Sugar-Binding Activity of Pea Lectin Enhances Heterologous Infection of Transgenic Alfalfa Plants by Rhizobium leguminosarum biovar viciae  

Abstract

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo Ϫ R. leguminosarum bv viciae strain that produced S. meliloti Nod factor. Bacteria belonging to the family Rhizobiaceae (Rhizobium, Bradyrhizobium, Azorhizobium, Mesorhizobium, and Sinorhizobium) induce the formation of nitrogenfixing nodules on their leguminous hosts. This symbiotic interaction, which is governed by sequential signal exchange between rhizobia and their symbiotic partners, exhibits a high degree of specificity, and a number of signal molecules involved in the initial stages of this specificity have been extensively studied. Nod factors are synthesized by the products of rhizobial nod genes, which are induced by plantsecreted molecules such as flavonoids (Hirsch, 1992; Long, 1996). The Sinorhizobium meliloti Nod factor consists of a variable-length N-acetylglucosamine oligomer with a C-16 acyl tail at the non-reducing end and a sulfate at the reducing end, whereas the Rhizobium leguminosarum bv viciae Nod factor has a C-18 fatty acyl residue and no sulfate (for review, see Schultze and Kondorosi, 1998). Nod factors are considered the main rhizobial inducËer molecules for nodulation because the purified molecules elicit, in a host-specific way, many of the plant responses observed in the early stages of nodule formation. These responses include changes in free calcium levels and ion balance, alterations in cytoskeletal organization and morphology of root hairs, the initiation of cortical cell divisions (Ccd), and the triggering of nodule development (Spaink et al., 1991; Truchet et al., 1991;

Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti

“…Since 1989, several studies using transgenic plants have lent support to this hypothesis because transgenic roots expressing heterologous lectins are capable of being nodulated by the heterologous rhizobial species (Diaz et al, 1989;Kijne et al, 1997;Hirsch, 1999). For this effect, the sugar-binding site of the lectin is required (Diaz et al, 2000;van Rhijn et al, 2001), and the latest results suggest that exopolysaccharide rather than Nod factors may be the critical rhizobial component for extension of the host range (van Rhijn et al, 2001). The lectins have been suggested to function as a glue, sticking the two partners together for Nod factors to operate, or as a cloaking device to avoid recognition as a pathogen, or as an element in signaling and friend recognition (Hirsch, 1999).…”

Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

“…Control plants and transgenic plants that expressed mutant pea lectin did not form nodules or infection threads when inoculated with low numbers of the same strain (171). Results such as these suggest that cell-cell contact and specific binding of compatible bacteria to root tips are important for infection and infection thread formation, because they result in the exposure of infectible root hair tips to the proper symbiont and hence to a high localized concentration of the Nod factors needed to trigger root hair curling and infection thread formation (79,171). A direct test of the importance of host lectins in promoting infection and invasion would be very interesting.…”

“…These lectins localize to root hair tips and are thought to help convey host-symbiont specificity by binding simultaneously to the plant cell wall and to saccharide moieties on the surfaces of compatible bacteria (41,43,79). A series of experiments in which a variety of transgenic plants expressed lectins from other species of legumes has shown that the presence of heterologous lectins often allows transgenic plants to respond to symbionts that are usually noncompatible, provided that the heterologous lectin can bind to the noncompatible bacteria and provided that the noncompatible bacteria make the proper Nod factor (42,79,171,172). For example, transgenic alfalfa that expressed pea lectin formed nodules and infection threads when inoculated with low numbers of R. leguminosarum bv.…”

ENOD40Gene Expression and Cytokinin Responses in the Nonnodulating, Nonmycorrhizal (Nod−Myc−) Mutant,Masym3, ofMelilotus albaDesr.

“…A Zeiss Axiophot light microscope in conjunction with epifluorescence was used for observation of attached fluorescent rhizobia. Roots inoculated with Rm1021/gusA were stained as previously described 35 and examined under bright field optics.…”

“…Afterwards, three-day-old wild-type M. alba seedlings of U389 and the Masym mutants were transferred to sterilized square dishes (Fisher Scientific International Inc., Hampton, NH) containing 1% Phytagar-solidified, one-quarter strength Hoagland's medium minus nitrogen (N), 34 and subsequently spot-or flood-inoculated with the wild-type controls, Sinorhizobium meliloti Rm1021 or Rm1021 carrying gusA, constructed as described earlier. 35 For studies of root hair deformation, only the wild-type M. alba and the Masym3 mutant, BT70, were examined. The roots were inoculated with either Rm1021 or the mutant strains, a Nod -S. meliloti (SL44: Cytokinin Triggers ENOD40 Expression in Melilotus DnodD1ABC) or a Nod -/pTZS + strain, which is SL44 carrying the trans-zeatin synthase gene of Agrobacterium tumefaciens.…”