Rhizobium Nodulation Signals Induce Calcium Changes and Actin Cytoskeleton Rearrangements in Plant Root Hair Cells

In the first stages of root nodule morphogenesis, there is evidence that the actin cytoskeleton participates in the formation of preinfection threads and induction of cortical cell divisions (Bakhuizen, 1988). It has been reported that actin becomes fragmented in curled root hairs fromViciae hirsuta when infected with Rhizobium (Ridge et al. 1992). Although Allen et al., (1994) have reported that in curled root hairs actin foci were observed close to the tip, when exposed to Nod factors, the question of how actin cytoskeleton responded to Nod factors, during the first minutes of exposure, remains unanswered. We are interested in analyzing this response using as a model, the symbiosis P. vulgaris-R. etli. On the other hand, it is a known fact that Ca2+ is indispensable for the infection process as suggested by the use of aureomycin. The increased fluorescence normally found at the root hair tip due to the presence of Ca2+ has suggested its possible role in the formation and growth of the infection thread (Sethi and Reporter 1981). Recent studies have explored ion movement as an indicator of root hair activity related to signal perception. It has been found that alfalfa root hairs displayed a depolarization of the cytoplasmic membrane in response to R. meliloti Nod factors (Ehrhardt et al., 1992; Felle et al., 1995; Kurkdjian, 1995). Albeit the ionic basis for this behaviour is unknown, there is some evidence that variable currents of H+ and Ca++ ions, are present outside root hairs from alfalfa (legume forming indeterminate nodules) which had been exposed to Nod factors (Allen et al., 1994). In alfalfa root hair cells, a calcium spiking response around the nucleus, that initiated 10 min after Nod factor exposure and remained up to 60 min has been reported (Ehrhardt et al., (1996). To elucidate the calcium response in root hair cells from a legume forming determinate nodules, P. vulgaris, we isolated and purified Nod factors from R. etli (Cardenas et al., 1995), and studied their effect on intracellular and extracellular Ca2+ current influxes in this legume. We used two approaches, dextran-linked calcium selective dyes injected directly into P. vulgaris root hairs, and an ion selective vibrating probe.