scholarly journals Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts

2021 ◽  
Author(s):  
Johannes W. Debler ◽  
Bernadette M. Henares ◽  
Robert C. Lee
Keyword(s):  
Broad Spectrum ◽  
Faba Bean ◽  
Fluorescent Protein ◽  
Transient Gene Expression ◽  
Low Frequency ◽  
Effector Proteins ◽  
Field Pea ◽  
Grain Legume ◽  
Pathogen Effectors ◽  
Green Fluorescent

Abstract Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

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.

Spectral Imaging for Separation of Fluorescent Signals from Autofluorescence

2000 ◽  
Vol 6 (S2) ◽  
pp. 838-839
Author(s):  
Richard M. Levenson
Keyword(s):  
Green Fluorescent Protein ◽  
Connective Tissue ◽  
Fluorescence Microscopy ◽  
Broad Spectrum ◽  
Fluorescent Protein ◽  
Fluorescence Signal ◽  
Post Processing ◽  
Neuronal Tissue ◽  
Green Fluorescent ◽  
Pathology Specimen

Autofluorescence , also known as adventitious fluorescence or background fluorescence, ofter poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can swamp the desired signal. Particularly difficult samples to image include many pathology specimen that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most (green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels which can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal.However, in many cases, these approaches are either infeasible or ineffective.

scholarly journals Necrosis-Inducing Proteins of Rhynchosporium commune, Effectors in Quantitative Disease Resistance

2012 ◽  
Vol 25 (10) ◽  
pp. 1314-1325 ◽  
Author(s):  
S. Kirsten ◽  
A. Navarro-Quezada ◽  
D. Penselin ◽  
C. Wenzel ◽  
A. Matern ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Quantitative Polymerase Chain Reaction ◽  
Fungal Spores ◽  
Growth Patterns ◽  
Effector Proteins ◽  
Defense Gene Expression ◽  
Host Genotype ◽  
Barley Cultivars ◽  
Green Fluorescent ◽  
The Impact

The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an “active” NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner.

scholarly journals pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

2015 ◽  
Vol 28 (2) ◽  
pp. 107-121 ◽  
Author(s):  
Xiaoyan Gong ◽  
Oscar Hurtado ◽  
Baohua Wang ◽  
Congqing Wu ◽  
Mihwa Yi ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Effector Proteins ◽  
Fungal Transformation ◽  
In Planta ◽  
Green Fluorescent

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their “directly fused” counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

scholarly journals Phytobacterial Type III Effectors HopX1, HopAB1 and HopF2 Enhance Sense-Post-Transcriptional Gene Silencing Independently of Plant R Gene-Effector Recognition

2011 ◽  
Vol 24 (8) ◽  
pp. 907-917 ◽  
Author(s):  
Panagiotis F. Sarris ◽  
Shang Gao ◽  
Konstantinos Karademiris ◽  
Hailing Jin ◽  
Kriton Kalantidis ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Pathogenic Bacteria ◽  
Fluorescent Protein ◽  
Effector Proteins ◽  
Transcriptional Gene Silencing ◽  
Type Iii ◽  
Post Transcriptional Gene Silencing ◽  
Rna Pathways ◽  
Green Fluorescent ◽  
Effector Recognition

Plant- and animal-pathogenic bacteria deploy a variable arsenal of type III effector proteins (T3EP) to manipulate host defense. Specific biochemical functions and molecular or subcellular targets have been demonstrated or proposed for a growing number of T3EP but remain unknown for the majority of them. Here, we show that transient expression of genes coding certain bacterial T3EP (HopAB1, HopX1, and HopF2), which did not elicit hypersensitive response (HR) in transgenic green fluorescent protein (GFP) Nicotiana benthamiana 16C line, enhanced the sense post-transcriptional gene silencing (S-PTGS) triggered by agrodelivery of a GFP-expressing cassette and the silencing enhancement could be blocked by two well-known viral silencing suppressors. Further analysis using genetic truncations and site-directed mutations showed that the receptor recognition domains of HopAB1 and HopX1 are not involved in enhancing silencing. Our studies provide new evidence that phytobacterial pathogen T3EP manipulate the plant small interfering RNA pathways by enhancing silencing efficiency in the absence of effector-triggered immunity signaling and suggest that phytopathogenic bacterial effectors affect host RNA silencing in yet other ways than previously described.

scholarly journals Bacterial Activity in the Rhizosphere Analyzed at the Single-Cell Level by Monitoring Ribosome Contents and Synthesis Rates

2000 ◽  
Vol 66 (2) ◽  
pp. 801-809 ◽  
Author(s):  
Cayo Ramos ◽  
Lars Mølbak ◽  
Søren Molin
Keyword(s):  
Growth Rate ◽  
Single Cell ◽  
Monitoring System ◽  
Fluorescent Protein ◽  
Transient Gene Expression ◽  
Single Cell Level ◽  
Cell Level ◽  
Gene Encoding ◽  
Rate Dependent ◽  
Green Fluorescent

ABSTRACT The growth activity of Pseudomonas putida cells colonizing the rhizosphere of barley seedlings was estimated at the single-cell level by monitoring ribosomal contents and synthesis rates. Ribosomal synthesis was monitored by using a system comprising a fusion of the ribosomal Escherichia coli rrnBP1 promoter to a gene encoding an unstable variant of the green fluorescent protein (Gfp). Gfp expression in a P. putida strain carrying this system inserted into the chromosome was strongly dependent on the growth phase and growth rate of the strain, and cells growing exponentially at rates of ≥0.17 h−1 emitted growth rate-dependent green fluorescence detectable at the single-cell level. The single-cell ribosomal contents were very heterogeneous, as determined by quantitative hybridization with fluorescently labeled rRNA probes in P. putida cells extracted from the rhizosphere of 1-day-old barley seedlings grown under sterile conditions. After this, cells extracted from the root system had ribosomal contents similar to those found in starved cells. There was a significant decrease in the ribosomal content of P. putida cells when bacteria were introduced into nonsterile bulk or rhizosphere soil, and the Gfp monitoring system was not induced in cells extracted from either of the two soil systems. The monitoring system used permitted nondestructive in situ detection of fast-growing bacterial microcolonies on the sloughing root sheath cells of 1- and 2-day-old barley seedlings grown under sterile conditions, which demonstrated that it may be possible to use the unstable Gfp marker for studies of transient gene expression in plant-microbe systems.

scholarly journals A vector carrying the GFP gene (Green fluorescent protein) as a yeast marker for fermentation processes

2000 ◽  
Vol 57 (4) ◽  
pp. 713-716 ◽  
Author(s):  
Luiz Humberto Gomes ◽  
Keila Maria Roncato Duarte ◽  
Felipe Gabriel Andrino ◽  
Ana Maria Brancalion Giacomelli ◽  
Flavio Cesar Almeida Tavares
Keyword(s):  
Green Fluorescent Protein ◽  
Early Detection ◽  
Fluorescent Protein ◽  
Low Frequency ◽  
Yeast Cells ◽  
Gfp Gene ◽  
Spoilage Yeasts ◽  
Adh2 Promoter ◽  
Pure Cultures ◽  
Green Fluorescent

Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3). Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.

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