical SMV composition.

In planta production and characterization of full-length human adipose triglyceride lipase

Zaikova Kseniia,정진성,김경록,구강모,김정선,이동욱 한국식물생명공학회 2022 Plant biotechnology reports Vol.16 No.6

Adipose triglyceride lipase (ATGL) is an enzyme that plays a critical role in the mobilization of stored triacylglycerol. Despite its importance in lipid metabolism, in-depth biochemical characterization of ATGL has been limited because the expression and purifcation of this enzyme are not feasible. In this study, we attempted to generate the full-length human ATGL in Nicotiana benthamiana. The ATGL expressed in N. benthamiana was highly soluble and stable. The purifed ATGL exhibited lipase activity in vitro. In conclusion, the full-length human ATGL could be reliably produced using a plant-based expression system, thereby enabling detailed biochemical characterization of this enzyme.