scholarly journals An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue

Biosensors ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 197
Author(s):  
Gabriele Panicucci ◽  
Sergio Iacopino ◽  
Elisa De Meo ◽  
Pierdomenico Perata ◽  
Daan A. Weits
Keyword(s):  
Fluorescent Protein ◽  
Narrow Range ◽  
Oxygen Deficiency ◽  
Plant Cells ◽  
Untranslated Regions ◽  
Plant Tissues ◽  
Reporter Activity ◽  
Hypoxic Conditions ◽  
Environmental Availability ◽  
Transcriptional Output

Oxygen levels in plant tissues may vary, depending on metabolism, diffusion barriers, and environmental availability. Current techniques to assess the oxic status of plant cells rely primarily on invasive microoptodes or Clark-type electrodes, which are not optimally suited for experiments that require high spatial and temporal resolution. In this case, a genetically encoded oxygen biosensor is required instead. This article reports the design, test, and optimization of a hypoxia-signaling reporter, based on five-time repeated hypoxia-responsive promoter elements (HRPE) driving the expression of different reporter proteins. Specifically, this study aimed to improve its performance as a reporter of hypoxic conditions by testing the effect of different untranslated regions (UTRs) at the 5′ end of the reporter coding sequence. Next, we characterized an optimized version of the HRPE promoter (HRPE-Ω) in terms of hypoxia sensitivity and time responsiveness. We also observed that severe oxygen deficiency counteracted the reporter activity due to inhibition of GFP maturation, which requires molecular oxygen. To overcome this limitation, we therefore employed an oxygen-independent UnaG fluorescent protein-coupled to an O2-dependent mCherry fluorophore under the control of the optimized HRPE-Ω promoter. Remarkably, this sensor, provided a different mCherry/UnaG ratiometric output depending on the externally imposed oxygen concentration, providing a solution to distinguish between different degrees of tissue hypoxia. Moreover, a ubiquitously expressed UnaG-mCherry fusion could be used to image oxygen concentrations directly, albeit at a narrow range. The luminescent and fluorescent hypoxia-reporters described here can readily be used to conduct studies that involve anaerobiosis in plants.

Direct protein delivery into intact plant cells using polyhistidine peptides

2021 ◽  
Author(s):  
Yoshino Tanaka ◽  
Yoshihiko Nanasato ◽  
Kousei Omura ◽  
Keita Endoh ◽  
Tsuyoshi Kawano ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fusion Proteins ◽  
Protein Delivery ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Plant Cells ◽  
Adenosine Monophosphate ◽  
Cell Penetrating Peptides ◽  
Intracellular Space ◽  
Intracellular Fluorescence

Abstract Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8–His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

scholarly journals Cytokinins Stimulate Plasmodesmatal Transport in Leaves

2021 ◽  
Vol 12 ◽  
Author(s):  
Wilson Horner ◽  
Jacob O. Brunkard
Keyword(s):  
Cell Walls ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Plant Biology ◽  
Negative Regulator ◽  
Cytokinin Signaling ◽  
Dynamic Changes ◽  
Cytokinin Receptors ◽  
Green Fluorescent ◽  
Development Regulation

Plant cells are connected by plasmodesmata (PD), nanoscopic channels in cell walls that allow diverse cytosolic molecules to move between neighboring cells. PD transport is tightly coordinated with physiology and development, although the range of signaling pathways that influence PD transport has not been comprehensively defined. Several plant hormones, including salicylic acid (SA) and auxin, are known to regulate PD transport, but the effects of other hormones have not been established. In this study, we provide evidence that cytokinins promote PD transport in leaves. Using a green fluorescent protein (GFP) movement assay in the epidermis of Nicotiana benthamiana, we have shown that PD transport significantly increases when leaves are supplied with exogenous cytokinins at physiologically relevant concentrations or when a positive regulator of cytokinin responses, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 5 (AHP5), is overexpressed. We then demonstrated that silencing cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE 3 (AHK3) or AHK4 or overexpressing a negative regulator of cytokinin signaling, AAHP6, significantly decreases PD transport. These results are supported by transcriptomic analysis of mutants with increased PD transport (ise1–4), which show signs of enhanced cytokinin signaling. We concluded that cytokinins contribute to dynamic changes in PD transport in plants, which will have implications in several aspects of plant biology, including meristem patterning and development, regulation of the sink-to-source transition, and phytohormone crosstalk.

Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: Fluorophore variant, actin binding domain, and promoter considerations

Cytoskeleton ◽  
2014 ◽  
Vol 71 (5) ◽  
pp. 311-327 ◽  
Author(s):  
Julia Dyachok ◽  
J. Alan Sparks ◽  
Fuqi Liao ◽  
Yuh-Shuh Wang ◽  
Elison B. Blancaflor
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Binding Domain ◽  
Actin Binding ◽  
Living Plant ◽  
Actin Binding Domain

scholarly journals Identification of a Protein from Rust Fungi Transferred from Haustoria into Infected Plant Cells

2005 ◽  
Vol 18 (11) ◽  
pp. 1130-1139 ◽  
Author(s):  
Eric Kemen ◽  
Ariane C. Kemen ◽  
Maryam Rafiqi ◽  
Uta Hempel ◽  
Kurt Mendgen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Polyclonal Antibodies ◽  
Rust Fungus ◽  
Infected Plant ◽  
Plant Cells ◽  
Host Cells ◽  
Nuclear Localization Signals ◽  
Uromyces Fabae ◽  
Host Cytoplasm ◽  
Functional Efficiency

The formation of haustoria is one of the hallmarks of the interaction of obligate biotrophic fungi with their host plants. In addition to their role in nutrient uptake, it is hypothesized that haustoria are actively involved in establishing and maintaining the biotrophic relationship. We have identified a 24.3-kDa protein that exhibited a very unusual allocation. Rust transferred protein 1 from Uromyces fabae (Uf-RTP1p) was not only detected in the host parasite interface, the extrahaustorial matrix, but also inside infected plant cells by immunofluorescence and electron microscopy. Uf-RTP1p does not exhibit any similarity to sequences currently listed in the public databases. However, we identified a homolog of Uf-RTP1p in the related rust fungus Uromyces striatus (Us-RTP1p). The localization of Uf-RTP1p and Us-RTP1p inside infected plant cells was confirmed, using four independently raised polyclonal antibodies. Depending on the developmental stage of haustoria, Uf-RTP1p was found in increasing amounts in host cells, including the host nucleus. Putative nuclear localization signals (NLS) were found in the predicted RTP1p sequences. However, functional efficiency could only be verified for the Uf-RTP1p NLS by means of green fluorescent protein fusions in transformed tobacco protoplasts. Western blot analysis indicated that Uf-RTP1p and Us-RTP1p most likely enter the host cell as N-glycosylated proteins. However, the mechanism by which they cross the extrahaustorial membrane and accumulate in the host cytoplasm is unknown. The localization of RTP1p suggests that it might play an important role in the maintenance of the biotrophic interaction.

scholarly journals Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein

2009 ◽  
Vol 191 (9) ◽  
pp. 3120-3131 ◽  
Author(s):  
Joanne E. Morello ◽  
Alan Collmer
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Effector Proteins ◽  
Type Iii ◽  
Native Promoter ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.

Rapid and massive green fluorescent protein production leads to formation of protein Y-bodies in plant cells

2012 ◽  
Vol 77 (6) ◽  
pp. 603-608 ◽  
Author(s):  
T. V. Komarova ◽  
E. V. Sheval ◽  
D. V. Pozdyshev ◽  
V. S. Kolesnikova ◽  
Yu. L. Dorokhov
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Production ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Green Fluorescent Protein Production ◽  
Green Fluorescent

2nd International Workshop on Desiccation-Tolerance and -Sensitivity of Seeds and Vegetative Plant Tissues

1997 ◽  
Vol 7 (2) ◽  
pp. 61-61
Author(s):  
Patricia Berjak ◽  
Daphne Osborne ◽  
Norman Pammenter
Keyword(s):  
South Africa ◽  
Desiccation Tolerance ◽  
International Workshop ◽  
Plant Cells ◽  
Cape Town ◽  
Plant Tissues ◽  
Vegetative Plant ◽  
The World

AbstractHow some plant cells can survive a period of desiccation to live again when other cells will die, was the theme of the recent Workshop in the mountain retreat of Franschhoek, near Cape Town, South Africa. Sixty-two biochemists and cell physiologists from around the world assembled to discuss these problems for the second time in three years.

scholarly journals Evidence for Agrobacterium-Induced Apoptosis in Maize Cells

2000 ◽  
Vol 13 (6) ◽  
pp. 649-657 ◽  
Author(s):  
Geneviève Hansen
Keyword(s):  
Cell Death ◽  
Dna Cleavage ◽  
Morphological Changes ◽  
Plant Cells ◽  
Plant Tissues ◽  
Apoptotic Pathway ◽  
Dicotyledonous Plant ◽  
Apoptotic Genes ◽  
Baculovirus P35 ◽  
Induced Apoptosis

Agrobacterium spp. can genetically transform most dicotyledonous plant cells whereas many monocot species are recalcitrant to Agrobacterium-mediated transformation. One major obstacle is that co-cultivation of Agrobacterium spp. with plant tissues often results in cell death. Report here is that, in maize tissues, this process resembles apoptosis, with characteristic DNA cleavage into oligonucleosomal fragments and morphological changes. Two anti-apoptotic genes from baculovirus, p35 and iap, had the ability to prevent the onset of apoptosis triggered by Agrobacterium spp. in maize tissues. p35 is reported to act as a direct inhibitor of a certain class of proteases (caspase) whereas iap may act upstream to prevent their activation. This evidence raises the possibility that caspase-like proteases may also be involved in the apoptotic pathway in plant cells.

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