Emile Duhoux

Hairy root nodulation of Casuarina glauca: a system for the study of symbiotic gene expression in an actinorhizal tree.

The purpose of this study was to establish a fast system for producing transgenic actinorhizal root nodules of Casuarina glauca. Agrobacterium rhizogenes strain A4RS carrying the p35S-gusA-int gene construct was used to induce hairy roots on hypocotyls of 3-week-old C. glauca seedlings. Three weeks after wounding, the original root system was excised, and composite plants consisting of transgenic roots on untransformed shoots were transferred to test tubes to be inoculated with Frankia. The actinorhizal nodules formed on transformed roots had the nitrogenase activity and morphology of untransformed nodules. beta-Glucuronidase (GUS) activity was examined in transgenic roots and nodules by fluorometric and histochemical assays. The results indicate that transgenic nodules generated with this root transformation system could facilitate the molecular study of symbiotic nitrogen fixation in actinorhizal trees.

Characterization of a Casuarina glauca Nodule-Specific Subtilisin-like Protease Gene, a Homolog of Alnus glutinosa ag12

In search of plant genes expressed during early interactions between Casuarina glauca and Frankia, we have isolated and characterized a C. glauca gene that has strong homology to subtilisin-like protease gene families of several plants including the actinorhizal nodulin gene ag12 of another actinorhizal plant, Alnus glutinosa. Based on the expression pattern of cg12 in the course of nodule development, it represents an early actinorhizal nodulin gene. Our results suggest that subtilisin-like proteases may be a common element in the process of infection of plant cells by Frankia in both Betulaceae (Alnus glutinosa) and Casuarinaceae (Casuarina glauca) symbioses.

Actinorhizal Symbioses: Recent Advances in Plant Molecular and Genetic Transformation Studies

Infection of actinorhizal plants roots by the actinomycete Frankia leads to the formation of a nitrogen-fixing root nodule (actinorhiza) consisting of multiple lobes, each of which is a modified lateral root. Actinorhiza development involves several specific steps, for example, root hair infection, prenodule formation, and initiation of lobe primordia from root pericycle. This article summarizes the latest development in the isolation and characterization of nodule-specific and -enhanced transcripts isolated from actinorhiza. The amino acid sequence derived from the nucleotide sequence of the cDNAs, in combination with localization data, showed that gene products are involved in nitrogen, carbon, and oxygen metabolism. Furthermore, some transcripts represented encoded gene products that might be part of infection and senescence mechanisms in actinorhiza. The article also reviews experiments designed to establish genetic transformation systems for actinorhizal plants. This research has led to the obtainment of transgenic plants of the Casuarinaceae family by using A. rhizogenes and A. tumefaciens . These new findings are discussed in view of future studies on actinorhizal symbiosis. As molecular and cellular studies on Casuarinaceae and Betulaceae are more advanced than on the other six actinorhizal plant families, we concentrate primarily on species within these two families.

Casuarina glauca prenodule cells display the same differentiation as the corresponding nodule cells

Recent phylogenetic studies have implied that all plants able to enter root nodule symbioses with nitrogen-fixing bacteria go back to a common ancestor (D.E. Soltis, P.S. Soltis, D.R. Morgan, S.M. Swensen, B.C. Mullin, J.M. Dowd, and P.G. Martin, Proc. Natl. Acad. Sci. USA, 92:2647-2651, 1995). However, nodules formed by plants from different groups are distinct in nodule organogenesis and structure. In most groups, nodule organogenesis involves the induction of cortical cell divisions. In legumes these divisions lead to the formation of a nodule primordium, while in non-legumes they lead to the formation of a so-called prenodule consisting of infected and uninfected cells. Nodule primordium formation does not involve prenodule cells, and the function of prenodules is not known. Here, we examine the differentiation of actinorhizal prenodule cells in comparison to nodule cells with regard to both symbionts. Our findings indicate that prenodules represent primitive symbiotic organs whose cell types display the same characteristics as their nodule counterparts. The results are discussed in the context of the evolution of root nodule symbioses.

The 35S promoter is not constitutively expressed in the transgenic tropical actinorhizal tree Casuarina glauca

The tropical nitrogen-fixing tree, Casuarina glauca Sieb. ex Spreng. was genetically transformed using Agrobacterium tumefaciens C58C1(pGV2260; pBIN19GUSINT). We report on the expression pattern conferred by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic C. glauca plants grown in vitro, and for one year in a greenhouse. Histochemical assays in shoots from in vitro plants revealed β-glucuronidase (GUS) staining in apical and axillary buds, and in nearly all tissues near the base of the stem. In roots, the CaMV 35S drove strong GUS expression in the apex and vascular tissue. In 1-year old plants grown in a greenhouse, the CaMV 35S promoter was highly active, except in peripheral suberized tissues. Transgenic C. glauca plants were nodulated by the actinomycete Frankia. Histochemical assays on vibratome sections of transgenic nodules demonstrated intense GUS activity in the vascular bundle, the phellogen, and in strands of uninfected cells filled with polyphenols. GUS expression was undetectable in Frankia-infected cells.

In vitro propagation of the nitrogen-fixing tree-legume Acacia mangium Willd.

In vitro propagation was initiated from 2-week-old and 7-month-old explants of Acacia mangium. Juvenile explants (2 week-old) of 5- to 10-mm lengths composed of two leaves were cultured on Murashige and Skoog (MS) medium containing 1.0 or 2.0 mg L-1 6-benzyladenine (BAP). After 6 weeks, most explants had formed a large cluster of 14–18 axillary shoots produced by prolific branching of the primary axillary shoot after elongation. The maximum multiplication rate (40) was obtained in the first subculture; the rate decreased to 10–20 in the second one. The mean length of shoots was not significantly affected by BAP concentrations during the subsequent cultures. Rooting ability of juvenile explants was greatly affected by BAP concentrations used in the multiplication medium. When both types of explants were multiplied on a MS medium containing 1.0 mg L-1 BAP and transferred to a half-strength MS medium containing 0.05 mg L-1 IBA, only 10% of the juvenile explants were rooted versus 70% of the 7-month-old explants. Rooted plants transferred onto artificial substrate were all nodulated, when inoculated with a specific Bradyrhizobium sp. strain.

Soybean (lbc3), Parasponia, and Trema Hemoglobin Gene Promoters Retain Symbiotic and Nonsymbiotic Specificity in Transgenic Casuarinaceae: Implications for Hemoglobin Gene Evolution and Root Nodule Symbioses

The purpose of this study was to compare the control of expression of legume and nonlegume hemoglobin genes. We used the Casuarina glauca and Allocasuarina verticillata transformation system to examine the properties of the soybean (lbc3), Parasponia andersonii, and Trema tomentosa hemoglobin gene promoters in actinorhizal plants. Expression of the hemoglobin promoters gus genes was examined by fluorometric and histochemical assays. The fluorometric assays in various organs showed that the soybean and P. andersonii promoters were most active in nodules whereas the T. tomentosa promoter gave a very high activity in roots. The histochemical study showed that GUS activity directed by the soybean and the P. andersonii gus chimeric genes appeared mainly confined to the infected cells of the C. glauca and A. verticillata nodules. The T. tomentosa hemoglobin promoter was primarily expressed in the roots cortex and vascular tissue. The results indicate that the soybean, P. andersonii, and T. tomentosa hemoglobin...

Le nodule actinorhizien

Summary Actinorhizal nodules or actinorhizae represent the most typical example of modified non pathogenic roots (nodule lobes), induced by signals synthetized by the microsymbiont. The formation of a nodule lobe occurs in four steps: infection of the root hair by Frankia, formation of the prenodule, initiation and nodule lobe infection. The nodule lobe comprises four distinct zones which are the result of the Frankia acropetal growth and of the differentiation of tissues originating from the apical meristem. In situ hybridization studies of the expression of nif genes of Frankia show that the zone of infected mature cells (zone III) is the zone where the nitrogen fixation is the greatest. Similitudes and differences with organogenesis of the nodules of legumes are displayed and the interest of this primitive symbiosis is emphasized.

Nitrogen-fixing potential of micropropagated clones of Acacia mangium inoculated with different Bradyrhizobium spp. strains

Five A. mangium seedlings of different shoot lengths were selected from a 600-seed screening experiment and micropropagated. Two-week-old rooted microcuttings of the 5 micropropagated clones were inoculated with 3 specific Bradyrhizobium spp. strains in 15 combinations. After 5 months of growth, nodule dry weight and shoot dry weight data showed significant effects of clone and Bradyrhizobium spp. strain. Clones RR-G1 and IR-M2 and Bradyrhizobium sp. Aust13c resulted in the highest dry-matter production and most efficient nodulation. No interaction was observed between clone and Bradyrhizobium spp. strain, which indicates that the Bradyrhizobium spp. strain and the host plant can be selected separately.

Choosing a reporter for gene expression studies in transgenic actinorhizal plants of the Casuarinaceae family

Transgenic Casuarinaceae and reporter genes provide valuable tools to study gene expression in transgenic actinorhizal nodules. In this paper, we discuss the use of s-glucuronidase for the histochemical localization and quantification of gene expression in transgenic plants of Allocasuarina verticillata and Casuarina glauca nodulated by the actinomycete Frankia. We also report on the genetic transformation of A. verticillata by the Agrobacterium tumefaciens strain C58C1(pGV2260) containing the 35S-mgfp5-ER construct encoding a modified green fluorescent protein of Aequorea victoria in a binary vector. The evolution of the GFP fluorescence was monitored through all stages of the regeneration process. The data indicate that GFP is not toxic in Casuarinaceae and that this reporter gene can be used for visual screening of transformed calli and transgenic plants. The fluorescence pattern of gfp provides a new tool for monitoring in vivo transgenc expression in actinorhizal plants.