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

Abstract

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.

A model for the development of the rhizobial and arbuscular mycorrhizal symbioses in legumes and its use to understand the roles of ethylene in the establishment of these two symbioses

“…The late stages of our model have been described by Brewin (1998), Timmers et al (1998, and Voroshilova et al (2001). Upon entering the cortex, the IT follows the path delineated by the PIT (Fig.…”

How rhizobial symbionts invade plants: the Sinorhizobium–Medicago model

“…Bacteria fail to differentiate or senesce prematurely in several mutants of the host plants M. truncatula and pea (Pisum sativum), but none of the genes carrying these mutations has yet been cloned [62][63][64]93,94 . In the pea sym13 mutant, symbiosome membranes are not closely associated with the bacteroid and the bacteroids senesce early 95 . Immunological detection of plant glycoproteins that are still attached to bacteroids isolated from pea nodules suggests that there is a close physical interaction between the symbiosome and bacteroid membranes, at least in indeterminate nodules where a single bacteroid is enclosed within each symbiosome membrane 96,97 .…”

Double Mutant Analysis of Sequential Functioning of Pea (Pisum sativum L.) Genes: Sym13, Sym33 and Sym40 During Symbiotic Nodule Development

“…В исследованиях гистологической организации клубеньков был использо-ван штамм VF39 gusA (const.). Данный штамм, любезно предоставленный У. Б. Прифер (Аахенский технический университет, Германия), характеризовался конститутив-ной экспрессией репортерного гена gusA (Voroshilova et al, 2001). Штамм 3841 (Wang et al, 1982) из коллекции Центра Джона Иннеса (Великобритания) был использо-ван в иммуноцитохимических экспериментах.…”

“…Для визуализации бактерий с использованием репор-терного гена (во время инфекционного процесса и во вре-мя развития поздних стадий клубенька), клубеньки окра-шивали по ранее описанной методике (Voroshilova et al, 2001) на 28 день после инокуляции. При изучении стадий клубенькообразования срезы клубеньков толщиной 70 мкм получали посредством вибротома VT 1000 S («Leica Microsystems Wetzlar GmbH», Германия).…”

“…До настоящего времени, самые крупные коллекции симбиотических мутантов были созданы для гороха (Borisov et al, 2004). С использованием мутантов из этих коллекций была предположена последовательность функционирования как ранних (Walker et al, 2000;Guinel, Geil, 2002;Tsyganov et al, 2002), так и поздних (Borisov et al, 1997;Morzhina et al, 2000;Voroshilova et al, 2001) симбиотических генов гороха.…”