Analysis of extracellular proteins by native polyacrylamide gel electrophoresis with infiltrated leaf tissue: application to pathogenesis-related proteins

1988 ◽  
Vol 66 (6) ◽  
pp. 1227-1229 ◽  
Author(s):  
Jean Grenier ◽  
François Côté ◽  
Alain Asselin
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Leaf Tissue ◽  
Polyacrylamide Gel ◽  
Simple Method ◽  
Pathogenesis Related Proteins ◽  
Intercellular Fluid ◽  
Pathogenesis Related ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Related Proteins

In addition to polyacrylamide gel electrophoretic analysis of intercellular fluid extracts, a simple method of detection of extracellular pathogenesis-related proteins was based on direct native polyacrylamide gel electrophoresis for acidic and basic proteins with leaf tissue infiltrated with 150 mM sucrose. This technique allowed for the detection of the complete set of tobacco pathogenesis-related proteins without having to extract the intercellular fluid by low-speed centrifugation. A major advantage of the technique is the capacity to observe the distribution of extracellular endogenous or exogenous proteins in the tissue directly subjected to electrophoresis.

Detection of 10 additional pathogenesis-related (b) proteins in intercellular fluid extracts from stressed 'Xanthi-nc' tobacco leaf tissue

1987 ◽  
Vol 65 (3) ◽  
pp. 476-481 ◽  
Author(s):  
Richard Hogue ◽  
Alain Asselin
Keyword(s):  
Gel Electrophoresis ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Leaf Tissue ◽  
Polyacrylamide Gel ◽  
Basic Proteins ◽  
Pathogenesis Related Proteins ◽  
Intercellular Fluid ◽  
Pathogenesis Related ◽  
Related Proteins

By using two-dimensional polyacrylamide gel electrophoresis, 10 additional pathogenesis-related proteins (b6c, b9c, b10a, b10b, b11a, b11b, b12, b13, b14, b15) were found in intercellular fluid extracts of stressed 'Xanthi-nc' tobacco leaf tissue. Proteins were identified as extracellular pathogenesis-related stress proteins by polyacrylamide gel electrophoresis analysis of three successive intercellular fluid extracts compared with homogenates before and after making intercellular fluid extracts. Four proteins (b12, b13, b14, b15) were only resolved by using native polyacrylamide gel electrophoresis for basic proteins in the first dimension gel and they were best extracted in 0.05 M Tris–HCl (pH 7.5) and 0.05 M CaCl2 as the infiltration buffer. The four basic proteins were found in intercellular fluid extracts from leaf tissue subjected to the same types of chemical inducers as previously described for tobacco pathogenesis-related proteins. Their accumulation was inhibited by basic amino acids or spermidine (1 mM) and they were resistant to endogenous and exogenous (trypsin, subtilisin) proteolysis. They did not bind to concanavalin A – Sepharose. These findings indicate that at least 23 proteins accumulate extracellularly after various types of stress in 'Xanthi-nc' tobacco green tissue. These proteins probably represent several groups or families of plant stress proteins.

Light-influenced extracellular accumulation of b (pathogenesis-related) proteins in Nicotiana green tissue induced by various chemicals or prolonged floating on water

1985 ◽  
Vol 63 (7) ◽  
pp. 1276-1283 ◽  
Author(s):  
Alain Asselin ◽  
Jean Grenier ◽  
François Côté
Keyword(s):  
Amino Acids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nicotiana Sylvestris ◽  
Protein Accumulation ◽  
Pathogenesis Related Proteins ◽  
Intercellular Fluid ◽  
Light Regimes ◽  
Green Tissue ◽  
Pathogenesis Related ◽  
Related Proteins

The extracellular accumulation of b (pathogenesis-related) proteins in Nicotiana tabacum cv. Xanthi-nc and cv. Samsun and in Nicotiana sylvestris was studied by floating leaf disks on solutions (50 μM – 5 mM) of putative inducers at pH 4 to 8 for 72 h. Intercellular fluid b proteins were detected only in green tissue, and optimal conditions required specific light regimes. Amino acids, organic acids, sugars, nucleotides, amines, vitamins, hormones, metals, and several other chemicals were compared with acetylsalicylate at 0.3 mM for the presence of b proteins in intercellular fluid extracts analyzed by native polyacrylamide gel electrophoresis. Several protein and non-protein amino acids and some of their derivatives were inducers in addition to thiamine, thiamine derivatives, and a few other chemicals at concentrations ranging from 200 μM to 5 mM. Various ways of introducing inducers into tissue (floating, injection, infiltration, root uptake) allowed b protein accumulation. Stimulation of b protein accumulation was observed when osmotica (sucrose at 200 mM) were added to inducers. Chelating agents, spermidine, lysine, and hydroquinone could inhibit b protein accumulation induced by L-serine, thiamine, L-α-aminoisobutyrate, and silver nitrate. Results with inducing chemicals and with prolonged floating of tissue on water, which was also conducive to b protein accumulation, support the hypothesis that b protein accumulation is a green tissue generalized response to various forms of prolonged stress.

scholarly journals Isolation of pathogenesis-related proteins from TMV-infected tobacco and their influence on infectivity of TMV

2010 ◽  
Vol 41 (No. 2) ◽  
pp. 52-57 ◽  
Author(s):  
M. Šindelářová ◽  
L. Šindelář
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Leaf Tissue ◽  
Exchange Chromatography ◽  
Pr Proteins ◽  
Inhibitory Influence ◽  
Pathogenesis Related Proteins ◽  
Molecular Weights ◽  
Pathogenesis Related ◽  
Related Proteins

The composition of pathogenesis-related proteins (PR-proteins) in the intercellular fluid (ICF) and leaf tissue of the hypersensitive tobacco cultivar Xanthi-nc inoculated with Tobacco mosaic virus (TMV), and their inhibitory influence on TMV multiplication were studied. The ICF PR-proteins of infected plants were separated after solubilisation by decreasing gradient of ammonium sulphate, the cell PR-proteins were separated after acidic homogenisation of leaf tissues. The ICF and cell PR-proteins were further purified by ion exchange chromatography on DEAE cellulose. Using discontinuous non-denaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions the PR-proteins were detected. Their molecular weights were estimated by SDS-PAGE. The ICF and cell proteins of infected leaves included PR-proteins of the molecular weights 15–16 kDa (Group 1), 27–28 kDa (Group 3: chitinases) and 36–40 kDa (Group 2a: &#1704-1,3-glucanases). Fractions with different PR-proteins were tested for their effect on infectivity of TMV. Particularly the PR3 and PR2a proteins seem to decrease the infectivity of TMV.

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