Nod factor-induced root hair curling: continuous polar growth towards the point of nod factor application

Abstract

A critical step in establishing a successful nitrogen-fixing symbiosis between rhizobia and legume plants is the entrapment of the bacteria between root hair cell walls, usually in characteristic 180 degrees to 360 degrees curls, shepherd's crooks, which are formed by the host's root hairs. Purified bacterial signal molecules, the nodulation factors (NFs), which are lipochitooligosaccharides, induce root hair deformation in the appropriate host legume and have been proposed to be a key player in eliciting root hair curling. However, for curling to occur, the presence of intact bacteria is thought to be essential. Here, we show that, when spot applied to one side of the growing Medicago truncatula root hair tip, purified NF alone is sufficient to induce reorientation of the root hair growth direction, or a full curl. Using wild-type M. truncatula containing the pMtENOD11::GUS construct, we demonstrate that MtENOD11::GUS is expressed after spot application. The data have been incorporated into a cell biological model, which explains the formation of shepherd's crook curls around NF-secreting rhizobia by continuous tip growth reorientation.

Figure 1.

The response of a growing M. truncatula root hair to a single spot application of 10^–9 m purified Nod factor (NF). Fifteen minutes after NF application, the reorientation of the root hair growth axis toward the site of application is already visible and becomes more pronounced at 30 min. As can be seen, root hair growth is continuous during and after reorientation, and the root hair diameter does not change. Bar = 15 μm.

Figure 2.

Iterative spot application of NF leads to iterative root hair growth axis reorientation, leading to root hair curling. a, Growing root hair after three successive spot applications of NF on different sides, showing that multiple applications lead to multiple growth axis reorientations. Bar = 30 μm. b, Growing root hair after three successive spot applications of NF on one side of the root hair tip, showing multiple reorientations of the root hair growth axis, leading to a partial root hair curl. Arrowheads point to the position of successive NF spot applications. c to f, Time series of root hair curling after single spot application. With a micropipette (c), a microdroplet of 10^–9 m Nod factor is applied to one side of the apical dome of a growing root hair (d). After 25 min, a clear reorientation of the root hair growth axis toward the side of application is visible (e), and in 50 min, a partial shepherd's crook is formed (f). Bar in b to f = 18 μm.

Figure 3.

Spot application controls showing that root hair reorientation is an NF-specific response. a, Spot application of 10^–9 m non-sulfated NF (from the Sinorhizobium meliloti NodH mutant) does not lead to growth reorientation. Bar = 30 μm. a1, At the moment of application; a2, 25 min after application. b, Growing M. truncatula wild-type root hair after spot application with 10^–6 m chitotetraose showing no growth axis reorientation. Bar = 18 μm. b1, At the moment of application; b2, 45 min after application. c, Spot application of 10^–9 m sulfated chitotetraose does not lead to growth reorientation. Bar = 30 μm. c1, Before application; c2, 30 min after application. d, Growing M. truncatula wild-type root hair after spot application of Millipore water (Millipore, Bedford, MA) showing no growth axis reorientation. Bar = 18 μm. d1, At the moment of application; d2, 55 min after application. Arrowheads point to the site of application.

Figure 4.

pMtENOD11::GUS expression is maintained in reorienting and branching wild-type M. truncatula root hairs after NF spot application. a, Transgenic M. truncatula wild-type root hair carrying the pMtENOD11::GUS fusion construct stained for β-glucuronidase (GUS) expression 120 min after spot application of 10^–9 m NF showing positive GUS staining of the cytoplasm. Bar = 30 μm. b, Transgenic M. truncatula wild-type root hair carrying the pMtENOD11::GUS fusion construct stained for GUS expression 75 min after spot application of 10^–9 m NF 60 μm below the growing tip, showing root hair branching at the site of application and GUS staining of the cytoplasm. Bar: 30 μm.

Figure 5.

Cartoon of the putative mechanism involved in root hair growth axis reorientation during curl formation around rhizobia. a, Growing root hair with a tip-focused calcium gradient in an area devoid of detectable actin filaments, a subapical fine F actin area (black lines), the nucleus (N) positioned at the base of the subapical fine F-actin area, and the vacuole (V). b, Bacterium attaches to the root hair tip and locally excretes NF, which induces a local calcium influx, leading to a gradual increase in [Ca²⁺]_c. c, High [Ca²⁺]_c; thus, the growth area, shifts toward the attached bacteria, redirecting the growth. Note that the new growth direction is the resultant of the NF-induced direction and the original growth axis. d, The enlarging bacterial colony also produces NF, thus shifting again the growth area toward itself. In the end, these continuously repeated growth axis reorientations give rise to a tight curl, entrapping the bacteria.