scholarly journals I see the light! Fluorescent proteins suitable for cell wall/apoplast targeting in Nicotiana benthamiana leaves

Plant Direct ◽  
2019 ◽  
Vol 3 (1) ◽  
pp. e00112 ◽  
Author(s):  
Angela Stoddard ◽  
Vivien Rolland
Keyword(s):  
Cell Wall ◽  
Nicotiana Benthamiana ◽  
Fluorescent Proteins ◽  
Cell Wall Apoplast

The transpiration stream in the leaf apoplast: water and solutes

1993 ◽  
Vol 341 (1295) ◽  
pp. 87-100 ◽  
Keyword(s):  
Cell Wall ◽  
Cell Membranes ◽  
Gradient Flow ◽  
Pressure Vessels ◽  
Water Soluble ◽  
Fluorescent Tracers ◽  
Transpiration Stream ◽  
Cell Wall Apoplast ◽  
Flow Through ◽  
High Concentrations

Flow of the transpiration stream in the lumen apoplast of the xylem appears hydrodynamically orthodox in being approximately described by the Hagen-Poiseuille Law, and by Murray’s Law for branching pipes. Flow may be followed in the major (supply) veins by labelling the stream with dye solutions. Progress of the dye in the stream into the minor (distribution) veins is obscured by surrounding tissues. Observations of the spread of fluorescent tracers from these veins in living leaves gave results that have been seriously misinterpreted to present a false view of the cell wall apoplast. Microscopy of the stabilized water-soluble fluorescent tracers moving out of the minor veins has revealed that: (i) the dye is separated from the water by filtration through cell membranes, and the water moves through the symplast; and (ii) the dye diffuses in the cell wall apoplast at rates 1/100 to 1/10 000 the rate of diffusion in water. As a consequence of (i), high concentrations of dye build up at sites called sumps. In grasses these sumps may be in the intercellular spaces outside the xylem. In dicotyledons these sumps are within the small tracheary elements. In fact, flow in the lumen apoplast is flow through leaky tubes, and is inadequately described by the Hagen-Poiseuille Law. Leaky tubes have a critical radius, below which (for a given pressure gradient) flow cannot occur. As a consequence of this, a wedge of xylem made up of vessels of different radii acts as a unit to concentrate dye tracers in a sump at its apex. Sumps may also be formed by evaporation of the water in the stream, especially at leaf margins. Investigations with the cryo-analytical scanning electron microscope of the natural ions of the transpiration stream reveal high concentrations of K, Cl, P and Ca in the stream in all the sizes of vessel and vein of sunflower leaves. These high concentrations appear to be produced, not by the mechanisms responsible for the formation of sumps of dyes, but by some other processes, probably occurring in the stem. The absence of sump formation by ions at the places where dyes form sumps is probably due to the more rapid penetration of the ions through the cell membranes. Reasons for the discrepancy between these measurements of salt concentrations in the stream and those obtained from sap expressed from leaves by pressure vessels are discussed. Implications of these facts for the design and interpretation of experiments with leaves are presented.

Determinations of dye diffusivities in the cell-wall apoplast of roots by a rapid method

1996 ◽  
Vol 134 (1) ◽  
pp. 1-4 ◽  
Author(s):  
C. BAYLISS ◽  
C. DER WEELE ◽  
M. J. CANNY
Keyword(s):  
Cell Wall ◽  
Rapid Method ◽  
Cell Wall Apoplast

scholarly journals Glycerol‐3‐phosphate acyltransferase 6 controls filamentous pathogen interactions and cell wall properties of the tomato and Nicotiana benthamiana leaf epidermis

2019 ◽  
Vol 223 (3) ◽  
pp. 1547-1559 ◽  
Author(s):  
Stuart Fawke ◽  
Thomas A. Torode ◽  
Anna Gogleva ◽  
Eric A. Fich ◽  
Iben Sørensen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Nicotiana Benthamiana ◽  
Leaf Epidermis ◽  
Wall Properties ◽  
Cell Wall Properties

scholarly journals Fluorescent labelling reveals spatial separation of potyvirus populations in mixed infected Nicotiana benthamiana plants

2003 ◽  
Vol 84 (10) ◽  
pp. 2871-2876 ◽  
Author(s):  
Christof Dietrich ◽  
Edgar Maiss
Keyword(s):  
Nicotiana Benthamiana ◽  
Fluorescent Proteins ◽  
Spatial Separation ◽  
Potato Virus X ◽  
Plum Pox Virus ◽  
Cdna Clones ◽  
Fluorescent Cell ◽  
Red Fluorescent Proteins ◽  
Large Numbers ◽  
Tobacco Vein Mottling Virus

The distribution of potyviruses in mixed infected Nicotiana benthamiana plants was investigated by using green and red fluorescent proteins (GFP, DsRed). Full-length cDNA clones of Plum pox virus (PPV-NAT-AgfpS; PPV-NAT-red), Tobacco vein mottling virus (TVMV-gfp; TVMV-red) and Clover yellow vein virus (ClYVV-GFP) expressing fluorescent proteins, referred to here as labelled viruses, were used to characterize the distribution of different potyviral populations (e.g. TVMV-gfp/PPV-NAT-red), as well as populations of identical, but differently labelled potyviruses (e.g. PPV-NAT-AgfpS/PPV-NAT-red) or in mixed infections of potyviruses with labelled Potato virus X (PVX). Plants infected by any of the PVX/potyvirus combinations exhibited synergistic symptoms and large numbers of cells were doubly infected. In contrast, co-infections of differently labelled potyvirus populations appeared non-synergistic and remained predominantly separate in the infected plants, independent of whether different viruses or identical but differently labelled viruses were co-infecting. Contact of differently labelled virus populations that exhibited spatial separation was restricted to a small number of cells at the border of different fluorescent cell clusters.

scholarly journals Glycerol phosphate acyltransferase 6 controls filamentous pathogen interactions and cell wall properties of the tomato and Nicotiana benthamiana leaf epidermis

2018 ◽  
Author(s):  
Stuart Fawke ◽  
Thomas A. Torode ◽  
Anna Gogleva ◽  
Eric A. Fich ◽  
Iben Sørensen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Nicotiana Benthamiana ◽  
Orthologous Gene ◽  
Outer Cell ◽  
Loss Of Function ◽  
Glycerol Phosphate ◽  
Disease Symptoms ◽  
Structure Morphology ◽  
Microbe Interactions ◽  
Pathogen Entry

ABSTRACTThe leaf epidermal wall is covered by a cuticle, composed of cutin and waxes, which protects against dehydration and constitutes a barrier against pathogen attack. Cutin monomers are formed by the transfer of 16- or 18-carbon fatty acids to glycerol by glycerol-3-phosphate acyltransferase (GPAT) enzymes, which facilitates their transport to the plant surface. Here we address the dual functionality of pathogen-inducible Glycerol phosphate acyltransferase 6 (GPAT6) in controlling pathogen entry and dehydration in leaves. Silencing of Nicotiana benthamiana NbGPAT6a increased leaf susceptibility to the oomycetes Phytophthora infestans and P. palmivora, whereas stable overexpression of NbGPAT6a-GFP rendered leaves more resistant to infection. A loss-of-function mutation of the orthologous gene in tomato (Solanum lycopersicum), SlGPAT6, similarly resulted in increased susceptibility of leaves to Phytophthora infection concomitant with altered intracellular infection structure morphology. Conversely, Botrytis cinerea disease symptoms were reduced. Modulation of GPAT6 expression predominantly altered the outer cell wall of leaf epidermal cells. The impaired cell wall-cuticle continuum of tomato gpat6-a mutants resulted in increased water loss and these plants had fewer stomata. Our work highlights a hitherto unknown role for GPAT6-generated cutin monomers in controlling epidermal cell properties that are integral to leaf-microbe interactions and limit dehydration.

scholarly journals Developing a Nicotiana benthamiana – Citrus tristeza virus model system to study superinfection exclusion

2020 ◽  
Vol 21 (2) ◽  
Author(s):  
Mariana Suguieda ◽  
Sung-Hwan Kang ◽  
Svetlana Yuryevna Folimonova
Keyword(s):  
Nicotiana Benthamiana ◽  
Fluorescent Proteins ◽  
Secondary Infection ◽  
Model System ◽  
Wild Type Virus ◽  
Protection Measures ◽  
Suitable Alternative ◽  
Superinfection Exclusion ◽  
Citrus Production ◽  
Citrus Tristeza

Citrus tristeza (CTV) is the causal agent of economically devasting diseases that affect  global citrus production. CTV has demonstrated superinfection exclusion (SIE), a phenomenon where a preexisting viral infection excludes a secondary infection by the same or closely related virus. This phenomenon has been used to develop cross-protection measures against CTV but there have been erratic results with this approach. Understanding of SIE by CTV on the molecular level has been challenging, especially due to time-consuming trials of over a year when citrus plants are used in the experiments. The purpose of this study was to develop a model system to study SIE by CTV by using a more amenable species, Nicotiana benthamiana, rather than citrus plants. cDNA constructs of CTV containing extra genes of the green or red fluorescent proteins (GFP/RFP) were engineered and transformed into Agrobacterium tumefaciens to visualize virus movement and SIE within the N. benthamiana plants by using the agro-infiltration approach for the virus constructs delivery.  The constructs carrying the full-length wild-type virus with the additional GFP- or RFP-encoding gene and the GFP-tagged hybrid constructs with deletion in the p33 gene were infiltrated with the agro-suspension and visualized under a microscope. The images captured were analyzed for peaks in grayscale values in the green or red channels along a line. The constructs used in the study behaved similarly in N. benthamiana and Citrus macrophylla but the trial time was significantly shortened with the N. benthamiana plants. Thus, N. benthamiana is a suitable alternative for studying SIE by CTV with the constructs in this study.

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