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Effect of Mutations in Pisum sativum L. Genes Blocking Different Stages of Nodule Development on the Expression of Late Symbiotic Genes in Rhizobium leguminosarum bv. viciae

In this report, the expression of late symbiotic genes (fnrN, fixN, and nifA) of Rhizobium leguminosarum bv. viciae was studied in nodules of mutant pea lines blocked at four successive stages of nodule development. Bacterial gene expression was analyzed in situ with transcriptional gusA reporter gene fusions. As a control, a constitutively expressed gusA gene was included. In the nodules of Nop^- (nodule persistence) mutants (mutant in gene sym13), which had not yet exhibited signs of premature senescence, the expression patterns observed were identical to those in wild-type nodules. Normal expression of fusions also occurred in nodules defective at the infection droplet differentiation stage (mutantin gene sym40) in which bacteria are endocytosed, but infection threads and infection droplets are hypertrophied. In contrast, in Itn^- (infection thread formation inside the nodule tissue) mutants (mutant gene sym33), in which there is no endocytosis of bacteria, expression of the constitutive fusion was only in infection threads and no activity was shown for the other fusions. From this it can be concluded that functionality of the plant gene Sym33, i.e., bacterial endocytosis, is a prerequisite for the expression of late symbiotic genes in the microsymbiont. No morphologically distinct interzone II--III could be detected in nodules blocked at the bacteroid differentiation stage (mutants in gene sym31). The constitutive fusion was expressed equally throughout the nodule tissue (except for the meristem), and the activity of fusions to late symbiotic genes increased gradually with a maximal expression level at the base of the nodule. This is consistent with an altered oxygen barrier previously reported for these nodules. By including double mutants, earlier results on sequential functioning of gene pairs sym33-sym40 and sym31-sym13 could be confirmed and it could be demonstrated that the developmental epistasis found at the morphological level also is reflected in the expression pattern of late symbiotic genes in the microsymbiont.