IMMUNOCYTOCHEMICAL ANALYSIS OF IMMUNOCYTOCHEMICAL ANALYSIS OF - - PowerPoint PPT Presentation

IMMUNOCYTOCHEMICAL ANALYSIS OF IMMUNOCYTOCHEMICAL ANALYSIS OF SPECIFIC MOLECULAR COMPONENTS SPECIFIC MOLECULAR COMPONENTS ORGANISATION OF EXTRACELLULAR ORGANISATION OF EXTRACELLULAR SYMBIOTIC COMPARTMENTS PROVIDED SYMBIOTIC COMPARTMENTS PROVIDED INTERACTION BETWEEN SYMBIOTIC INTERACTION BETWEEN SYMBIOTIC PARTNERS DURING DEVELOPMENT OF ROOT PARTNERS DURING DEVELOPMENT OF ROOT NODULES OF PEA ( NODULES OF PEA (PISUM SATIVUM PISUM SATIVUM L.) L.)

EU EU -

• Russia: Prospects for Cooperation in Biotechnology in the

Russia: Prospects for Cooperation in Biotechnology in the Seventh Framework Seventh Framework Programme Programme, , Saint Saint-

• Petersburg, Russia, June 6

Petersburg, Russia, June 6-

• 8 2006.

8 2006.

Pea root nodule Pea root nodule morphology morphology

I II III N IT

I – meristem II – infection zone III – nitrogen- fixing zone N – nucleus IT – infection threads

Topology of Infection Threads and Symbiosomes

(A) (B) (C)

Brewin N.J. Plant cell wall remodelling in the Rhizobium-legume symbiosis Critical Reviews in Plant Sciences 23: 293-316 (2004)

D E

Immunolocalisation Immunolocalisation of

• f rhizobial

rhizobial lipopolysaccharide lipopolysaccharide (MAC57) and (MAC57) and arabinogalactan arabinogalactan protein protein-

• extensin

extensin (MAC265) (MAC265) in single and double pea mutants and in single and double pea mutants and wt wt line SGE line SGE

МАС 57 МАС 265 МАС 57 МАС 265 МАС 57 МАС 265 МАС 265 МАС 57

SGE (wt) SGEFix-1 (sym40) SGEFix-2 (sym33) RBT3 (sym33, sym40)

Immunogold Immunogold localisation localisation of

• f arabinogalactan

arabinogalactan protein protein-

• extensins

extensins (MAC265) in a single pea mutant SGEFix (MAC265) in a single pea mutant SGEFix-

• 2

2 ( (sym33 sym33) ( ) (cw

cw – – cell wall, cell wall, b b – – bacteria, bacteria, ecm ecm – – extracellular extracellular matrix matrix)

)

cw cw b b b b b b b b b b ecm ecm

Immunogold Immunogold localisation localisation of

• f arabinogalactan

arabinogalactan protein protein-

• extensin

extensin (MAC265) within intercellular spaces in a single (MAC265) within intercellular spaces in a single pea mutant SGEFix pea mutant SGEFix-

• 1 (

1 (sym40 sym40) ( ) (cw

cw – – cell wall, cell wall, is is – – intercellular intercellular space, space, v v -

• vacuole

vacuole)

)

cw cw cw cw is is v v v v

Targetted Targetted secretion of the matrix glycoprotein secretion of the matrix glycoprotein (MAC265) (MAC265) into infection threads of double pea mutant RBT3 ( into infection threads of double pea mutant RBT3 (sym33, sym33, sym40 sym40) ( ) (cw

cw – – cell wall, cell wall, cv cv – – cytoplasmic cytoplasmic vesicles, vesicles, b b – – bacteria, bacteria, ecm ecm – – extracellular extracellular matrix, matrix, v v -

• vacuole

vacuole)

)

cw cw ecm ecm b b cv cv cv cv cv cv v v

Dynamic model for infection thread growth Dynamic model for infection thread growth

Brewin N.J. Pods and Nods: a new look at symbiotic nitrogen fixing Biologist 49 (3): 1-5 (2002)

Absence of Absence of arabinogalactan arabinogalactan protein protein-

• extensin

extensin (MAC265) (MAC265) label in some infection threads of single and double pea label in some infection threads of single and double pea mutants ( mutants (cw

cw – – cell wall, cell wall, ecm ecm – – extracellular extracellular matrix, matrix, b b – – bacteria bacteria)

)

cw cw ecm ecm b b b b cw cw cw cw ecm ecm b b b b b b b b b b

RBT3 (sym33, sym40) RisFixV (sym42)

Immunolocalisation Immunolocalisation of de

• f de-
• esterified

esterified pectin (JIM5) in cell pectin (JIM5) in cell wall of single and double pea mutants wall of single and double pea mutants (

(cw

cw – – cell wall, cell wall, b b – – bacteria, bacteria, ecm ecm – – extracellular extracellular matrix matrix)

)

cw cw b b b b ecm ecm cw cw b b b b b b ecm ecm

RBT4 (sym33, sym42) RisFixV (sym42)

Secretion of pectin (JIM5) around degrading Secretion of pectin (JIM5) around degrading bacteroids bacteroids in senescent infected cells in single pea in senescent infected cells in single pea mutant mutant RisFixV RisFixV ( (sym 42 sym 42) ( ) (B

B – – bacteroids bacteroids, , cyt cyt – – cytoplasm cytoplasm)

)

B B B B B B B B B B cyt cyt

Conclusions Conclusions

• The increase of AGP

The increase of AGP-

• extensin

extensin synthesis in synthesis in intercellular spaces in mutant genotypes of pea intercellular spaces in mutant genotypes of pea was revealed as well as the intensification of was revealed as well as the intensification of defense reactions in the form of peroxide defense reactions in the form of peroxide-

• driven

driven cross cross-

• linking of AGP

linking of AGP-

• extensin

extensin. .

• In single mutant

In single mutant RisFixV RisFixV ( (sym42 sym42) it was ) it was recognized the intensification of defense reaction recognized the intensification of defense reaction in the form of in the form of callose callose synthesis around cell wall of synthesis around cell wall of both infection threads and plant infected cell as both infection threads and plant infected cell as well as synthesis of well as synthesis of pectic pectic components around components around degrading degrading bacteroids bacteroids in senescent infected cells. in senescent infected cells.

Anna V. Anna V. Khodorenko Khodorenko, Viktor E. , Viktor E. Tsyganov Tsyganov, , Alexey Alexey Y.

• Y. Borisov

Borisov, Igor A. , Igor A. Tikhonovich Tikhonovich

All All-

• Russia Research Institute for Agricultural Microbiology,

Russia Research Institute for Agricultural Microbiology, St. St.-

• Petersburg, Russia

Petersburg, Russia

Nicholas J. Brewin

John Innes Centre, Norwich, UK

This work was supported by the INTAS (YSF 04-83-3196), the Federal science agency of Russia (РИ-19.0/002/140), the Russian Foundation for Basic Research (05-04-49105-а)