Initial work of Dr. Noel Thomas Keen on phytoalexin elicitor activity of carbohydrate elicitors from Phytophthora cell walls along with Dr. Yoshikawa

1. Molecular basis of host pathogen interaction
“Phytoalexin elicitor activity of carbohydrates from Phytophthora
megasperma f.sp. glycinea and other sources”
Indian Agricultural Research Institute
Division of Plant Pathology
Speaker - M. Ashajyothi, 10863
Ph.D Scholar

2. Background
• Gene for gene hypothesis - H.H.Flor (1946)
• Protein for protein hypothesis - Vanderplank (1978)
• Elicitor- receptor model - Albersheim (1975)
• Term Elicitors - The molecules that signal plants to begin synthesizing
phytoalexins
• Plant origin - Plant enzymes & unsaturated fatty acids : Arachidonic acid
• Microbe origin - Peptides, enzymes : Poly galacturonases & Pectate lyases,
Polysaccharides or glycoconjugates

3. Hypothesis
Phytophthora avr gene Carbohydrate elicitors
Soybean R gene R gene products
• Albersheim et al. Isolated Hepta-1,3-glucoside from the culture medium and
from isolated cell walls of Pmg.
• Yoshikawa et al. incubation of isolated cell walls from Pmg with soybean β-1,3-
endoglucanase enzyme released water-soluble carbohydrates with potent
elicitor activity.
Resistance

4. Noel Thomas Keen
Pioneer in the molecular plant pathology

5. Soybean – Pmg pathosystem
• Soybean plants exemplifies a gene-for-gene host-parasite relationship, and
single gene resistance in the host is expressed by the postinfectional
production of phytoalexins called 'glyceollin‘.
• Hepta-1,3-glucoside in the cell wall of the fungus Phytophthora
megasperma f.sp. glycinea is an elicitor of phytoalexins in soybean
cotyledons.
• The heptaglucoside elicitor was structurally characterized, and the
structure confirmed by chemical synthesis.
• These results provided the first compelling evidence that plants have the
capacity to recognize structurally defined oligosaccharides as regulatory
molecules.

6. Objectives
1. Isolate and partially characterize the carbohydrate elicitor released
by soybean endoglucanase.
2. Compare the elicitor activity of the various polysaccharides thus far
isolated from Pmg with structurally related carbohydrates.
3. Assess which elicitor(s) is of probable physiological significance in the
plant pathogen interaction.
General methods
 Phytophthora megasperma f.sp. glycinea (Race 1,2,3,4,5 ) – cell wall isolation -
tested for the binding of FITC Con A
 Soybean Cultivars : Harosoy (rps) / Near isogenic Harosoy 63 (Rpsa)

7. • Elicitor Preparation:
Purified hyphal cell walls (0.6 g)
(Incubation)
60ml 10 mM Na-acetate (pH 5.3) containing 20 - 50 ug/ ml of soybean
endoglucanase (after 50 to 80 min)
Centrifuge and take supernatant pass through Millipore filters (0.22um)
Dialyzed against water and brought to pH 4.4 with dilute Hcl
dialyzed against dH20 and lyophilized, yielding white amorphous powders.
To remove endoglucanase pass through 0.7- x 5.0-cm columns of CM Bio-gel A
Washing with the acetate buffer was then concentrated to approximately 0.05% of
the original volume in vacuo

8.  Release of Glycopeptide Elicitors from Cell Walls with Pronase.
Elicitor Bioassays
Cotyledon Bioassay:
Six cotyledons freshly harvested from 8- to 9-d-old Harosoy 63 plants
Wounded on the undersurfaces
Serial dilutions of the elicitor solutions
Rifampicin and penicillin were added to 10 and 300ug ml-‘
Solutions were placed onto the cut Surfaces and incubated at room temperature
under lights for 24 h
Take five representative cotyledons added to 20 ml of dH20 which was thoroughly mixed,
and glyceollin was immediately determined at 285 nm.

9. • Hypocotyl Bioassay:
Solutions of elicitors introduced into 1-cm hypocotyl wounds on intact 6-d-old
soybean plants grown in 10-cm pots.
Plants were covered with plastic bags and incubated for 18 to 24 h at 22°C in a
lighted growth chamber.
The wounded areas were then excised, weighed, and placed into 18-mm test tubes
with 6 ml of 95% ethanol.
Tubes were placed in a boiling water bath for 1 min, and the fluids were immediately
decanted into a second tube which was taken to dryness In vacuo at 50°C.
The entire contents of the dried tube were dissolved in 0.15 ml of CHCl3
Further estimation of glyciollin

10. Findings
 Isolation and Partial Characterzation of the Elicitor Released from Walls by
Glucanase
• Reaction products from Pmg cell walls incubated with purified soybean
endoglucanase chromatographed on S-200 Sephacryl columns as two
carbohydrate peaks.
• Reincubation of the fraction I carbohydrate did not alter its elution position
from the S-200 Sephacryl column.
• It is not further degraded by the endoglucanase and that fraction II is not a
degradation product of fraction I.
S-200 Sephacryl columnsSize exclusion chromatography

11. • Analysis of the sugars by acid hydrolysis showed that fraction I elicitors
contained glucose and mannose as the major neutral sugars.
• Preparations from two different isolates of race I gave a glucose/mannose ratio
of 55% to 45% ± 1%.
• A single race 4 sample gave glucose/mannose at 52% to 48%, whereas a single
race 7 sample yielded glucose/mannose at 98 to 1, with 1% arabinose.
• Release of Glycopeptide elicitors
• Chromatography of the products liberated by pronase led to the recovery of
heterogeneous peaks containing carbohydrate and protein
 A at 280 nm Protein
• A at 625 nm carbohydrate

12. Elicitor Activty
• The purified fraction I glucomannan released from Pmg cell walls by
endoglucanase was the most active elicitor of glyceollin.
• A glucomannan extracellular polysaccharide from the bacterial pathogen
Pseudomonas syringae pv. glycinea exhibited trace activity in the cotyledon
bioassay.
• The glucan elicitor and glycopeptides : 10-fold less active
• Intracellular Pmg oligosaccharide mycolaminaran : 100-fold less active.
• Laminarin gave activity comparable to mycolaminaran.
• Arachidonic acid reported to be a phytoalexin elicitor in potato was devoid of
similar activity in soybean cotyledons.
• Chitin and chitosan are elicitors in other plants and exhibited activity in
soybean cotyledons, but only at relatively high concentrations.

13. Elicitor Activity of Purified Glucomannans and Crude
Glucomannans in Soybean Hypocotyls

14. Fluorescein isothiocyanate (FITC)- concanavalin A (Con A) Staining
of fungal cell walls
Glucomannan is of interest because it gives a pronounced Con A precipitin test
and it indicate that the glucosyl residues are present in predominantly β -1,3
linkages.
• Glucomannans represent unique components of the cell wall of the fungus,
and they are different from the carbohydrates of the cell surface glycoproteins
and the β glucan fraction.
• The elicitor activity of carbohydrate polymers in soybean therefore requires
the presence of specific linkages, a conclusion consistent with the hypothesis
that elicitor activity is mediated by specific receptors in soybean cells.

15. • The cell surface glycoproteins and glucomannans are major candidates for
the fungus elicitors.
• They are surface molecules on the Pmg cell wall that would facilitate their
possible detection by plant cells.
• The glucomannans are the most active elicitors released within minutes, and
appear to give race-specific elicitation of glyceollin in soybean hypocotyls.
• These factors all suggest that they may be the physiologically important
elicitors in the relatively rapid interactions which are known to occur
between soybean cells and infecting fungus hyphae.
Conclusion

16. Thank you