[Regulatory genes of garden pea (Pisum sativum L.) controlling the development of nitrogen-fixing nodules and arbuscular mycorrhiza: a review of basic and applied aspects].

The review sums up the long experience of the authors and other researchers in studying the genetic system of garden pea (Pisum sativum L.), which controls sthe development of nitrogen-fixing symbiosis and arbuscular mycorrhiza. A justified phenotypic classification of pea mutants is presented. Progress in identifying and cloning symbiotic genes is adequately reflected. The feasibility of using double inoculation as a means of increasing the plant productivity is demonstrated, in which the potential of a tripartite symbiotic system (pea plants-root nodule bacteria-arbuscular mycorrhiza) is mobilized.

Developmental downregulation of rhizobial genes as a function of symbiosome differentiation in symbiotic root nodules of Pisum sativum.

• The expression of nodA and dctA genes of Rhizobium leguminosarum bv. viciae has been studied in mutant nodules of pea (Pisum sativum L.), blocked at the following developmental stages: infection thread development inside the nodule (Itn); infection droplet differentiation (Idd); bacteroid differentiation after endocytosis (Bad); and nodule persistence (Nop). • With the use of reporter fusions to these symbiotic bacterial genes it was shown that both nodA and dctA were expressed at all developmental stages, with a pattern similar to that of constitutive, symbiosis-unrelated genes. • As well as two constitutively expressed genes, both nodA and dctA genes seemed to be subjected to gradual downregulation in nodule bacteria, correlating with the stage of bacteroid differentiation reached. No such effect was observed for the symbiotic, oxygen-regulated fixN gene. The bacteroid development stage also appeared to be related to the ability of bacteria that have been subjected to endocytosis to resume free-living vegetative growth. • The results support the suggestion that bacteroid differentiation into a nitrogen-fixing, organelle-like form, is a gradual process involving several stages, each controlled by different plant genes.

Genetic dissection of the initiation of the infection process and nodule tissue development in the Rhizobium-pea (Pisum sativum L.) symbiosis.

Twelve non-nodulating pea (Pisum sativum L.) mutants were studied to identify the blocks in nodule tissue development. In nine, the reason for the lack of infection thread (IT) development was studied; this had been characterized previously in the other three mutants. With respect to IT development, mutants in gene sym7 are interrupted at the stage of colonization of the pocket in the curled root hair (Crh- phenotype), mutants in genes sym37 and sym38 are blocked at the stage of IT growth in the root hair cell (Ith- phenotype) and mutants in gene sym34 at the stage of IT growth inside root cortex cells (Itr- phenotype). With respect to nodule tissue development, mutants in genes sym7, sym14 and sym35 were shown to be blocked at the stage of cortical cell divisions (Ccd- phenotype), mutants in gene sym34 are halted at the stage of nodule primordium (NP) development (Npd- phenotype) and mutants in genes sym37 and sym38 are arrested at the stage of nodule meristem development (Nmd- phenotype). Thus, the sequential functioning of the genes Sym37, Sym38 and the gene Sym34 apparently differs in the infection process and during nodule tissue development. Based on these data, a scheme is suggested for the sequential functioning of early pea symbiotic genes in the two developmental processes: infection and nodule tissue formation.

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 (mutant in 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.