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 ( PISUM SATIVUM PISUM SATIVUM L.) L.) NODULES OF PEA ( EU - - Russia: Prospects for Cooperation in Biotechnology in the Russia: Prospects for Cooperation in Biotechnology in the EU Seventh Framework Programme Programme, , Seventh Framework Saint- -Petersburg, Russia, June 6 Petersburg, Russia, June 6- -8 2006. 8 2006. Saint
Pea root nodule Pea root nodule morphology morphology I – meristem II – infection I zone II III III – nitrogen- fixing zone N – nucleus IT IT – infection N threads
Topology of Infection Threads and Symbiosomes (A) D E (B) (C) Brewin N.J. Plant cell wall remodelling in the Rhizobium -legume symbiosis Critical Reviews in Plant Sciences 23: 293-316 (2004)
Immunolocalisation of of rhizobial rhizobial lipopolysaccharide lipopolysaccharide Immunolocalisation (MAC57) and arabinogalactan arabinogalactan protein protein- -extensin extensin (MAC265) (MAC265) (MAC57) and in single and double pea mutants and wt wt line SGE line SGE in single and double pea mutants and МАС 57 МАС 57 МАС 57 МАС 57 МАС 265 МАС 265 МАС 265 МАС 265 SGE ( wt ) SGEFix-1 ( sym40 ) SGEFix-2 ( sym33 ) RBT3 ( sym33, sym40 )
Immunogold localisation localisation of of arabinogalactan arabinogalactan protein protein- - Immunogold extensins (MAC265) in a single pea mutant SGEFix (MAC265) in a single pea mutant SGEFix - -2 2 extensins - - ( sym33 sym33 ) ( ) ( cw ) ( matrix ) cw – – cell wall, cell wall, b b – – bacteria, bacteria, ecm ecm – – extracellular extracellular matrix b b b b b b ecm ecm cw cw b b b b
Immunogold localisation localisation of of arabinogalactan arabinogalactan protein protein- - Immunogold extensin (MAC265) within intercellular spaces in a single (MAC265) within intercellular spaces in a single extensin pea mutant SGEFix- -1 ( 1 ( sym40 sym40 ) ( ) ( cw pea mutant SGEFix cw – – cell wall, cell wall, is is – – intercellular intercellular ) vacuole ) space, v space, v - - vacuole is is v v cw cw v v cw cw
Targetted secretion of the matrix glycoprotein secretion of the matrix glycoprotein (MAC265) (MAC265) Targetted into infection threads of double pea mutant RBT3 ( sym33, sym33, into infection threads of double pea mutant RBT3 ( sym40 ) ( ) ( cw sym40 cw – – cell wall, cell wall, cv cv – – cytoplasmic cytoplasmic vesicles, vesicles, b b – – bacteria, bacteria, ecm ecm ) vacuole ) – extracellular extracellular matrix, matrix, v v - - vacuole – cw cv cw cv cv cv cv cv ecm ecm v v b b
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 arabinogalactan arabinogalactan protein protein- -extensin extensin (MAC265) (MAC265) Absence of label in some infection threads of single and double pea label in some infection threads of single and double pea mutants ( cw ) mutants ( bacteria ) cw – – cell wall, cell wall, ecm ecm – – extracellular extracellular matrix, matrix, b b – – bacteria b b cw cw b b b b ecm ecm b b b b b b ecm ecm b b cw cw cw cw RBT3 ( sym33, sym40 ) RisFixV ( sym42 )
Immunolocalisation Immunolocalisation of de of de- -esterified esterified pectin (JIM5) in cell pectin (JIM5) in cell wall of single and double pea mutants ( ( cw wall of single and double pea mutants cw – – cell wall, cell wall, b b – – ) matrix ) bacteria, ecm ecm – – extracellular extracellular matrix bacteria, b b b b ecm ecm b b ecm ecm b b cw cw b b cw cw RBT4 ( sym33, sym42 ) RisFixV ( sym42 )
Secretion of pectin (JIM5) around degrading Secretion of pectin (JIM5) around degrading bacteroids in senescent infected cells in single pea in senescent infected cells in single pea bacteroids mutant RisFixV RisFixV ( ( sym 42 sym 42 ) ( ) ( B ) mutant cytoplasm ) B – – bacteroids bacteroids, , cyt cyt – – cytoplasm B B B B cyt cyt B B B B B B
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- -driven driven defense reactions in the form of peroxide cross- -linking of AGP linking of AGP- -extensin extensin. . cross � 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 callose callose synthesis around cell wall of synthesis around cell wall of in the form of both infection threads and plant infected cell as both infection threads and plant infected cell as well as synthesis of pectic pectic components around components around well as synthesis of degrading bacteroids bacteroids in senescent infected cells. in senescent infected cells. degrading
Anna V. Khodorenko Khodorenko, Viktor E. , Viktor E. Tsyganov Tsyganov, , Alexey Alexey Anna V. Y. Borisov Borisov, Igor A. , Igor A. Tikhonovich Tikhonovich Y. 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 )
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