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Pisum sativum symbiotic mutant SGEFix−-2 carries the sym33-3 allele of the gene Sym33, encoding transcription factor PsCYCLOPS/PsIPD3. Previously, strong host cell defence reactions were identified in nodules of this mutant. In the present study, new manifestations of defence reactions were revealed in 28-day-old white nodules in which bacterial release had occurred. These nodules were investigated using histochemical staining of pectin and suberin and by immunogold localisation of three components of pectin: highly methyl-esterified homogalacturonan (HG) recognised by monoclonal antibody JIM7, low methyl-esterified HG recognised by JIM5 and linear (1–4)-β-D-galactan side-chain of rhamnogalacturonan I (RG I) recognised by LM5. In the mutant, but not in the wild-type, cell wall material was deposited around the vacuole in the uninfected cells, in cells containing infection threads and in the infected cells. The deposits around the vacuole were marked with JIM7 and LM5 antibodies but not with JIM5, suggesting that they contain newly formed cell wall material. Deposition was accompanied by suberin accumulation. This is the first report that deposition of cell wall material around the vacuole may be associated with the defence reaction in ineffective nodules. In addition, hypertrophic infection droplets labelled with JIM7 were identified. In the matrix of some infection threads, RG I recognised a pectic gel component. Callose deposits in the cell walls and in the walls of infection threads were occasionally observed. The observations suggest that an important function of transcriptional factor CYCLOPS/IPD3 is the suppression of defence reactions during establishment of the legume-rhizobial symbiosis. [ABSTRACT FROM AUTHOR]

Infection of host cells by nitrogen-fixing soil bacteria, known as rhizobia, involves the progressive remodelling of the plant–microbe interface. This process was examined by using monoclonal antibodies to study the subcellular localisation of pectins and arabinogalactan proteins (AGPs) in wild-type and ineffective nodules of Pisum sativum and Medicago truncatula. The highly methylesterified homogalacturonan (HG), detected by monoclonal antibody JIM7, showed a uniform localisation in the cell wall, regardless of the cell type in nodules of P. sativum and M. truncatula. Low methylesterified HG, recognised by JIM5, was detected mainly in the walls of infection threads in nodules of both species. The galactan side chain of rhamnogalacturonan I (RG-I), recognised by LM5, was present in the nodule meristem in both species and in the infection thread walls in P. sativum, but not in M. truncatula. The membrane-anchored AGP recognised by JIM1 was observed on the plasma membrane in nodules of P. sativum and M. truncatula. In P. sativum, the AGP epitope recognised by JIM1 was present on mature symbiosome membranes of wild-type nodules, but JIM1 labelling was absent from symbiosome membranes in the mutant Sprint-2Fix− (sym31) with undifferentiated bacteroids, suggesting a possible involvement of AGP in the maturation of symbiosomes. Thus, the common and species-specific traits of cell wall remodelling during nodule differentiation were demonstrated. [ABSTRACT FROM AUTHOR]

Plant symbiotic mutants are useful tool to uncover the molecular-genetic mechanisms of nodule senescence. The pea (Pisum sativum L.) mutants SGEFix−-1 (sym40), SGEFix−-3 (sym26), and SGEFix−-7 (sym27) display an early nodule senescence phenotype, whereas the mutant SGEFix−-2 (sym33) does not show premature degradation of symbiotic structures, but its nodules show an enhanced immune response. The nodules of these mutants were compared with each other and with those of the wild-type SGE line using seven marker genes that are known to be activated during nodule senescence. In wild-type SGE nodules, transcript levels of all of the senescence-associated genes were highest at 6 weeks after inoculation (WAI). The senescence-associated genes showed higher transcript abundance in mutant nodules than in wild-type nodules at 2 WAI and attained maximum levels in the mutant nodules at 4 WAI. Immunolocalization analyses showed that the ethylene precursor 1-aminocyclopropane-1-carboxylate accumulated earlier in the mutant nodules than in wild-type nodules. Together, these results showed that nodule senescence was activated in ineffective nodules blocked at different developmental stages in pea lines that harbor mutations in four symbiotic genes. [ABSTRACT FROM AUTHOR]