Hassen Gherbi

SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria

Root endosymbioses vitally contribute to plant nutrition and fitness worldwide. Nitrogen-fixing root nodulation, confined to four plant orders, encompasses two distinct types of associations, the interaction of legumes (Fabales) with rhizobia bacteria and actinorhizal symbioses, where the bacterial symbionts are actinomycetes of the genus Frankia. Although several genetic components of the host–symbiont interaction have been identified in legumes, the genetic basis of actinorhiza formation is unknown. Here, we show that the receptor-like kinase gene SymRK, which is required for nodulation in legumes, is also necessary for actinorhiza formation in the tree Casuarina glauca. This indicates that both types of nodulation symbiosis share genetic components. Like several other legume genes involved in the interaction with rhizobia, SymRK is also required for the interaction with arbuscular mycorrhiza (AM) fungi. We show that SymRK is involved in AM formation in C. glauca as well and can restore both nodulation and AM symbioses in a Lotus japonicus symrk mutant. Taken together, our results demonstrate that SymRK functions as a vital component of the genetic basis for both plant–fungal and plant–bacterial endosymbioses and is conserved between legumes and actinorhiza-forming Fagales.

Transcriptomics of Actinorhizal Symbioses Reveals Homologs of the Whole Common Symbiotic Signaling Cascade

Comparative transcriptomics of two actinorhizal symbiotic plants, Casuarina glauca and Alnus glutinosa, was used to gain insight into their symbiotic programs triggered following contact with the nitrogen-fixing actinobacterium Frankia. Approximately 14,000 unigenes were recovered in roots and 3-week-old nodules of each of the two species. A transcriptomic array was designed to monitor changes in expression levels between roots and nodules, enabling the identification of up- and down-regulated genes as well as root- and nodule-specific genes. The expression levels of several genes emblematic of symbiosis were confirmed by quantitative polymerase chain reaction. As expected, several genes related to carbon and nitrogen exchange, defense against pathogens, or stress resistance were strongly regulated. Furthermore, homolog genes of the common and nodule-specific signaling pathways known in legumes were identified in the two actinorhizal symbiotic plants. The conservation of the host plant signaling pathway is all the more surprising in light of the lack of canonical nod genes in the genomes of its bacterial symbiont, Frankia. The evolutionary pattern emerging from these studies reinforces the hypothesis of a common genetic ancestor of the Fabid (Eurosid I) nodulating clade with a genetic predisposition for nodulation.

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.

The Independent Acquisition of Plant Root Nitrogen-Fixing Symbiosis in Fabids Recruited the Same Genetic Pathway for Nodule Organogenesis

Only species belonging to the Fabid clade, limited to four classes and ten families of Angiosperms, are able to form nitrogen-fixing root nodule symbioses (RNS) with soil bacteria. This concerns plants of the legume family (Fabaceae) and Parasponia (Cannabaceae) associated with the Gram-negative proteobacteria collectively called rhizobia and actinorhizal plants associated with the Gram-positive actinomycetes of the genus Frankia. Calcium and calmodulin-dependent protein kinase (CCaMK) is a key component of the common signaling pathway leading to both rhizobial and arbuscular mycorrhizal symbioses (AM) and plays a central role in cross-signaling between root nodule organogenesis and infection processes. Here, we show that CCaMK is also needed for successful actinorhiza formation and interaction with AM fungi in the actinorhizal tree Casuarina glauca and is also able to restore both nodulation and AM symbioses in a Medicago truncatula ccamk mutant. Besides, we expressed auto-active CgCCaMK lacking the auto-inhibitory/CaM domain in two actinorhizal species: C. glauca (Casuarinaceae), which develops an intracellular infection pathway, and Discaria trinervis (Rhamnaceae) which is characterized by an ancestral intercellular infection mechanism. In both species, we found induction of nodulation independent of Frankia similar to response to the activation of CCaMK in the rhizobia-legume symbiosis and conclude that the regulation of actinorhiza organogenesis is conserved regardless of the infection mode. It has been suggested that rhizobial and actinorhizal symbioses originated from a common ancestor with several independent evolutionary origins. Our findings are consistent with the recruitment of a similar genetic pathway governing rhizobial and Frankia nodule organogenesis.

Symbiotic Signaling in Actinorhizal Symbioses

Actinorhizal symbioses are mutualistic associations between plants belonging to eight angiosperm families and soil bacteria of the genus Frankia. These interactions lead to the formation of new root organs, actinorhizal nodules, where the bacteria are hosted and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. It involves an elaborate signaling between both partners of the symbiosis. In recent years, our knowledge of this signaling pathway has increased tremendously thanks to a series of technical breakthroughs including the sequencing of three Frankia genomes [1] and the implementation of RNA silencing technology for two actinorhizal species. In this review, we describe all these recent advances, current researches on symbiotic signaling in actinorhizal symbioses and give some potential future research directions.

Post-Transcriptional Gene Silencing in the Root System of the Actinorhizal Tree Allocasuarina verticillata

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.

Agrobacterium tumefaciens gene transfer to Casuarina glauca, a tropical nitrogen-fixing tree

Abstract Transgenic calli of the tropical tree Casuarina glauca were produced using Agrobacterium tumefaciens -mediated gene transfer. Hypocotyls, cotyledons and epicotyls were excised from 30–60-day old Casuarina seedlings and cocultivated with Agrobacterium strain C58C1(pGV2260) containing the binary vector BIN19GUSINT. Transformed calli were selected on nutrient medium supplemented with 0.5 μM NAA, 2.5 μM BA and 50 mg/l kanamycin. Some of the factors influencing T-DNA transfer to C. glauca explants were studied. Optimal transformation rates were obtained when explants from 45-day old seedlings were cocultivated for 3 days in the presence of 25 μM acetosyringone. Transgenic buds differentiated on 10% of the calli grown on transformed epicotyls. Evidence for genetic transformation was obtained by β-glucuronidase assay, PCR and Southern hybridization.

Actinorhizal root nodule symbioses: what is signalling telling on the origins of nodulation?

Two groups of bacteria are able to induce the formation of nitrogen-fixing nodules: proteobacteria called rhizobia, which associate with Legumes or Parasponia and actinobateria from the genus Frankia which are able to interact with ∼220 species belonging to eight families called actinorhizal plants. Legumes and different lineages of actinorhizal plants differ in bacterial partners, nodule organogenesis and infection patterns and have independent evolutionary origins. However, recent technical achievements are revealing a variety of conserved signalling molecules and gene networks. Actinorhizal interactions display several primitive features and thus provide the ideal opportunity to determine the minimal molecular toolkit needed to build a nodule and to understand the evolution of root nodule symbioses.

Functional Analysis of the Metallothionein Gene cgMT1 Isolated from the Actinorhizal Tree Casuarina glauca

cgMT1 is a metallothionein (MT)-like gene that was isolated from a cDNA library of young nitrogen-fixing nodules resulting from the symbiotic interaction between Frankia spp. and the actinorhizal tree Casuarina glauca. cgMT1 is highly transcribed in the lateral roots and nitrogen-fixing cells of actinorhizal nodules; it encodes a class I type 1 MT. To obtain insight into the function of cgMT1, we studied factors regulating the expression of the MT promoter region (PcgMT1) using a beta-glucuronidase (gus) fusion approach in transgenic plants of Arabidopsis thaliana. We found that copper, zinc, and cadmium ions had no significant effect on the regulation of PcgMT1-gus expression whereas wounding and H2O2 treatments led to an increase in reporter gene activity in transgenic leaves. Strong PcgMT1-gus expression also was observed when transgenic plants were inoculated with a virulent strain of the bacterial pathogen Xanthomonas campestris pv. campestris. Transgenic Arabidopsis plants expressing cgMT1 under the control of the constitutive 35S promoter were characterized by reduced accumulation of H2O2 when leaves were wounded and by increased susceptibility to the bacterial pathogen X. campestris. These results suggest that cgMT1 could play a role during the oxidative response linked to biotic and abiotic stresses.

Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation

Nitrogen-fixing root nodulation is confined to four plant orders, including > 14,000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation.