Cellular localization and cytochemical probing of resistance reactions to arbuscular mycorrhizal fungi in a ‘locus a’ myc? mutant of Pisum sativum L.

Pisum sativum L. myc^– mutants which fail to form arbuscular mycorrhiza have recently been identified amongst nod^– mutants (Duc et al., 1989, Plant Sci. 60, 215–222). The reason for this resistance to symbiotic fungi has been investigated in the case of a locus a mutant (P2) inoculated with Glomus mosseae (Nicol. and Gerd.) Gerd, and Trappe. The fungal symbiont formed viable appressoria in contact with the root surface but its development was stopped at the root epidermis. Abundant material was deposited on the inner face of root cell walls adjacent to the appressoria in the P2 mutant, but not in the wild-genotype parent cultivar (Frisson) forming a symbiotic mycorrhizal infection. Fluorescence, histochemical, cytochemical and immunocytological approaches were used to characterize the paramural deposits in epidermal and hypodermal cells of the mutant. Strong fluorescence under blue light indicated the accumulation of phenolic compounds although polymers like lignin or suberin were not localized. Proteins and glycoproteins were homogeneously distributed within the paramural deposits. In the latter, the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) reaction for 1,4-polysaccharide detection showed a heterogeneous composition with electron-dense points surrounded by non-reactive material, but cytological tests for cellulose and pectin gave weak responses as compared to epidermal and hypodermal walls of the wild genotype. -1,3-Glucans indicative of callose were detected by in-situ immunolocalization in the paramural deposits below appressoria on mutant roots, but not in walls of the wild genotype. Thus, appressorium formation by G. mosseae on roots of the locus a P. sativum mutant elicits wall modifications usually associated with activation of defence responses to pathogens. It is proposed that this locus must be involved in a key event in symbiotic infection processes in P. sativum, and the possible role of complex regulatory interactions between symbiosis and defence genes in endomycorrhiza development is discussed.Abbreviations DAPI 4,6-diamino-2-phenylindole - FDA fluo-rescein diacetate - PATAg periodic acid-thiocarbohydrazide-silver proteinate The authors are grateful to C. Arnould for technical assistance, K. Niehaus for the purified Sirofluor, K. Roberts for the AFRC JIM5 antibody and J. Lherminier (INRA, Dijon, France), for useful discussion. This collaborative research programme was financially supported by MRT, INRA, EPR-Bourgogne (grant to A.G., Contrat de Plan project 3060A), EEC COST ACTION 8.10 (Endomycorrhizas) and the National Research Council of Italy, Special Project RAISA, Sub-project N.2, Paper N. 801