Nod Factor Induced Changes in Ca in Root Hairs of Pea Mutants

Ehrhardt et al. (1996) first demonstrated that Nod factors could induce periodic oscillations in calcium (calcium spiking) around the nuclear region of legume root hairs. On alfalfa, this required the appropriate (Sinorhizobium meliloti-made) Nod factor because the Nod factor from R. I. viciae had no effect. The host specific sulfation of the terminal reducing glucosamine was necessary for induction of Ca spiking. In Phaseolus bean, Nod factor induced a change in Ca at the root tip and subsequently an oscillation in intracytoplasmic Ca levels (Cardennas et al. 1999).

In pea, Nod factor-induced changes in intracellular Ca were analyzed using the calcium-sensitive fluorescent dye Oregon green-dextran, which was microinjected into young growing root hairs on lateral roots. The dye was excited at 488 nm and emitted fluorescent light was passed through a 515 nm-long pass filter. Sets of images were collected at 5 s intervals and the data obtained were calculated using the average pixel intensity for regions of the root hair. The Nod factor (at 10"9 M) clearly induced Ca spiking (Figure 1) and this occurred in all the cells that were imaged. This induction of Ca spiking occurred if the Nod factor lacked the nodE-determined CI 8:4 acyl group and the nodi-determined acetyl group. To determine the minimal Nod factor structure that would induce a response, we tested the effects of unsubstituted chitin backbone of the Nod factor, which consists of an oligomer of four or five 7V-acetyl glucosamine residues. At relatively fi ft high concentrations (10" -10" M), such chitin oligomers induced abnormal Ca spiking. Thus, although calcium spikes were observed, they were less frequent and more sporadic than those induced by Nod factor. Further details are described by Walker et al. (2000).

We analyzed the effect of Nod factor on intracellular calcium in the root hairs of several pea nodulation-defective mutants that do not show infections. Normal calcium spiking occurred in mutants carrying non-nodulating alleles of sym2/1, sym7 or sym9. In contrast, no calcium spiking was induced in peas carrying mutant alleles of sym8, symlO or syml9 (Walker et al. 2000). Representative traces from some mutants are shown in Figure 1.

WT: Pre Nod factor

WT + Chitin pentaose WT + Nod factor sym8 + Nod factor

3 sym9 + Nod factor

symlO + Nod factor

5 min

Figure 1. Calcium spiking induced by Nod factors. The traces show measurements of fluorescence in root hairs either before additions (top trace) or 20-25 min after the additions of chitin pentaose (10"6 M) or Nod factors (10-9 M). Representative traces obtained with only three of the mutants tested are shown. Further details are described in Walker et al (2000).

These results suggest that induction of calcium spiking occurs downstream of events that require the sym8, symlO and syml9 gene products, but upstream of events that require the sym2A, sym7 and sym9 gene products. If the other phenotypes of pea mutants carrying mutations at these loci are considered, it is possible to place the mutations into an order that could correspond to the relative order of function of the different gene products (Figure 2). Thus, plants carrying sym2A can induce root hair curling and infection foci (Geurts et al. 1997, and see below), whereas sym 7

mutants induce root hair deformation but no infections (Markwei, LaRue 1992). Therefore sym2A is likely to influence events after sym7, and since the sym.9 mutant induces little or no root hair deformation, this is likely to be upstream of sym7 and sym2A. Therefore after calcium spiking, the events are likely to occur in the order determined by sym9, sym7 and sym2A. Of the three loci that are required for calcium spiking, two {sym8 and syml9) have phenotypes that are, as yet, indistinguishable; pea lines carrying mutations at these loci do not induce root hair deformation and do not induce early nodulin gene expression (Albrechts et al. 1998; Schneider et al. 1999).

Nod —► c„m in <—+ sym8 Calcium 0 _ 7 __ Infected factor^ Syml°-: syml9^ spiking ^ sym9-+sym7 syrn2^ ^

Mycorrhizae ^ Mycorrhizal infection

Figure 2. Model showing proposed sequence of early nodulation signaling events in relation to mutations blocking nodulating and infection thread growth in pea.

In previous work, Due et al. (1989) and Albrecht et al. (1998) demonstrated that non-nodulating peas carrying alleles of sym8, sym9 and syml9 were also defective for mycorrhizal infections. In contrast, symlO mutants established a normal mycorrhizal symbiosis. Taking these observations into account together with the data on calcium spiking, it seems likely that the sym 10 gene product plays a role that is upstream of sym8 and syml9 (Figure 2). Therefore, of those pea mutant loci characterized, it is likely that symlO is involved in the earliest step in nodulation signaling.

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