Didier Hérouart

Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction.

Reactive oxygen species are produced as an early event in plant defense response against avirulent pathogens. We show here that alfalfa responds to infection with Sinorhizobium meliloti by production of superoxide and hydrogen peroxide. This similarity in the early response to infection by pathogenic and symbiotic bacteria addresses the question of which mechanism rhizobia use to counteract the plant defense response.

Expression of the Bacterial Catalase Genes During Sinorhizobium meliloti-Medicago sativa Symbiosis and Their Crucial Role During the Infection Process

Sinorhizobium meliloti possesses three distinct catalases to cope with oxidative stress: two monofunctional catalases (KatA and KatC) and one bifunctional catalase-peroxydase (KatB). The katB gene is constitutively expressed during growth in batch culture and is not induced under oxidative stress conditions. In contrast, the expression of katA and katC genes is mainly regulated at the transcription level in these conditions. A differential expression of kat genes was observed during the development of the nodule. A high expression of katA gene was detected in bacteroids, suggesting that the nitrogen-fixation process induces a strong oxidative stress. In contrast, bacteria express katB and katC genes and not the H2O2-inducible katA gene in infection threads despite the detection of H2O2 around the bacteria. A katB katC double mutant nodulated poorly and displayed abnormal infection. After nonefficient release into plant cells, bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Our results indicate that these two catalases are essential for the establishment of the symbiosis. They also suggest that the bacteria are in a nonexponential growth phase in infection threads and corroborate previous studies on the growth rate of bacteria inside the plant.

Critical protective role of bacterial superoxide dismutase in Rhizobium–legume symbiosis

In nitrogen‐poor soils, rhizobia elicit nodule formation on legume roots, within which they differentiate into bacteroids that fix atmospheric nitrogen. Protection against reactive oxygen species (ROS) was anticipated to play an important role in Rhizobium–legume symbiosis because nitrogenase is extremely oxygen sensitive. We deleted the sodA gene encoding the sole cytoplasmic superoxide dismutase (SOD) of Sinorhizobium meliloti. The resulting mutant, deficient in superoxide dismutase, grew almost normally and was only moderately sensitive to oxidative stress when free living. In contrast, its symbiotic properties in alfalfa were drastically affected. Nitrogen‐fixing ability was severely impaired. More strikingly, most SOD‐deficient bacteria did not reach the differentiation stage of nitrogen‐fixing bacteroids. The SOD‐deficient mutant nodulated poorly and displayed abnormal infection. After release into plant cells, a large number of bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Thus, bacterial SOD plays a key protective role in the symbiotic process.

Reactive oxygen species, nitric oxide and glutathione: a key role in the establishment of the legume-Rhizobium symbiosis?

Reactive oxygen species are generated in the first steps of the Rhizobium–legume symbiosis. Superoxide radicals and hydrogen peroxide have been detected in infection threads and there is also evidence of the presence of nitric oxide in young alfalfa nodules. Moreover, rhizobial mutants, with a reduced antioxidant defense, exhibit an impaired capacity to nodulate. The oxidative burst generated in response to symbiotic infection can be consistent with rhizobia being initially perceived as invaders by the plant; in this framework, it may be correlated with the existence of abortive infections. However, the burst appears to be also involved in the expression of early nodulins associated with successful infections. Thus, in parallel to its involvement in defense processes, a positive role for the oxidative burst (including nitric oxide) in the establishment of the symbiotic interaction can also be proposed. The burst could trigger the expression of plant and/or bacterial genes which are essential for the nodulation process. In this framework, glutathione and homoglutathione could be key intermediates for gene expression, via the modification of the redox balance. Thus, the oxidative burst may have a dual role in the establishment of the symbiosis.

H2O2 Is Required for Optimal Establishment of the Medicago sativa/Sinorhizobium meliloti Symbiosis

The symbiotic interaction between Medicago sativa and Sinorhizobium meliloti RmkatB(++) overexpressing the housekeeping catalase katB is delayed, and this delay is combined with an enlargement of infection threads. This result provides evidence that H(2)O(2) is required for optimal progression of infection threads through the root hairs and plant cell layers.

γ-Glutamyl Transpeptidase in Transgenic Tobacco Plants. Cellular Localization, Processing, and Biochemical Properties

γ-Glutamyl transpeptidase (γ-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation in the γ-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for γ-GT was overexpressed in tobacco (Nicotiana tabacum) plants. A high level of the membrane-bound γ-GT activity was localized outside the cell in transgenic plants. The overproduced enzyme was characterized by a high affinity to GSH and was cleaved post-translationally in two unequal subunits. Thus, Arabidopsis γ-GT is similar to the mammalian enzymes in enzymatic properties, post-translational processing, and cellular localization, suggesting analogous biological functions as a key enzyme in the catabolism of GSH.

Identification of new up-regulated genes under drought stress in soybean nodules

Legumes/rhizobium biological N(2) fixation (BNF) is dramatically affected under abiotic stress such as drought, salt, cold and heavy metal stresses. Nodule response to drought stress at the molecular level was analysed using soybean (Glycine max) and Bradyrhizobium japonicum as a model, since this symbiotic partnership is extremely sensitive to this stress. To gain insight into molecular mechanisms involved in drought-induced BNF inhibition, we have constructed a SSH (Suppression Subtractive Hybridisation) cDNA library from nodular tissue of plants irrigated at field capacity or plants water deprived for 5 days. Sequence analysis of the first set of 128 non redundant ESTs using protein databases and the Blastx program indicated that 70% of ESTs could be classified into putative known functions. Using reverse northern hybridization, 56 ESTs were validated as up-regulated genes in response to drought. Interestingly, only a few of them had been previously described as involved in plant response to drought, therefore most of the ESTs could be considered as new markers of drought stress. Here we discuss the potential role of some of these up-regulated genes in response to drought. Our analysis focused on two genes, encoding respectively a ferritin and a metallothionein, which are known to be involved in homeostasis and detoxification of metals and in response to oxidative stress. Their spatiotemporal expression patterns showed a high accumulation of transcripts restricted to infected cells of nodules in response to drought.

Tissue-Specific Activity of Two Manganese Superoxide Dismutase Promoters in Transgenic Tobacco

In eukaryotes, manganese superoxide dismutase is a nuclear-encoded protein that scavenges superoxide radicals in the mitochondrial matrix. We have isolated two manganese superoxide dismutase genes from Nicotiana plumbaginifolia L. and fused the 5[prime] upstream regulatory region of these genes to the [beta]-glucuronidase reporter gene. The two gene fusions displayed a differential tissue specificity in transgenic tobacco (Nicotiana tabacum). Promoter activity of the SodA1 gene fusion was found in the pollen, middle layer, and stomium of anthers, but was usually undetectable in vegetative organs of mature plants. The SodA2 gene fusion was expressed in the leaves, stems, roots, and flowers. SodA2 promoter activity was most prominent in the vascular bundles, stomata, axillary buds, pericycle, stomium, and pollen. Histochemical analysis of succinate dehydrogenase activity suggested that the spatial expression of the two gene fusions is generally correlated with mitochondrial respiratory activity.

Plant genes involved in harbouring symbiotic rhizobia or pathogenic nematodes

The establishment and development of plant-microorganism interactions involve impressive transcriptomic reprogramming of target plant genes. The symbiont (Sinorhizobium meliloti) and the root knot-nematode pathogen (Meloidogyne incognita) induce the formation of new root organs, the nodule and the gall, respectively. Using laser-assisted microdissection, we specifically monitored, at the cell level, Medicago gene expression in nodule zone II cells, which are preparing to receive rhizobia, and in gall giant and surrounding cells, which play an essential role in nematode feeding and constitute the typical root swollen structure, respectively. We revealed an important reprogramming of hormone pathways and C1 metabolism in both interactions, which may play key roles in nodule and gall neoformation, rhizobia endocytosis and nematode feeding. Common functions targeted by rhizobia and nematodes were mainly down-regulated, whereas the specificity of the interaction appeared to involve up-regulated genes. Our transcriptomic results provide powerful datasets to unravel the mechanisms involved in the accommodation of rhizobia and root-knot nematodes. Moreover, they raise the question of host specificity and the evolution of plant infection mechanisms by a symbiont and a pathogen.

Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco.

Superoxide dismutases (SODs) play a key role in the cellular defense against reactive oxygen species. To study the transcriptional regulation at the cellular level, the promoter of the Nicotiana plumbaginifolia cytosolic gene encoding Cu/ZnSOD (SODCc) was fused to the [beta]-glucuronidase (GUS) reporter gene (gusA) and analyzed in transgenic tobacco plants. The promoter was highly active in vascular bundles of leaves and stems, where it is confined to phloem cells. In flowers, GUS activity was detected in ovules and pollen grains, in pigmented tissues of petals, and in vascular tissue of ovaries and anthers. In response to treatment with the superoxide-generating herbicide paraquat, very strong GUS stainng was observed in photosynthetically active cells of leaves and in some epidermal root cells of seedlings. The expression of the SODCc-gusA was also induced in seedlings after heat shock and chilling and after treatment with sulfhydryl antioxidants such as reduced glutathione and cysteine. It is postulated that SODCc expression is directly linked to a cell-specific production of excess superoxide radicals in the cytosol.