Saleela Philip-Hollingsworth

Natural endophytic association between Rhizobium leguminosarum bv. trifolii and rice roots and assessment of its potential to promote rice growth

For over 7 centuries, production of rice (Oryza sativa L.) in Egypt has benefited from rotation with Egyptian berseem clover (Trifolium alexandrinum). The nitrogen supplied by this rotation replaces 25- 33% of the recommended rate of fertilizer-N application for rice production. This benefit to the rice cannot be explained solely by an increased availability of fixed N through mineralization of N- rich clover crop residues. Since rice normally supports a diverse microbial community of internal root colonists, we have examined the possibility that the clover symbiont, Rhizobium leguminosarum bv. trifolii colonizes rice roots endophytically in fields where these crops are rotated, and if so, whether this novel plant-microbe association benefits rice growth. MPN plant infection studies were performed on macerates of surface-sterilized rice roots inoculated on T. alexandrinum as the legume trap host. The results indicated that the root interior of rice grown in fields rotated with clover in the Nile Delta contained ∼106 clover-nodulating rhizobial endophytes g fresh weight of root. Plant tests plus microscopical, cultural, biochemical, and molecular structure studies indicated that the numerically dominant isolates of clover-nodulating rice endophytes represent 3 – 4 authentic strains of R. leguminosarum bv. trifolii that were Nod Fix on berseem clover. Pure cultures of selected strains were able to colonize the interior of rice roots grown under gnotobiotic conditions. These rice endophytes were reisolated from surface-sterilized roots and shown by molecular methods to be the same as the original inoculant strains, thus verifying Kochs postulates. Two endophytic strains of R. leguminosarum bv. trifolii significantly increased shoot and root growth of rice in growth chamber experiments, and grain yield plus agronomic fertilizer N-use efficiency of Giza-175 hybrid rice in a field inoculation experiment conducted in the Nile Delta. Thus, fields where rice has been grown in rotation with clover since antiquity contain Fix strains of R. leguminosarum bv. trifolii that naturally colonize the rice root interior, and these true rhizobial endophytes have the potential to promote rice growth and productivity under laboratory and field conditions.

Mutation or Increased Copy Number of nodE Has No Effect on the Spectrum of Chitolipooligosaccharide Nod Factors Made By Rhizobiumleguminosarum bv. trifolii

The bacterial gene nodE is the key determinant of host specificity in the Rhizobium leguminosarum-legume symbiosis and has been proposed to determine unique polyunsaturated fatty acyl moieties in chitolipooligosaccharides (CLOS) made by the bacterial symbiont. We evaluated nodE function by examining CLOS structures made by wild-type R. leguminosarum bv. trifolii ANU843, an isogenic nodE::Tn5 mutant, and a recombinant strain containing multiple copies of the pSym nod region of ANU843. 1H-NMR, electrospray ionization mass spectrometry, fast atom bombardment mass spectrometry, flame ionization detection-gas chromatography, gas chromatography/mass spectrometry, and high performance liquid chromatography/UV photodiode array analyses revealed that these bacterial strains made the same spectrum of CLOS species. We also found that ions in the mass spectra which were originally assigned to nodE-dependent CLOS species containing unique polyunsaturated fatty acids (Spaink, H. P., Bloemberg, G. V., van Brussel, A. A. N., Lugtenberg, B. J. J., van der Drift, K. M. G. M., Haverkamp, J., and Thomas-Oates, J. E.(1995) Mol. Plant-Microbe Interact. 8, 155-164) were actually due to sodium adducts of the major nodE-independent CLOS species. No evidence for nodE-dependent CLOSs was found for these strains. These results indicate a need to revise the current model to explain how nodE determines host range in the R. leguminosarum- legume symbiosis.

Subnanomolar concentrations of membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii are fully capable of eliciting symbiosis-related responses on white clover

Axenic seedling bioassays were performed on white clover, vetch, and alfalfa to assess the variety and dose responses of biological activities exhibited by membrane chitolipooligosaccharides (CLOSs) from wild type Rhizobium leguminosarum bv. trifolii ANU843. Subnanomolar concentrations of CLOSs induced deformation of root hairs (Had) and increased the number of foci of cortical cell divisions (Ccd) in white clover, some of which developed into nodule meristems. In contrast, ANU843 CLOSs were unable to induce Had in alfalfa and required a 104-fold higher threshold concentration to induce this response in vetch. Also, ANU843 CLOSs were not mitogenic on either of these non-host legumes. In addition, CLOS action also increased chitinase activity in white clover root exudate. Thus, the membrane CLOSs from wild type R. leguminosarum bv. trifolii are fully capable of eliciting various symbiosis-related responses in white clover in the same concentration range as extracellular CLOSs of other rhizobia on their respective legume hosts. These results and our earlier studies indicate that membrane CLOSs represent one of many different classes of bioactive metabolites made by R. leguminosarum bv. trifolii which elicit more intense symbiosis-related responses in white clover than in other legumes. Therefore, CLOSs evidently play an important role in symbiotic development, but they may not be the sole determinant of host-range in the Rhizobium-clover symbiosis.

Root hair deforming and nodule initiating factors from Rhizobium trifolii

Axenic legume root hairs typically remain straight. Early in the infection process, Rhizobium secretes compounds that induce various morphological changes in root hairs, including enhanced differentiation (leading to greater hair density per root surface area), elongation, various deformations including helices, branches, bulbs, and curlings including the 360° tight curl (so called “shepherd crooks”) at hair tips (Yao & Vincent 1969). It has been proposed that these deformations may represent altered synthesis and/or stabilization of the root hair cell wall during growth (for reviews, see Bauer 1981; Dazzo & Hubbell 1982; Halverson & Stacey 1986; Rolfe & Gresshoff 1988).

Isolation, characterization, and structural elucidation of a “nod signal” excreted by Rhizobium trifolii ANU843 which induces root hair branching and nodule-like primordia in axenic white clover seedlings

Rhizobium excretes various metabolites which are biologically active on its leguminous host. For example, a substituted tetrasaccharide excreted by R. meliloti has been reported to induce alfalfa-specific root hair branching and control host specificity in this symbiosis (3). We study the excreted metabolites from the clover symbiont, R. trifolii, which are responsible for inducing the morphological changes that occur during infection and nodulation by this bacterial symbiont. We are particularly interested in the metabolites whose product ion/export require pSym nod genes; hence, these are called “nod signals”. Our general strategy has been to grow wild-type R. trifolii ANU843 on defined Bill medium with or without flavones to enhance nod gene expression, fractionate the culture supernatant, and perform axenic seedling bioassays on the purified excreted metabolites. Our results to date have shown that wild-type R. trifolii excretes a diversity of bacterial factors capable of affecting development of the root nodule symbiosis (1,2this paper, Fig. 1).

Analysis of extracellular polysaccharides of Azospirillum brasilense

Bacteria of the genus Azospirillum live in association with the Graminae. Azospirillum has a growth-promoting effect due to nitrogen-fixation, phytohormone production and improved water household of the plant. Azospirillum is able to attach to plant roots (Umali-Garcia et al. 1980) and A. brasilense Sp245 can even invade into the root cortex. In plant bacteria interactions, signalling is an important factor. There are different signal molecules known in other microbe-plant interactions: β 1–2 glucans in the interaction of Agrobacterium and Rhizobium for respectively tumor-formation and nodule-induction; hepta-saccharides which act as elicitors for the plant-defense mechanisms against plant pathogens; extracellular polysaccharides (EPS) necessary for the formation of nitrogen-fixing nodules on legumes by Rhizobium. The production of EPS by Azospirillum was shown by the fluorescent dye Calcofluor. In the F. A. Janssens Memorial Laboratory for Genetics, recent interest is focussed on the genetics of the synthesis of this EPS by Azospirillum. Tn5 induced A. brasilense mutants that are are affected in Calcofluor fluorescence have already been isolated (Michiels et al., 1988a; 1990). Some A. brasilense genes are able to correct R. meliloti exo mutations. In this way, the R. meliloti exoB, exoC, exoG, exoM, exoN, exoP and exoK mutants have been genetically complemented by A. brasilense DNA. The exoB, exoC1 and exoM correcting loci are located on the 90 MDa plasmid of A. brasilense Sp7 (Michiels et al., 1988b; Marc Eyers, personal communication), whereas the exoC2, exoG, exoK, exoN and exoP correcting loci are located on the chromosome (Marc Eyers, personal communicaton). The A. brasilense exoB en exoC1 mutants have been constructed by marker-exchange (Michiels et al. 1988b).

Recent studies on the Rhizobium-legume symbiosis

This brief paper summarizes our research progress since the 7th International Congress on Nitrogen Fixation at Koln, West Germany. We published studies reported at that meeting on the structural similarities and differences between the capsular acidic heteropolysaccharide (CPS) of Rhizobium trifolii and R. leguminosarum (4), and the effect of interspecies transfer of Rhizobium host-specific nodulation genes on CPS structure and in situ binding by trifoliin A lectin (5). We also published on the application of computer-aided image analysis for studies of the Rhizobiurn-legume symbiosis (2). Interesting measurements reported in that paper include: (i) R. leguminosarum bacteroids are 7.2 times the volume of vegetative bacteria within pea nodules, (ii) the average vertex angle of root hair branching induced by R. trifolii on white clover is 67°, (iii) R. trifolii rotates its flagella at 330 rpm and swims at 53 µm/sec in the external root environment of agar less Fahraeus slide cultures of white clover, (iv) inoculation of white clover roots with R. trifolii leads to a 35% increase in cytoplasmic streaming in root hairs, and (v) white clover root hairs elongate during active growth at an average rate of 38 µm/nr.

Early Recognition Signals in the Rhizobium Trifolii-White Clover Symbiosis

Rhizobium is a bacterial symbiont which infects root hairs of legumes, inducing the formation of symbiotic root nodules which fix atmospheric nitrogen into ammonia fertilizer for the host plant. A hallmark of the root nodule symbiosis is a high degree of host specificity which restricts the range of legume hosts infected by the bacterium. We are studying surface and extracellular molecules of R. trifolii which interact with white clover root hairs as a model of cell-cell communication in this plant-bacterial interaction.

Structural Requirements for Uptake and Bioactivity of Rhizobium Chitolipooligosaccharides in Legume Roots as Revealed by Synthetic Analogs and Fluorescent Probes

We study the structure / function relationships of chitolipooligosaccharides (CLOSs) made by authentic wild type R. leguminosarum bv. trifolii ANU843 and their role in symbiotic development with white clover. Previously, we found that ANU843 accumulates a large (22+) family of CLOS glycolipids primarily in its membranes, and this structural diversity is unaltered in the ANU297nodE::Tn5 mutant derivative [from M. Djordjevic], despite its change in legume host range (Philip-Hollingsworth et al. 1995). Nevertheless, (sub)nano-molar concentrations of ANU843 membrane CLOSs fully elicit symbiosis-related responses in white clover roots, including Had and Ccd leading to nodule primordia, modulation of growth dynamics, localized disruption of crystalline wall architecture, and development of new infection sites in root hairs, and elevated chitinase activity in root exudate (Dazzo et al. 1996; Orgambide et al. 1996). Here, we prepared fluorescent-tagged NBD derivatives and synthetic analogs of ANU843 CLOSs to define their structural requirements for uptake and mitogenicity in living root cells, using laser scanning confocal microscopy and axenic seedling bioassays. We found that (i) NBD-CLOS conjugates are still Had+ and Ccd+, and are rapidly internalized within cell membranes and nuclei of living clover root hairs and cortical cells; (ii) the structural requirements for uptake and biological activity of CLOSs are directly correlated; and (iii) the smallest CLOS analog still internalized and mitogenic on both clover and alfalfa is a N-fattyacylglucosamine, without an essential requirement for oligomerization or functionalization of the polar head group or unsaturation of the fatty acyl tail (Philip-Hollingsworth et al. 1997).