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First published online October 29, 2004; 10.1104/pp.104.049064 Plant Physiology 136:3692-3702 (2004) © 2004 American Society of Plant Biologists nip, a Symbiotic Medicago truncatula Mutant That Forms Root Nodules with Aberrant Infection Threads and Plant Defense-Like Response1Department of Biological Sciences, University of North Texas, Denton, Texas 76203–5220 (H.V., R.D.); Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711 (J.G.H., D.J.S); and Department of Plant Pathology, University of California, Davis, California 95616 (R.V.P., D.R.C.)
To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses during symbiotic interactions.
The symbiosis that develops between leguminous plants and soil rhizobia to form a nitrogen-fixing root nodule is a complex and unique interaction. The interaction begins with an exchange of signals between rhizobia and plants in the rhizosphere. Flavonoid compounds released by the host plant stimulate the expression of nod genes in an appropriate rhizobial species, resulting in the production of bacterial Nod factors. Nod factors, lipochitooligosaccharide molecules, are able to induce specific responses in the host plant, including root hair deformation and cortical cell division. Infection conduits called infection threads originate in deformed root hairs that curl to form a so-called shepherd's crook and facilitate entry of rhizobia into the root. Infection thread initiation and growth require living rhizobia that are synthesizing specific Nod factors (Ardourel et al., 1994  ; Limpens et al., 2003). Most infection threads abort in root epidermal cells, with only a small fraction of the infections proceeding to the interior of the root (Vasse et al., 1993; Penmetsa and Cook, 1997). For the infection threads that penetrate the interior of the root, the course by which they proceed is predetermined by cytoplasmic bridges or preinfection threads; these allow infection threads to pass through outer cortical cells toward the inside of the root (Kijne, 1992; van Brussel et al., 1992). When infection threads reach cells in the newly formed nodule primordium, they become confined to the intercellular space between cells (Kijne, 1992). As an infection thread ceases growth, irregular structures including unwalled droplets of infection thread matrix material containing rhizobia are formed. These are engulfed by the plant host plasma membrane, forming symbiosomes in a process resembling endocytosis. The peribacteroid membrane surrounding the symbiosome is initially formed by the plasma membrane of the host cell and expands by fusion with newly synthesized vesicles carrying peribacteroid membrane nodule-specific proteins (Brewin, 1998). Within symbiosomes, the rhizobia differentiate into bacteroids that are capable of fixing nitrogen. Reviews on legume root nodule development and the rhizobial infection process are available (Brewin, 1991; Hirsch, 1992; Kijne, 1992; Brewin, 1998; Gage and Margolin, 2000; Gage, 2004).
The means by which invading rhizobia are able to avoid triggering most of the legume root's host defense mechanisms are not understood. In a normal legume-rhizobia interaction, there are some detectable plant defense responses. Very localized hypersensitive-like reactions accompany aborted infections in epidermal cells and may be part of the plant host's autoregulation of nodule number (Vasse et al., 1993
Genes and processes involved in lateral root formation may also be involved in nodule formation. Like nodule development, lateral root development is influenced by hormones as well as the nutritional status of the plant. Unlike nodules that develop from cortical cell divisions, lateral roots usually develop from divisions in the pericycle. Both types of divisions take place opposite a protoxylem pole, but lateral roots have a central arrangement of vascular bundles, whereas nodules have peripheral vascular bundles (Hirsch, 1992
In 1990, Medicago truncatula was proposed as a model legume, particularly for studying the Rhizobium-legume symbiosis, because of its small genome size (approximately 500 Mbp, n = 8), self-compatibility, relatively short generation time, ability to be transformed, and the well-characterized nature of its microsymbiont, Sinorhizobium meliloti (Barker et al., 1990 Here, we describe the isolation of a novel M. truncatula mutant, nip, and present data on its phenotype. nip plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. It has an abnormal defense-like response in root nodules as well as defects in lateral root development.
Isolation of the Recessive nip Mutant
Ethyl methane sulfonate-generated M. truncatula plants from the A17 genetic background were screened for nodulation phenotypes in the M2 generation by inoculating them with an S. meliloti strain containing the constitutive hemA::lacZ construct (Boivin et al., 1990
nip was back-crossed twice (BC2), utilizing the male-sterile tap mutation in the wild-type A17 background (Penmetsa and Cook, 2000
Representative wild-type and nip BC2 plants grown in an aeroponic chamber were photographed at 15 dpi (Fig. 1, A and B). nip roots showed variation in root length from plant to plant and were observed to have abnormal lateral roots. Unlike wild-type plants that had clearly emerged lateral roots (Fig. 1C), most nip plants had either no lateral roots, although lateral root primordia were visible (Fig. 1D), or had shorter lateral roots (Fig. 1B) than wild type (Fig. 1A). Instead of effective, pink, leghemoglobin-containing nodules as were found in wild type (Fig. 1E), nip developed straw-colored root nodules/bumps containing a brown pigment (Fig. 1F), suggesting an accumulation of polyphenolic compounds. Cosegregation of the lateral root defect with the nodulation defect was found in all nip F2 progeny tested in all crosses.
nip Nodules Accumulate Polyphenolic Compounds and Have Large, Proliferating Infection Threads That Rarely Release Rhizobia When grown without nitrate and in the presence of compatible rhizobia, nip plants exhibited reduced vigor with chlorotic leaves that are characteristic of nitrogen deficiency. These symptoms were alleviated by supplementation with nitrate fertilizer and are indicative of a defect in the ability to form an effective nitrogen-fixing symbiosis. Growth of nip plants in the presence of nitrate fertilizer had no effect on the lateral root phenotype (data not shown). To evaluate nip's nodulation phenotype, nip plants were grown in an aeroponic system and inoculated with S. meliloti carrying the hemA::lacZ fusion. Staining with X-Gal at 15 dpi revealed a proliferation of S. meliloti inside numerous infection threads in the nodule primordia (Fig. 2, A and B). The nip nodule primordia emerged from the root only after prolonged nodule development times, at or after 25 dpi. In contrast, emerged elongated wild-type A17 nodules were filled with the blue-staining S. meliloti at 15 dpi.
In order to more closely examine the extent of infection, nip and wild-type nodules were stained with nucleic acid-binding dye SYTO-13 and examined by laser-scanning confocal microscopy (LSCM). Stained rhizobia fluoresced in the green spectrum using this technique, while polyphenolics and cell walls fluoresced in the red spectrum, pseudocolored blue in the figure to enhance detail (Fig. 3). By 13 dpi, fully mature wild-type nodules had emerged from the surface of the primary root and infected cells were characterized by high rhizobial cellular occupancy (Fig. 3A). In contrast, despite an examination of over 160 nip nodules, we failed to find clear examples of infected host cells in the nip mutant. As was seen using the hemA::lacZ marker and X-Gal staining, infection thread numbers were found to be substantially higher in nip (Fig. 3B), and LSCM revealed abnormal infection thread morphology in nip nodules. Infection threads, including the original infection thread that originates in the root epidermis, were thickened relative to wild-type threads with abnormal bulbous protrusions (Fig. 3C) that became more pronounced over time with an increase in diameter of the threads and the size of the protrusions (Fig. 3D). nip nodules were found to emerge from the root epidermis only late in development, and were also found to have large areas of autofluorescing cells, suggesting an induction of the plant defense response. The autofluorescing cells were localized near cells with infection threads, but did not themselves contain threads.
To determine if the autofluorescence and the accumulation of brown pigment represented an accumulation of polyphenolic compounds, nodules were stained with potassium permanganate using a method that results in blue precipitates at sites of polyphenols (Fig. 2, C and D). Individual cells in the nodule cortex of nip nodules stained heavily for polyphenolics, with other cell types displaying no staining (Fig. 2C). In contrast, in wild-type A17 nodules, no polyphenolic staining was detected in any of the cells in the nodule (Fig. 2D). To further explore the nip nodule phenotype and examine the interface between the infection threads and plant host cells in detail, studies using transmission electron microscopy (TEM) on 15 and 21 dpi nip nodules were carried out. No differences between the 15 and 21 dpi nodules were observed and data from 15 dpi nodules are presented here. Wild-type nodules developed normally, and by 15 dpi had produced fully mature nodules exhibiting the typical developmental zones: meristem, prefixation zone (Fig. 4A), interzone, and nitrogen-fixation zone (Fig. 4, B and I). In contrast, nip nodules commonly remained unemerged at this stage and exhibited no distinct developmental zones; rather, the nodule interior most closely resembled an enlarged prefixation zone (Fig. 4, C and E). Infection droplets in the wild-type nodule were confined to a narrow zone below the meristem, were generally small, and exhibited normal release of bacteria (Fig. 4A), whereas droplets in nip were seen throughout the nodule interior and were often enlarged with excess matrix material (Fig. 4C). Wild-type infection threads were generally narrow and grew in a linear pattern through cells (Fig. 4B). In contrast, nip infection threads were much more numerous, often unusually wide and irregularly shaped (Fig. 4D), and commonly grew in a serpentine pattern (Fig. 4E). At high magnification, wild-type infection droplets (Fig. 4F) were similar in appearance to nip infection droplets, but nip infection droplets rarely released bacteria into host cells. In one instance, limited release of bacteria was seen in one cell of a nip nodule (Fig. 4H), but the bacteria were small and undifferentiated when compared to the mature fixation zone of a wild-type nodule (Fig. 4I). At this stage, nip plants were severely nitrogen stressed and it is unlikely that this limited infection could have progressed to a normally infected cell. In addition to abnormal threads, nip nodules also exhibited abnormal membrane profiles within vacuoles in the uninfected nodule periphery, several cell layers removed from the threads. Membrane profiles were seen in the vacuoles and were suggestive of cell death in these regions (Fig. 4, J and K). These cells correspond to the region in Figure 3 that contained autofluorescent compounds and to the region in Figure 2 that stained for polyphenolics.
nip Plants Have Normal Shoots and Abnormal Lateral Roots Growth characteristics were investigated by growing F3 plants derived from the BC2 generation and from crosses into ecotype A20, using the aeroponic system with nitrate-free media in the presence of rhizobia. The results are shown in Tables II and III. In plants propagated from BC2, nip primary shoots were found to be approximately the same size as wild type at 5 and 10 dpi, and averaged 75% of wild type at 15 dpi, presumably reflecting a nitrogen deficiency (Table II).
As was noted above (see Fig. 1), in F3 plants derived from the BC2 population, nip plants showed root abnormalities. To further investigate the nip root phenotype, nip plants with the most normal-looking root systems were crossed to ecotype A20. Several F2 plants resulting from this cross were observed that had primary roots of similar length to wild type and possessed lateral roots. To determine the correlation between altered lateral root development with symbiotic phenotype, 47 individual F3 progeny from these F2 plants were examined for their root phenotypes (Table III). The F3 progeny had roots that averaged a similar length as wild type, 18.4 ± 4.4 cm, compared to 19.7 ± 1.7 cm for A17, and 18.6 ± 3.5 cm for A20. However, only 11, or approximately 25% of the 47 individual F3 plants, had lateral roots, although, as before, lateral root primordia were evident. When present, the lateral roots occurred at a lower frequency than wild type (Table III) and were shorter in length compared to wild type (data not shown). This strongly suggests that the lateral root defect in nip is incompletely penetrant.
Previous studies have shown that the plant hormone ethylene plays a central role in regulation of successful infections as well as root growth in M. truncatula (Penmetsa and Cook, 1997
nip Nodule Gene Expression Studies Indicate a Block to Nodule Differentiation and Elevated Levels of a Transcript Associated with Plant Defense
To evaluate the progression of nip nodule development at the transcriptional level, the expression of four genes associated with nodule development and two genes associated with plant host defense were investigated in nip and wild-type nodulating roots. The ENOD40 gene is correlated with formation of nodule and lateral root primordia (Crespi et al., 1994
Cytological evaluation demonstrated an unusual plant defense-like response in nip nodules (Figs. 2C, 3, and 4, J and K). Therefore, expression of two genes associated with plant host defense was studied in nodulating nip and wild-type roots. Elevated levels of pathogenesis-related protein-10 (PR-10) and Phe ammonia lyase (PAL) are both associated with plant host defense and plant stress (Hammond-Kosack and Jones, 2000 ). PR-10 mRNA accumulation in nip nodulating roots was found to be comparable to wild-type nodulating roots. In contrast, PAL expression was found to increase dramatically during nip nodule development compared to wild type. Because PAL encodes the enzyme catalyzing the first step in phenylpropanoid metabolism, the elevated levels of PAL mRNA are consistent with the accumulation of polyphenolics detected in nip nodules.
Although nip has a different phenotype from other legume mutants studied by several groups in the Medicago scientific community, it is possible that it is allelic to another mutant. To test this possibility, nip was crossed to other mutants and two to six independent F1 progeny from each cross were scored for nodulation phenotype. Of particular interest were mutants that are able to initiate nodulation but are defective in rhizobial invasion or later steps in the symbiosis. Genetic crosses of the nip mutant to each of several M. truncatula mutants, lin, rit, bit, TE7 (Mtsym1), dnf1, dnf2, dnf3, dnf4, dnf5, dnf6, or dnf7 (Benaben, 1994
We identified a novel symbiotic mutant in M. truncatula called nip. The monogenic and recessive nip mutant is able to initiate nodule development, but is not competent to attain functional nitrogen-fixing nodules when grown in the presence of the compatible Rhizobium sp., S. meliloti. As our studies using light, confocal and electron microscopy show, the nip mutant is capable of developing nodule primordia and initiating rhizobial invasion through plant-derived infection threads. However, nip infection threads are thicker than wild type and characterized by abnormal bulbous protrusions and unusual branching. The block to nodule development is most likely at release of rhizobia from infection threads and endocytosis into the host cytoplasm. The nip mutant is very slightly leaky, rarely allowing release of rhizobia into host cells, but neither the rhizobia nor the host cells appeared to differentiate in response to release. Our data do not allow us to distinguish whether the infection thread characteristics observed are a consequence of the failure to release rhizobia from the threads or if infection thread characteristics, including size, proliferation, or biochemical constitution, cause the rhizobia not to be released.
nip nodules show evidence of an abnormal plant host defense-like response. We noted that the cells with the defense-like response, those that stain for polyphenolics, are autofluorescent and with a vacuolar accumulation of membrane fragments, are only a subset of cells within the nodule and are generally adjacent to cells not undergoing a defense-like response. This feature of defense-like response in nip resembles the hypersensitive response, where cells undergoing programmed cell death are interspersed among living cells (Hammond-Kosack and Jones, 2000
Ethylene is a plant hormone that has been implicated in regulation of plant defense, rhizobial infections, and root growth (Penmetsa and Cook, 1997
nip plants showed an approximate 2-fold increase in nodule number as compared to wild-type plants (e.g. see Fig. 5). Similar increases in nodule number have been noted in studies with other legume and rhizobial mutants that form ineffective nodules (Dickstein et al., 1988
Nodule-specific marker genes for nodule primodia (ENOD40) and infection thread formation (MtN12) were expressed in nodulating nip root systems. Genes that are associated with nodule organogenesis (ENOD2, ENOD8) were not. Lack of ENOD2 expression in nip nodules is consistent with recent studies that showed nodule-like structures formed in M. truncatula in response to S. meliloti exo mutants are associated with an absence of ENOD2 expression (Mitra and Long, 2004
The nip nodule phenotype is similar in some respects to defective nodules elicited by other mutations in either the bacterial or legume symbiotic partners. Both S. meliloti EPS I-deficient mutants and LPS-deficient R. leguminosarum mutants induce nodule structures on alfalfa and pea, respectively, showing signs of a host defense reaction. Thickened infection thread-like structures containing rhizobia were observed in intercellular spaces in the nodule structures, with some rhizobia able to form symbiosomes, although at a higher frequency than was observed in the nip mutant (Niehaus et al., 1993
The nip nodule phenotype is also similar to that of other M. truncatula symbiotic mutants, especially the lin and Mtsym1 (TE7) mutants. lin forms nodule primordia in which infection thread development halts at the root epidermis, apparently before the blockage in nip. Similar to nip, nodulating lin root systems express genes associated with nodule primordium formation but not the ENOD2 and ENOD8 genes associated with nodule differentiation (Kuppusamy et al., 2004 Because of the diverse effects of the nip mutation on infection thread morphology, nodule differentiation, marker gene expression, polyphenolic accumulation, and lateral root growth, we speculate that NIP may have a regulatory role in root and nodule development. NIP is also required to suppress a defense-like response. It is unclear whether NIP is a direct suppressor of the defense-like response or whether the infection thread proliferation and failure to release rhizobia resulting from NIP loss of function triggers the response. The molecular identification of NIP should further elucidate the intersection between symbiotic nodule and lateral root development and may yield insight into aspects of the balance between symbiosis and host defense.
Plant Material and Growth Conditions
Medicago truncatula A17 wild-type plants were germinated and grown in aeroponic growth chambers misted with an inorganic nutrient media lacking NH4NO3 (Lullien et al., 1987 For the ethylene inhibitor and precursor studies, plant growth and nodulation studies were performed on agar plates. The same media were used with the appropriate AVG or ACC concentrations and solidified with 1% w/v phytagar (Invitrogen, Carlsbad, CA). The root portion was shaded by covering the bottom half of plates with aluminum foil. Growth conditions were the same as for aeroponic growth.
S. meliloti/pXLDG4 containing the hemA::lacZ reporter was visualized on plant roots as described (Boivin et al., 1990
For LSCM studies, nodulated roots were prepared and imaged as described (Haynes et al., 2004
Detection of polyphenolics was accomplished by staining with potassium permanganate to visualize polyphenols as described (Vasse et al., 1993
Nodules were produced aeroponically as described and harvested at 5, 10, 15, and 21 dpi. Nodulated roots were harvested into a solution of 4% formaldehyde and 1% glutaraldehyde in 80 mM PIPES (pH 7.0). Wild-type nodules were dissected from the root system and cut longitudinally to aid in infiltration. nip nodules were generally unemerged and formed in clusters, so nodulated portions of the root were cut into small segments for fixation. Nodules were vacuum infiltrated in fixative 4 x 2 min and fixed overnight at 4°C with rotation. Nodules were rinsed 3 x 15 min with distilled water, stained 4 h with 1% OsO4 (aq), rinsed 3 x 15 min with distilled water, dehydrated in a graded series of acetone, and infiltrated with a graded series of Epon Araldite resin (Mollenhauer, 1964
RNA was extracted and northern blots prepared with 20 µg RNA for each sample as previously described (Dickstein et al., 2002
Mutants were crossed into the male sterile M. truncatula A17 mutant (MtAp) as previously described (Penmetsa and Cook, 2000
We gratefully acknowledge Kate VandenBosch, in whose lab some mutant screening was performed; Colby Starker, Giles Oldroyd, Sharon Long, Kate VandenBosch, Thierry Huguet, and Jeanne Harris for seed of various M. truncatula nodulation and nitrogen-fixation mutants; Jannon Fuchs and Harry Schwark for help with the vibratome; Kate VandenBosch, Joe Clouse, and Pascal Gamas for cDNA clones; Jeanne Harris for insightful discussions; and Kirk Czymmek and Carol Carlson for assistance with confocal microscopy. Received July 8, 2004; returned for revision August 12, 2004; accepted August 23, 2004.
1 This work was supported by University of North Texas Faculty Research Funds (to R.D.), the University of Delaware Research Foundation, and a National Institutes of Health BRIN (no. RR16472–02) to the Delaware Biotechnology Institute (to D.J.S).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.049064. * Corresponding author; e-mail beccad{at}unt.edu; fax 940–565–3821.
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ABSTRACT
RESULTS
-(2-aminoethoxyvinyl)-Gly (AVG) and in the presence of the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC). The results, shown in 
