On the other hand, alfalfa responds to infection with Sinorhizobium meliloti by production of O2" and H2O2. O2" was detected in infection threads and in infected cells in nodules up to nine days old. H2O2 production was seen in walls and infection threads of infected cells (zone II). H2O2 was not found in the meristematic zone I and fixation zone III (Santos et al. 2001). Thus, alfalfa produces a prolonged oxidative burst in response to S. meliloti infection. This is reminiscent of the oxidative burst occurring as an early response in plant defense reactions towards pathogens. To investigate the role of the microsymbiont superoxide dismutase (SOD) in protecting against oxidative stress in the symbiotic process, the sodA gene - encoding the sole cytoplasmic SOD of S. meliloti - was isolated and a null mutant was constructed. The resulting mutant, deficient in SOD activity, was able to grow normally and was only moderately sensitive to oxidative stress when free living. In contrast, its symbiotic properties in alfalfa were drastically affected (Santos et al. 2000). The SOD-deficient mutant nodulated poorly and displayed abnormal infection. Electron microscopy showed that bacteroid differentiation was blocked in most nodules at the level of infection zone II. In some cases, the bacteria were released into the cytoplasm without a peribacteroid membrane and did not differentiate. In other cases, the infection threads aborted and the bacteria degenerated without release (Santos et al. 2000).
As the SOD activity leads to the formation of H2O2, which has been detected in the infection threads, the symbiotic behavior of S. meliloti mutants lacking catalase activity was also investigated. S. meliloti contains three catalase genes, named katA, katC and katB, which encode two monofunctional catalases and a bifunctional catalase/peroxidase, respectively (Sigaud et al. 1999). Mutants lacking one of the catalases were not affected in their symbiotic capacities. However, a katA/katC double mutant exhibited a reduced efficiency of nodulation compared to the wild type. Other double mutants (katA/katB and katB/katC) and a triple mutant are currently being constructed and their nodulation capacities will be tested. Moreover, experiments using promoter-lacZ fusions showed that sodA was strongly expressed in infection threads, whereas a differential expression of the catalase genes was observed. This further confirms that the bacteria have to face an oxidative challenge during the infection process. In this framework, it must be noted that a bacterial gene, encoding a protein with a sequence similar to those of peroxiredoxins, has been isolated along with other genes induced during symbiosis (Oke, Long 1999).
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