Genes induced during early response to Meloidogyne incognita in roots of resistant and susceptible alfalfa cultivars

Plant Science 161 (2001) 289– 299 www.elsevier.com/locate/plantsci

Genes induced during early response to Meloidogyne incognita in roots of resistant and susceptible alfalfa cultivars Carol Potenza a, Stephen H. Thomas b, Champa Sengupta-Gopalan a,* b

a Department of Agronomy and Horticulture, New Mexico State Uni6ersity, Las Cruces, NM 88003, USA Department of Entomology, Plant Pathology and Weed Science, New Mexico State Uni6ersity, Las Cruces, NM 88003, USA

Received 3 January 2001; received in revised form 23 March 2001; accepted 23 March 2001

Abstract A cDNA library made to RNA from roots of Meloidogyne incognita (root-knot nematode) susceptible alfalfa cv. Lahontan seedlings 72 h after root-knot nematode inoculation was differentially screened with cDNA made from uninoculated control and M. incognita infested (72 h) root RNA. Of the six cDNAs isolated, the deduced amino acid sequences of four showed significant homology to sequences present in the databank, while two were pioneer sequences. The four cDNAs with matches to known sequences include those for glycine-rich protein, the gluconeogenic pathway enzyme phosphoenolpyruvate carboxykinase, an isoflavone reductase-like protein, and metallothionein. We have followed the expression of these genes during the course of nematode infection in both the susceptible and resistant host and also in different plant organs. Based on these analyses, the genes induced early in nematode infection are related either to metabolic pathways or to stress/defense. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glycine-rich protein; Isoflavone reductase; Metallothionein; Nematode-induced genes; Phosphoenolpyruvate carboxykinase; Root-knot nematodes

1. Introduction Root-knot nematodes (RKN) are phytopathogenic obligate endoparasites that infect many plant species and cause millions of dollars of damage to agricultural crops annually [1,2]. Meloidogyne incognita, the most prevalent root-knot nematode species, infects roots as a second stage juvenile (J2) and moves intercellularly between cortical cells to the root apex. Once in the apex, the nematode enters the developing vascular cylinder and, upon reaching the zone of cell elongation, ultimately becomes sedentary within the root. At this point, one or more of the plant cells situated around the head of the J2 are stimulated by repeated stylet probing to undergo mitosis without cytokinesis and differentiate into multi-nucleated giant cells * Corresponding author. Tel.: + 1-505-6465784; fax: +1-5056466041. E-mail address: [email protected] (C. Sengupta-Gopalan).

[3,4]. The giant cells act as sinks, diverting plant nutrients to provide metabolic energy for the nematode [4]. Frequently, cortical cell division also occurs concurrently in the area immediately surrounding the sedentary nematode and the giant cells, forming the characteristic root galls or knots. Ultimately, the shunting of nutrients from the plant to the nematode reduces plant growth and crop yield. Giant cell development and gall formation are important parts of the complex physiological interaction that occurs between the plant and RKN as the parasitic relationship progresses. It has been proposed that proteins secreted by the nematode induce and maintain the feeding site [2] during the life of the nematode, while other unidentified proteins or degradation products may function as elicitors of plant resistance responses [5]. Because of the established relationship that exists between the host plant and the nematode, the susceptible

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and resistant responses of host plants have been the subject of speculation and investigation. These responses have been studied at both the morphological and molecular levels. A number of resistant responses to RKN have been observed, including: hypersensitive responses in tomato and potato [6– 9], which interfere with feeding site establishment; induction of known plant defense responses; and exclusion of the nematode from the vascular cylinder by unknown mechanisms [10]. Molecular studies have identified changes in abundance of translatable mRNA species during nematode invasion and feeding site establishment [10,11]. Numerous studies have focused on the identification of new plant genes specifically involved in susceptible and resistant interactions with sedentary endoparasitic nematodes. By differential screening of a PCR-based cDNA library, Lambert and Williamson [12] identified several cDNAs specific for nematode-infected tomato roots, one of which had sequence similarity to members of the soybean trypsin inhibitor gene family [13] and might have implications in defense against pathogens. In tobacco, RKN feeding induced expression of a gene encoding extensin, which is localized in the intercellular spaces in and around the nematode feeding site and may also have a role in defense [14]. Key plant cell-cycle markers, such as the mitotic cyclin cyc1At and the cyclin-dependent kinase cdc2a [15] are activated during establishment of the feeding site. Wilson et al. [16] found that the transcripts for the large subunit of RNA polymerase II and a plasmalemma H+-ATPase were among those exhibiting upregulated expression in giant cells of tomato [16,17]. An Arabidopsis thaliana promoter tagging strategy has identified upregulation of an enzyme involved in the pentose phosphate pathway, suggesting that nematode feeding site establishment may cause metabolic reprogramming of primary biochemical pathways [18]. A number of isolated genes are believed to be induced in response to the expansion and maintenance of giant cells, and may play a role in intracellular osmotic adjustments. These include Lemmi9 in tomato, which has sequence similarity to late embryogenesis-abundant proteins [19,20], and a tobacco gene, TobRB7, which encodes a putative water channel [21,22]. Many of the molecular studies detailing plant– RKN interactions have addressed responses in tomato and tobacco [12,14–17] and, more re-

cently, A. thaliana [18,23]. Alfalfa represents a unique system in which to examine the molecular interaction between M. incognita and its host [10]. A novel feature of the resistance response in alfalfa is that it lacks the typical hypersensitive response to RKN infection, as has been described in tobacco and tomato. We have chosen to work with an RKN-susceptible alfalfa variety, Lahontan, for isolation of plant genes whose expression is modified by RKN infection at early time points within the interaction. The focus of this paper is to identify and characterize some of the alfalfa genes that are induced in response to M. incognita infection and study their expression pattern in both susceptible and resistant alfalfa hosts during the early periods of plant–nematode interaction.

2. Materials and methods

2.1. Nematode inoculum Nematode inoculum was obtained from a population of Meloidogyne incognita (Kofoid and White) Chitwood host race 3, isolated from cotton in Dona Ana County, NM, USA and maintained in the greenhouse on Lycopersicon esculentum Mill. cv. Rutgers. Inoculum consisted of freshly hatched juveniles (J2) collected from nematode-infested tomato plants growing in aerated hydroponic cultures [24]. Freshly hatched J2 were recovered twice daily from the hydroponic culture, concentrated on a 30 mm pore size nylon sieve, and resuspended in water at 2000 nematodes/ml.

2.2. Plant material Alfalfa (Medicago sati6a) cv. Lahontan, an RKN-susceptible cultivar, and cv. Moapa 69, an RKN-resistant cultivar, were used in this study. Alfalfa cultivars were grown in Magenta boxes as previously described [10]. Ten days after alfalfa seedling emergence, the soil in each Magenta box was infested with 2×104 M. incognita J2, suspended in 10 ml of distilled water. Controls were mock-inoculated with 10 ml of water. Eight Magenta boxes of each cultivar were inoculated with J2, and eight were mock-inoculated with water. Two inoculated and two mock-inoculated boxes of each cultivar were harvested at each time point. Each time point was evaluated at least twice. The

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seedlings were then placed in a growth chamber at 28°C, as previously described [10]. Plants were harvested 24, 48, 72, and 96 h after inoculation. Roots from the inoculated and the control plants were washed free of soil, gently dried and sectioned 1– 3 cm above the growth tip, snap-frozen in liquid nitrogen, and stored at − 80°C for future use. To verify nematode infection, roots from 10 inoculated and 10 control plants from each time point were randomly removed at each harvest, washed, stained with acid fuchsin and examined microscopically for the presence of M. incognita. Alfalfa plant tissues, other than roots, were harvested from mature alfalfa cv. Malone. Tissue was immediately snap-frozen in liquid nitrogen and stored at − 80°C for future use.

2.3. Library construction and screening A cDNA library made to poly(A)+ RNA isolated from roots of alfalfa c.v. Lahontan seedlings 72 h after RKN inoculation, was constructed in lgt11 by Stratagene (La Jolla, CA). The insert size of the clones varied from 0.5 kilobases (kb) to 4.0 kb, with average insert size of 1.9 kb. The library contained 1× 107 independent clones, and was titered at 1×109 pfu/ml after a single amplification. The library (50 000 plaques) was differentially screened using two magnetically anchored cDNA libraries from uninfected control and 72 h RKN-infected root RNA. Poly(A)+ RNA from infected and control roots was bound to two separate populations of Dynabead poly(T)+ magnetic beads (DYNAL; Oslo, Norway) according to the manufacturer’s instructions. First-strand cDNA was synthesized to the bound RNA using MMLV reverse transcriptase (Stratagene), creating a cDNA library anchored to magnetic beads. Second-strand DNA was then synthesized from the solid-phase cDNA libraries by random priming accompanied by incorporation of radiolabeled 32PdCTP according to the manufacturer’s instructions (DYNAL). The labeled second strand was melted off of the bead libraries and used to differentially screen the lgt11 cDNA library. For differential screening, duplicate filters were made to the phage library growing on bacterial plates and incubated with an equal number of counts from either the control root RNA probe or the infected root RNA probe. The filters were then exposed to X-ray film, and the signal intensity of individual phage plaque

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was compared. The plaques showing differential hybridization patterns with the two probes were selected and subjected to further screening until unique phage were isolated that showed differential hybridization with the two probes.

2.4. RNA isolation and analysis Total RNA was isolated from the roots of both the susceptible and resistant host by using the LiCl precipitation procedure [25]. RNA was fractionated in 1% agarose/formaldehyde gels by standard methods [26], transferred to Hybond-N nylon membrane (Amersham; Arlington, IL), and fixed by baking at 80°C for 2 h. DNA probes were prepared from plasmid inserts isolated from agarose using GenElute Columns (Supelco; Bellefonte, PA) and labeled by the random priming method [26]. All filters were pre-hybridized for a minimum of 4 h and hybridized for 16– 20 h in 50% formamide, 5× SSC (1× SSC is 0.15 M NaCl, 0.015 M sodium citrate), 5× Denhardt’s solution, 5 mM sodium phosphate, pH 7.0, 0.1% SDS, 0.1 mg/ml of denatured herring sperm DNA at 42°C. After hybridization, the filters were washed twice with 2× SSC, 0.1% SDS at 42°C for 30 min each, followed by two washes with 0.2× SSC, 0.1% SDS for 15 min each and exposed to X-ray film. The bands were quantified using a BioImage Intelligent Quantifier. All experiments were performed three times, and only a representative experiment in each category is shown here.

2.5. Time-course analysis of RNA le6els RNA isolated from the infected or uninfected root tips of both the resistant and susceptible alfalfa at different time points following inoculation with the J2s was subjected to Northern blot analysis followed by autoradiography. The hybridization signals were quantified using the BioImage Intelligent Quantifier, and the signals were standardized against the ethidium bromide (EtBr)-stained 28S rRNA band. The values of the hybridization signal between the control uninfected sample and the infected sample for each time point were compared, and the ratio of infected versus control was plotted. Thus, a ratio of 1 would imply that the signals for the control and infected root tip were identical. Three time-course experiments were performed, and the results of a representative experiment are presented here.

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2.6. Genomic DNA blot analysis Alfalfa genomic DNA was prepared from 10 day old nematode-free susceptible and resistant cultivars according to the method described by Murray and Thompson [27]. Genomic DNA was digested to completion with HindIII, and 10 mg of DNA per lane was separated on 0.8% agarose gels, transferred to Hybond-N nylon membranes (Amersham), and fixed by baking at 80°C for 2 h. DNA probes were prepared as previously described, and prehybridization, hybridization, and washing occurred as described above.

2.7. DNA sequencing and sequence analysis Nucleotide sequences were determined by the dideoxynucleotide chain-termination method using the Licor 4200 automated sequencer and dye-labeled primers. Both strands of the cDNA clones were sequenced. Sequence similarity searches for the selected cDNA clones were carried out using NCBI non-redundant database GenBank BLASTn and BLASTx programs [28].

3. Results

3.1. Isolation and sequence analysis of cDNAs differentially expressed in control 6s. RKN-infected root tissue Thirteen differentially expressed cDNAs were chosen from the primary library screen, and six of these cDNAs that were selected for further purification exhibited differential hybridization pattern in additional rounds of library screening. These clones were selected for sequence analysis and were given the name Mia (Mi — Meloidogyne incognita; a — alfalfa) and numbered sequentially. All of these clones showed a higher level of hybridization with the probe from 72 h infected root RNA when compared to the probe from uninfected control root RNA in the differential screens. The six cDNAs were sequenced and subjected to sequence similarity searches using the NCBI database. The Mia-1 deduced amino acid sequence showed a high similarity to GenBank database sequences within a class of glycine-rich RNAbinding proteins. The second differentially selected

cDNA, Mia-2, showed high sequence similarity to plant phosphoenolpyruvate carboxykinase (PCK) sequences whereas the predicted Mia-3 amino acid sequence showed similarity to plant isoflavone reductase-like, or IRL, NAD(P)H-dependent oxidoreductases. The Mia-4 deduced amino acid sequence aligned well to plant type 1 metallothionein proteins. The translated sequences from Mia-5 and Mia-6 did not align with plant sequences within the GenBank database. Mia-5, which consisted of a 1700 base pair (bp) insert and Mia-6, which consisted of a 1100 bp insert, showed no homology at the nucleotide or amino acid sequence level to anything in the database. Initial characterization and homology profiles of the 6 cDNAs are presented inTable 1.

3.2. Expression of the differentially selected genes 72 h post-RKN inoculation To confirm the differential hybridization pattern of the Mia genes for RNA from uninfected and infected roots, the selected cDNAs were used as probes in Northern blot hybridization on RNA from infected and uninfected roots. The hybridization pattern for the differentially selected genes, Mia-1–Mia-4, in the infected and uninfected roots of the RKN susceptible and resistant alfalfa plants is shown in Fig. 1. Mia-1, Mia-2, and Mia-3 all showed increased hybridization to RNA from RKN-inoculated susceptible and resistant alfalfa compared to controls. The hybridization pattern Table 1 Gene identification of Mia cDNAs Clone name Gene identification (GenBank c) Mia-1 (AF191305) Mia-2 (AF212109) Mia-3 (AF201458) Mia-4 (AF189766)

Mia-5 Mia-6

Postulated function

Glycine-rich RNA maturation RNA-binding protein Phosphoenolpyruvate First enzyme in carboxykinase gluconeogenic pathway Isoflavone NAD(P) H-dependent reductase-like oxidoreductase protein Metallothionein-like Heavy metal protein tolerance; multiple stress-related function Pioneer Unknown Pioneer Unknown

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Fig. 1. RNA expression patterns of the differentially selected cDNAs in susceptible and resistant alfalfa roots ( −) and ( + ) RKN at 72 h post-inoculation. Total RNA was isolated 72 h post-inoculation from alfalfa roots mock-inoculated or inoculated with J2 of RKN. Twenty micrograms of total RNA were size-separated on a 1% MOPS formaldehyde agarose gel and transferred to nylon. The blot was sequentially hybridized with 32P-labeled Mia probes (as indicated) and autoradiographed. The size of the hybridizing bands was determined by including standard molecular weight markers in the same gel. The hybridization patterns were measured using BioImage Intelligent Quantifier (B.I. Systems Corp.), standardized based on ethidium bromide staining of the rRNA bands, and presented as bar graphs of integrated intensities on the right. Susceptible alfalfa (susc.); resistant alfalfa (res.); control roots (con.); inoculated roots (inoc).

of Mia-4 did not differ significantly with RKN inoculation in resistant plants, but showed increased expression in RKN-inoculated susceptible plants. Mia-5 and Mia-6 did not show any detectable hybridization signal when total RNA was used on RNA blots, even after prolonged exposure of the autoradiograms. We did not pursue using poly(A+ ) RNA for Northern blot hybridization with the Mia-5 and Mia-6 clones as probes because of the prohibitively large amount of tissue necessary for isolation of poly(A+ ) RNA.

3.3. Expression pattern of Mia genes during the early periods of susceptible and resistant plant interaction with RKN Since Mia-1, Mia-2, Mia-3, and Mia-4 showed a differential expression pattern at the 72 h time point between infected and control roots in one or

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both of the susceptible and resistant plants, the expression pattern of these genes was studied over a longer period of time following infection. A correlation in the hybridization pattern with specific events in the infection process could shed light on the possible role of these Mia genes. The hybridization signals to RNA from infected roots were compared to the signal with the RNA from control roots at the different points with the different Mia probes, and the ratios were plotted as shown in Fig. 2. Hybridization signal analysis for Mia-1 in the susceptible host showed a threefold increase in the signal ratio, which peaked at 48 h followed by a drop to the basal level by 96 h. Under similar conditions, the ratios obtained for the resistant host remained around 2 for the entire experimental period. Mia-2 showed almost identical hybridization patterns for both the susceptible and the resistant hosts (Fig. 2). The ratio gradually increased until it peaked at 72 h, after which the ratio decreased. The decrease was more significant for the susceptible host than the resistant host. Mia-3 showed a somewhat different profile between the susceptible and resistant hosts (Fig. 2). While the ratio in the susceptible host remained constant for all the time points except for a slight increase at 72 h post-infection, the ratio in the resistant host increased dramatically after the 48 h time point through the end of the experimental period. Mia-4 expression in infected roots of the resistant host showed a small but consistent decrease from the control during the entire 96 h experimental period (Fig. 2). This was very different from the Mia-4 expression pattern in infected susceptible roots, which peaked at the 72 h timepoint (twofold higher) and dropped back to the control level by 96 h. Taken together, the results suggest that Mia-1, Mia-2, Mia-3, and Mia-4 show changes in the expression pattern during the first 96 h following infection with RKN, and these changes may reflect specific functional responses in the alfalfa–RKN interaction.

3.4. Transcript abundance of Mia genes in different organs of the alfalfa plant Since it has been postulated that a plant– parasite interaction may involve recruitment of fundamental plant systems and biochemical pathways, the expression pattern of the Mia genes in different plant organs was investigated. RNA isolated

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from different plant parts were subjected to Northern blot analysis using the different Mia cDNAs as probes (Fig. 3). Mia-1 showed hybridization to RNA from all organs except the seeds, with the highest hybridization signal occurring for the control roots. Longer film exposures

Fig. 3. RNA expression patterns of the Mia genes. Total RNA was isolated from selected alfalfa tissues, and 10 mg of the RNA were size-separated on a 1% MOPS formaldehyde agarose gel. After transfer of the RNA to nylon membrane, the membrane was sequentially hybridized with each of the selected 32P-labeled Mia cDNAs and autoradiographed. Ethidium bromide staining of the 28S ribosomal RNA is shown for loading comparison. Lanes: pod (p), bud (b), flower (fl), nodule (n), leaf (l), hypocotyl (hy), cotyledon (c), seed (s), epicotyl (e), control root (r).

showed low but detectable levels of transcript present in the seed (data not shown). Mia-2 showed selective hybridization with the strongest signals for cotyledon and nodule RNA. The root and pod showed some hybridization, whereas the leaves, flowers and buds showed faint hybridization on longer exposure of the autoradiogram (data not shown). Transcripts hybridizing to Mia3 showed a more ubiquitous distribution, though the levels were fairly low in the cotyledons and seeds. The highest level of expression was in the pod, followed by the root. Mia-4 showed the highest level of expression in the roots but showed some expression pattern in all plant organs examined.

Fig. 2. Expression pattern of the Mia genes over the course of 96 h post-inoculation. RNA (20 mg) isolated from inoculated and uninoculated roots of both the resistant and susceptible alfalfa cultivars at the times indicated were subjected to quantitative Northern blot analysis using the Mia cDNAs as probes. The blot was also probed with a 28S ribosomal RNA (rRNA) gene for load standardization. The hybridizing bands were quantified using BioImage Intelligent Quantifier and standardized against the rRNA band. The ratio of the signal for the infected versus the uninfected control root RNA was plotted. Each experiment was performed at least twice, and control measurements were normalized to 1. Susceptible: ; resistant: 2.

3.5. Analysis of genomic DNA for the Mia genes in RKN susceptible and resistant alfalfa culti6ars Genomic Southern blot analysis was performed to estimate the number of genes for each of the four Mia cDNAs from above. Genomic DNA isolated from both the resistant and susceptible cultivars was digested with HindIII and subjected to Southern blot hybridization. The pattern of hybridization obtained with a 500 bp EcoRI fragment from the Mia-1 cDNA clone, which includes the start of translation and N-terminal conserved

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coding region, was similar in the susceptible and resistant alfalfa cultivars. Both cultivars showed two major and several minor hybridizing bands (Fig. 4). A Mia-2 700 bp 5% EcoRI fragment, which includes the 5% untranslated region and the start of translation, when used as a probe, showed a less complex pattern of hybridization compared to the profile obtained with Mia-1 (Fig. 4). Only one major band of 11.0 kb and a minor band of 8.1 kb hybridized in both cases. Mia-3 hybridized to two bands of molecular weight 5.5 and 4.5 kb in both cultivars (Fig. 4). These results suggest that these three Mia genes are encoded by members of small gene families. Mia-4 showed the most complex pattern of hybridization in both the susceptible and resistant alfalfa cultivars and the patterns showed major differences between the two cultivars (Fig. 4). The complete coding region, including the 5% and 3% untranslated regions, were used for Southern analysis. The results for Mia-4 suggest a large, multigene family for metallothioneins in the alfalfa cultivars analyzed. Genomic Southern blot analysis for the Mia genes was also performed with alfalfa DNA digested with other restriction enzymes (data not shown), essentially confirming that Mia-1, 2 and 3 are encoded by small gene families and Mia-4 by a relatively larger gene family. Neither Mia-5 nor Mia-6 hybridized to plant genomic DNA (data not shown), suggesting that these are not plant genes and may represent nematode genes.

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4. Discussion We have cloned and studied six cDNAs that showed a differential hybridization pattern between the probes made to total RNA from control and infected alfalfa roots when screened against a cDNA library made to RNA from infected roots of the susceptible host. After sequencing, the Mia clones were identified by GenBank sequence analysis, and four were found to be similar to genes with known functions in other processes unrelated to nematode/plant interaction. The four genes, Mia-1 through Mia-4, aligned most closely to plant genes and could be classified as: (1) defense or stress-related genes and (2) genes related to metabolic pathways. Two of the sequences, Mia-5 and Mia-6, though selected based on differential hybridization from the cDNA library to RNA from infected roots, did not hybridize to alfalfa root RNA or to alfalfa genomic DNA. They also did not align with any significance to any sequences in the GenBank Database. We speculate that Mia-5 and Mia-6 may represent RNA of nematode origin that was included in the cDNA library. The Mia-1 deduced amino acid sequence has a strong similarity to glycine-rich RNA-binding proteins (RBPs) in plants. The amino terminus of the Mia-1 sequence contains the two well-defined RNA-binding domains, Ribonucleoprotein (RNP) consensus-1 and -2 [29]. Because RNA usually exists as a complex with proteins forming ribonu-

Fig. 4. Genomic Southern analysis of the selected Mia genes. Genomic DNA was isolated from both nematode susceptible and resistant alfalfa. Restriction enzyme digestion was performed using HindIII, and 10 mg of the digested genomic DNA was size-separated on a 0.8% agarose gel. The fractionated DNA was transferred to nylon membrane and probed with 32P-labeled Mia gene probes as indicated. The molecular weight for each hybridizing fragment is shown. SH: susceptible, HindIII; RH: resistant, HindIII.

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cleoprotein particles, RNA-processing proteins play an essential role in RNA maturation and may have an important role in gene regulation [30,31]. RBP exhibits a high affinity for single-stranded nucleotides [32]. RBP transcript has been shown to be induced by light- and temperature-entrained circadian clocks in Sinapis alba and A. thaliana [33,34], by abscisic acid or water stress in maize [35], by acute hypersensitive response in tobacco [36], and by wounding in carrot [37]. Mia-1 gene(s) appeared to be strongly induced with nematode infection in both the susceptible and resistant alfalfa roots. While high levels of the transcript were maintained throughout the 96 h following infection in the resistant host, the level peaked at 48 h in the susceptible host and then gradually diminished to control levels at 96 h post-inoculation. Mia-1 induction in alfalfa may result from non-specific wounding by nematodes during the infection process since the expression of the gene is altered in both alfalfa cultivars, and prior studies in carrot and tobacco have shown wound and defense-related induction of the glycine-rich RNA-binding proteins [36,37]. However, since the drop in the level of Mia-1 transcript coincides with the entry of the nematodes into the vascular tissue in the susceptible host, it is possible that the expression of this gene is related to the presence of the nematodes in the intercellular spaces of the cortical cells in the root tips of both the resistant and susceptible host. The nucleotide and deduced amino acid sequences of Mia-2 best resemble the phosphoenolpyruvate carboxykinase (PCK) cDNAs from different plants [38–40]. PCK is an ATP-dependent enzyme and the first enzyme in the gluconeogenic pathway in plants, a pathway that allows the synthesis of sugars from three-carbon compounds. PCK, a cytosolic enzyme, catalyzes the conversion of malate into phosphoenolpyruvate (PEP) via oxaloacetate in lipid-mobilizing tissues. Enzymes within the gluconeogenic pathway then convert PEP into sugars for use in starch synthesis. Mia-2 is most highly expressed in cotyledons where PCK, as part of the gluconeogenic pathway, is active in the conversion of stored lipids into sugar to support growth until photosynthesis begins. In cotyledons, sucrose is the major end product of this pathway. Most interestingly, Mia-2 appears to be expressed very highly in alfalfa root-nodules and may have a role related to the storage of excess

phloem sugars as starch for use in maintaining the bacteroids. In a plant–nematode system, Jones and Payne [41] reported accumulation of starch in tomato gall cells and syncytial cells due to infection by the false root-knot nematode, Nacobbus aberrans. The induction of Mia-2 by M. incognita infection of a plant is only the second report of the modification in expression of a gene encoding an enzyme in a primary metabolic pathway by nematodes. Using promoter trapping in A. thaliana, Favery et al. [18] isolated a gene encoding for D-ribulose-5-phosphate-3-epimerase (RPE), a key enzyme in the oxidative pentose phosphate pathway, which they proposed to be essential to nematode feeding site initiation. In A. thaliana, GUS-tagged plants infected with M. incognita showed that the RPE promoter activated the GUS transgene 24–48 h after giant cell initiation [18]. In alfalfa, the expression of Mia-2 peaks at 72 h post-infection, probably at a stage preceding the initiation of giant cells in the susceptible host. The induction of Mia-2 in resistant plants suggests that sugar-related metabolic pathways may be recruited without specificity in stressed plants, or metabolic reprogramming by the nematode may begin as soon as the nematode enters the root in both the susceptible and resistant plants. A search of the GenBank database showed that the deduced amino acid sequence of Mia-3 shares considerable similarity with isoflavone reductaselike protein (IRL) and isoflavone reductases (IFRs) from legumes [42,43]. IFRs are cytosolic, NAD(P)H-dependent oxidoreductases found in legumes that participate in the production of the defense-related molecules, phytoalexins [43]. IRL genes studied include a potato homologue that is expressed during pollen tube growth and can be induced in the pistils by touch [44], oxidative stress in A. thaliana [42], UV irradiation, wounding and pathogen infection in grapefruit [45], and sulfur starvation in maize [46]. Unlike the function of the leguminous IFRs, the function of IRLs is not clear. Suggestions for the function of these genes can be deduced from the study of their expression and from the presence of the putative NAD(P)Hbinding domain. This domain suggests that these proteins are all oxidoreductases, and, although diverse, the expression pattern suggests a role in defense against oxidative stress [42,44,46] with possible involvement in the production of antioxi-

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dant protectants. Such a role in defense against oxidative stress could also apply to nematode infection of roots because oxygen radicals are natural by-products of wounding and defense reactions [47,48]. Also, the induction of antioxidants such as catalases during nematode–plant interaction has been described [49], and it is likely that Mia-3 functionally can be grouped along with catalase and peroxidase. Prolonged induction of Mia-3 in the resistant host would strongly suggest a defense function. Genomic Southern analysis suggests that Mia-3 hybridizes to members of a small gene family in alfalfa, with two or three members. Because Mia-3 appears to be constitutively expressed in most plant parts, it may also serve a more general function than defense or response to wounding. Understanding the very high expression of Mia-3 in pod compared to root tissue may help explain the function of this gene with regards to nematode infection. The sequence of Mia-4 aligned strongly with the genes encoding metallothionein-like (MT) proteins from plants [50]. These genes are characterized by highly conserved arrays of cysteine residues at the C and N termini. Whereas inducible responses to heavy metals have been found for some plant MT-like genes, including two isoforms of MT-like genes in A. thaliana [51] and an MT-like gene in Triticum aesti6um L. [52], MT genes have been shown to be induced by senescence [53] and due to infection of A. thaliana with incompatible strains of Peronospora parasitica and Pseudomonas syringae [54]. Whereas sequestration of metal ions released due to macromolecular degradation during senescence and hypersensitive responses in incompatible reactions (with pathogens) is a viable function for MT-like genes in plants, it may be more likely that metallothioneins are synthesized as protectants against the effects of oxidative damage. It has been proposed that MTs in animal tissues have a role in protection against the effects of reactive oxygen species by acting as antioxidants since MTs are potent scavengers of hydroxyl radicals [55,56]. Mia-4, like Mia-3 and catalases [49], may play a role in scavenging reactive oxygen species resulting from the interaction between the nematodes and the susceptible host. Although Mia-4 was selected based on differential screening in the RKN infected system, Northern analysis at the selected time points did not show a clear

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picture of induction of this gene in the system. Since the roots showed the highest level of expression of Mia-4 gene(s), it is possible that any localized and small increase in the level of Mia-4 transcripts in the infected roots of the susceptible host remains undetected due to the high basal levels of the transcript in the uninfected roots. The importance of characterizing differentially expressed genes within plant– pathogenic nematode systems must be emphasized. Although real progress has been made in understanding specific single genes involved in gene-for-gene recognition in many plant–parasite models, most interactions do not fall into this category. Instead, the relationships between a host and its various parasites are more complicated, involving interactions that have evolved over time, and involving an intricate array of genes. Whereas only four members of an induced pool of host genes in a plant/M. incognita system are presented here, data are beginning to accumulate that may give us a better understanding of the plant– parasitic nematode interaction and show us how we can control this important agricultural pest. Future work will focus on localizing the site of induction of these genes at a cellular level.

Acknowledgements The research described here was funded by grant from the Southwest Consortium for Plant Genetics and by the Agricultural Experiment Station at New Mexico State University.

References [1] J.N. Sasser, Root-knot nematodes: a global menace to crop production, Plant Dis. 64 (1980) 36 –41. [2] V.M. Williamson, R.S. Hussey, Nematode pathogenesis and resistance in plants, Plant Cell 8 (1996) 1735 – 1745. [3] U. Wyss, F.M.W. Grundler, A. Munch, The parasitic behavior of 2nd-stage juveniles of Meloidogyne incognita in the roots of Arabidopsis thaliana, Nematologica 38 (1992) 98 –111. [4] M.A. McClure, Meloidogyne incognita: a metabolic sink, J. Nematol. 9 (1977) 88 –90. [5] V.H. Dropkin, The necrotic reaction of tomatoes and other hosts resistant to Meloidogyne: reversal by temperature, Phytopathology 59 (1969) 1632 –1637. [6] S.W. Hutchenson, Current concepts of active defense in plants, Annu. Rev. Phytopathol. 36 (1998) 59 – 90.

298

C. Potenza et al. / Plant Science 161 (2001) 289–299

[7] D.T. Kaplan, N.T. Kee, Mechanisms conferring plant incompatibility to nematodes, Revue de Nematologie 3 (1980) 123 –134. [8] R.E. Paulson, J.M. Webster, Ultrastructure of the hypersensitive resction in roots of tomato, Lycopersicon esculentum L., to infection by the root-knot nematode, Meloidogyne incognita, Physiol. Plant Pathol. 2 (1992) 227–234. [9] R.M. Edens, S.C. Anand, R.I. Bolla, Enzymes of the phenylpropanoid pathway in soybean infected with Meloidogyne incognita or Heterodera glycines, J. Nematol. 27 (1995) 292 –303. [10] C.L. Potenza, S.T. Thomas, E.A. Higgins, C. SenguptaGopalan, Early root response to Meloidogyne incognita in resistant and susceptible alfalfa cultivars, J. Nematol. 28 (1996) 475 – 484. [11] K.E. Hammond-Kosack, H.J. Atkinson, D.J. Bowles, Changes in abundance of translatable mRNA species in potato roots and leaves following root invasion by cystnematode G. rostochiensis pathotypes, Biol. Mol. Plant Path. 37 (1990) 339 –354. [12] K.N. Lambert, V.M. Williamson, cDNA library construction from small amounts of RNA using paramagnetic beads and PCR, Nucleic Acid Res. 21 (1993) 775 –776. [13] C.A. Ryan, Protease inhibitors in plants: genes for improving defenses against insects and pathogens, Annu. Rev. Phytopathol. 28 (1990) 425 – 449. [14] A. Niebel, J. De Almeida Engler, C. Tire, G. Engler, M. Van Montagu, G. Gheyson, Induction patterns of an extensin gene in tobacco upon nematode infection, Plant Cell 5 (1993) 1697 –1710. [15] A. Niebel, J. de Almeida-Engler, A. Hemerly, P. Ferreira, D. Inze, M. Van Montagu, G. Gheysen, Induction of cdc2a and cycAt expression in Arabidopsis thaliana during early phases of nematode-induced feeding cell formation, Plant J. 10 (1996) 1037 –1043. [16] M.A. Wilson, D.M. Bird, E. van der Knaap, A comprehensive subtractive cDNA cloning approach to identify nematode-induced transcripts in tomato, Phytopathology 84 (1994) 299 –303. [17] D.M. Bird, M.A. Wilson, DNA sequence and expression analysis of root-knot nematode-elecited giant cell transcripts, Mol. Plant –Microbe Interact. 7 (1994) 419 –424. [18] B. Favery, P. Lecomte, N. Gil, N. Bechtold, D. Bouchez, A. Dalmasso, P. Abad, RPE, a plant gene involved in early developmental steps of nematode feeding cells, EMBO J. 17 (1998) 6799 –6811. [19] G.A. Galau, H.Y.-C. Wang, D.W. Hughes, Cotton Lea5 and Lea14 encode atypical late embryogenesis-abundant proteins, Plant Physiol. 101 (1993) 695 –696. [20] W. Van der Eycken, J. de Almeida Engler, D. Inze, M. Van Montagu, G. Gheysen, A molecular study of rootknot nematode-induced feeding sites, Plant J. 9 (1996) 45– 54. [21] Y.T. Yamamoto, C.G. Taylor, G.N. Acedo, C.L. Cheng, M.A. Conkling, Characterization of cis-acting sequences regulating root-specific gene expression in tobacco, Plant Cell 3 (1991) 371 –382. [22] C.H. Opperman, C.G. Taylor, M.A. Conkling, Root-knot nematode-directed expression of a plant root-specific gene, Science 263 (1994) 221 –223.

[23] P.C. Sijmons, F.M.W. Grundler, N. von Mende, P.R. Burrows, U. Wyss, Arabidopsis thaliana as a new model host for plant-parasitic nematodes, Plant J. 1 (1991) 245–254. [24] K.N. Lambert, E.C. Tedford, E.P. Caswell, V.M. Williamson, A system for continuous production of root-knot nematode juveniles in hydroponic culture, Phytopathology 82 (1992) 512 – 515. [25] S.C. De Vries, J. Springer, J.G.H. Wessels, Diversity of abundant mRNA sequences and patterns of protein sunthesis in etiolated and greened pea seedlings, Planta 156 (1982) 129 – 135. [26] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [27] M.G. Murray, W.F. Thompson, Rapid isolation of high molecular weight plant DNA, Nucleic Acids Res. 8 (1980) 4321– 4325. [28] S.F. Altschul, W. Fish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403 –410. [29] G. Dreyfuss, M.S. Swanson, S. Pinol-Roma, Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation, Trends Biochem. Sci. 13 (1988) 86– 91. [30] M.M. Alba, F.A. Culianez-Macia, A. Goday, M.A. Freire, B. Nadal, M. Pages, The maize RNA-binding protein, MA16, is a nucleolar protein located in the dense fibrillar component, Plant J. 6 (1994) 825 –834. [31] S. Hanano, M. Sugita, M. Sugiura, Isolation of a novel RNA-binding protein and its association with a large ribonucleoprotein particle present in the nucleoplasm of tobacco cells, Plant Mol. Biol. 31 (1996) 57 – 68. [32] M.A. Freire, M. Pages, Functional characteristics of the maize RNA-binding protein MA16, Plant Mol. Biol. 29 (1995) 797 –807. [33] C.D. Carpenter, J.A. Kreps, A.E. Simon, Genes encoding glycine-rich Arabidopsis thaliana proteins with RNAbinding motifs are influenced by cold treatment and an endogenous circadian rhythm, Plant Physiol. 104 (1994) 1015–1025. [34] C. Heintzen, M. Siegbert, R. Fischer, S. Kappeler, K. Apel, D. Staiger, A light- and temperature-entrained circadian clock controls expression of transcripts encoding nuclear proteins with homology to RNA-binding proteins in meristematic tissue, Plant J. 5 (1994) 799 – 813. [35] J. Gomez, D. Sanchez-Martinez, V. Steifel, J. Rigau, P. Puigdomenech, M. Pages, A gene induced by plant hormone abscisic acid in response to water stress encodes a glycine-rich protein, Nature 334 (1988) 262 – 264. [36] S.M.S. Naqvi, K.-S. Park, S.-Y. Yi, H.-W. Lee, S.H. Bok, D. Choi, A glycine-rich RNA-binding protein gene is differentially expressed during acute hypersensetive response following Tobacco Mosaic Virus infection in tobacco, Plant Mol. Biol. 37 (1998) 571 – 576. [37] A. Sturm, A wound-inducible glycine-rich protein from Daucus carota with homolgy to single-stranded nucleic acid-binding proteins, Plant Physiol. 99 (1992) 1689 – 1692.

C. Potenza et al. / Plant Science 161 (2001) 289–299

[38] K. Dae-Jae, S.M. Smith, Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression, Plant Mol. Biol. 26 (1994) 423–434. [39] J. Saez-Vasquez, M. Raynal, M. Delseny, A rapeseed cold-inducible transcript encodes a phosphoenolpyruvate carboxykinase, Plant Physiol. 109 (1995) 611 –618. [40] P.M. Finnegan, J.N. Burnell, Isolation and sequence analysis of cDNAs encoding phosphoenolpyruvate carboxykinase from the PCK-type C4 grass Urochloa panicoides, Plant Mol. Biol. 27 (1995) 365 –376. [41] M.G. Jones, H.L. Payne, The structure of syncytia induced by the phytoparasitic nematode Nacobbus aberrans in tomato roots, and the possible role of plasmodesmata in their nutrition, J. Cell Sci. 23 (1977) 299–313. [42] E. Babiychuk, S. Kushnir, E. Belles-Boix, M. Van Montagu, D. Inze, Arabidopsis thaliana NADPH oxidoreductase homologs confer tolerance of yeasts toward the thiol-oxidizing drug diamide, J. Biol. Chem. 270 (1995) 26224–26231. [43] N.L. Paiva, R. Edwards, Y. Sun, G. Hrazdina, R.A. Dixon, Stress responses in alfalfa (Medicago sati6a L.) 11. Molecular cloning and exression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis, Plant Mol. Biol. 17 (1991) 653 –667. [44] G.J. van Eldik, R.K. Ruiter, P.H.W.N. Colla, M.M.A. van Herpen, J.A.M. Schrauwen, G.J. Williams, Expression of an isoflavone reductase-like gene enhanced by pollen tube growth in pistils of Solanum tuberosum, Plant Mol. Biol. 33 (1997) 923 –929. [45] A. Lers, S. Burd, E. Lomaniec, S. Droby, E. Chalutz, The expression of a grapefruit encoding an isoflavone reductase-like protein is induced in response to UV irradiation, Plant Mol. Biol. 36 (1998) 847 – 856. [46] S. Petrucco, A. Bolchi, C. Foroni, R. Percudani, G.L. Rossi, S. Ottonello, A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reduc-

.

[47]

[48]

[49]

[50] [51]

[52]

[53]

[54]

[55]

[56]

299

tases is activated in response to sulfur starvation, Plant Cell 8 (1996) 69 – 80. A. Levine, R. Tenhaken, R. Dixon, C. Lamb, H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response, Cell 79 (1994) 583 – 593. M. Orozco Cardenas, C.A. Ryan, Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway, Proc. Natl. Acad. Sci. 96 (1999) 6553 – 6557. A. Niebel, K. Heungens, N. Barthels, D. Inze, M. Van Montagu, G. Gheysen, Characterization of a pathogeninduced potato catalase and its systemic expression upon nematode and bacterial infection, Mol. Plant –Microbe Interact. 8 (1995) 371 –378. N.J. Robinson, A.M. Tommey, C. Kuske, P.J. Jackson, Plant metallothioneins, Biochem. J. 295 (1993) 1 – 10. J. Zhou, P.B. Goldsborough, Functional homologs of fungal metallothionein genes from Arabidopsis, Plant Cell 6 (1994) 875 –884. K.C. Snowden, R.C. Gardner, Five genes induced by aluminum in wheat (Triticum aesti6um L.) roots, Plant Physiol. 103 (1993) 855 –861. V. Buchanan-Wollaston, C. Ainsworth, Leaf senescence in Brassica napus: cloning of senescence related genes by subtractive hybridisation, Plant Mol. Biol. 33 (1997) 821–834. A. Butt, C. Mousley, K. Morris, J. Beynon, C. Can, E. Holub, J.T. Greenberg, V. Buchanan-Wollaston, Differential expression of a senescence-enhanced metallothionein gene in Arabidopsis in response to isolates of Peronospora parasitica and Pseudomonas syringae, Plant J. 16 (1998) 209 –221. L. Chubatsu, R. Meneghini, Metallothionein protects DNA from oxidative damage, Biochem. J. 291 (1993) 193–198. T. Muira, S. Muraoka, T. Ogiso, Antioxidant activity of metallothionein compared with reduced glutathione, Life Sci. 60 (1997) 301 –309.