Parasitism of the cyst nematode Heterodera schachtii is characterized by the formation of syncytial feeding structures in the host root. Syncytia are formed by the fusion of root cells, accompanied by local cell wall degradation, fusion of protoplasts and hypertrophy. Expansins are cell wall-loosening proteins involved in growth and cell wall disassembly. In this study, we analysed whether members of the expansin gene family are specifically and developmentally regulated during syncytium formation in the roots of Arabidopsis thaliana. We used PCR to screen a cDNA library of 5–7-day-old syncytia for expansin transcripts with primers differentiating between 26 α- and three β-expansin cDNAs. AtEXPA1, AtEXPA3, AtEXPA4, AtEXPA6, AtEXPA8, AtEXPA10, AtEXPA15, AtEXPA16, AtEXPA20 and AtEXPB3 could be amplified from the library. In a semi-quantitative RT-PCR and a Genechip analysis AtEXPA3, AtEXPA6, AtEXPA8, AtEXPA10 and AtEXPA16 were found to be upregulated specifically in syncytia, but not to be transcribed in surrounding root tissue. Histological analyses were performed with the aid of promoter::GUS lines and in situ RT-PCR. Results from both approaches supported the specific expression pattern. Among the specifically expressed genes, AtEXPA3 and AtEXPA16 turned out to be of special interest as they are shoot-specific in uninfected plants. We conclude that syncytium formation involves the specific regulation of expansin genes, indicating that the encoded expansins take part in cell growth and cell wall disassembly during syncytium formation.
Sedentary plant-parasitic cyst nematodes of the genera Heterodera and Globodera cause substantial damage to a variety of crop plants such as soybean, potato, sugar beet and wheat. After root penetration the nematodes induce changes in the vascular cylinder and in the entire system of water, mineral and assimilate transport (Grundler and Böckenhoff, 1997). Infective second-stage juveniles invade the roots of host plants where they induce a syncytial feeding structure. Syncytium formation is supposed to be triggered by secretions released through the nematode stylet into a single initial root cell, which then fuses with neighbouring cells – a process that requires local cell wall dissolutions and subsequent fusion of the protoplasts (Golinowski et al., 1996; Wyss and Grundler, 1992). In Arabidopsis, which has also been successfully established as a model plant also in plant nematology (Sijmons et al., 1991), Heterodera schachtii induces syncytia within the root central cylinder. The cell walls of syncytia undergo remarkable changes, which were described by Golinowski et al. (1996) and Grundler et al. (1998). With syncytium formation the outer syncytial cell walls are expanded and thickened, whereas at the interface to xylem vessels elaborate cell wall ingrowths are formed. Syncytia develop by the fusion of root cells through partial cell wall degradation. Openings between syncytial elements are formed via two different mechanisms: at the beginning of syncytium development, plasmodesmata between the initial cell and neighbouring cells are widened by gradual cell wall dissolution; in more advanced syncytia, the affected cell walls between two cells expand and become bent before being degraded without the involvement of plasmodesmata (Grundler et al., 1998). This process of cell fusion and cell expansion is suggested to be based on the activity of cell wall-modifying agents such as hydrolytic enzymes and expansins.
Grundler et al. (1998) gave indirect evidence for cell wall-degrading enzymes within developing syncytia by detecting the precipitations of liberated reducing sugars close to cell wall openings. Goellner et al. (2001) found five β-1,4-endoglucanases (NtCel2, NtCel4, NtCel5, NtCel7 and NtCel8) to be upregulated in tobacco roots upon infection by both the tobacco cyst nematode (Globodera tabacum) and the root-knot nematode (Meloidogyne incognita). Root-knot nematodes induce giant cells as feeding structures within a root gall. Using differential display Vercauteren et al. (2002) identified DiDi 9C-12, a putative pectin acetylesterase homolog that was found to be upregulated in syncytia and giant cells. Mitchum et al. (2004) infected transgenic tobacco and Arabidopsis plants carrying an AtCel1::GUS construct with H. schachtii (Arabidopsis), G. tabacum (tobacco) and M. incognita, and found that the AtCel1 promoter was activated only at the beginning of giant-cell formation. Using genome-wide expression profiling, Jammes et al. (2005) found seven EXPA and two EXPB to be upregulated in galls induced by M. incognita in the roots of Arabidopsis. Golecki et al. (2002) reported on the upregulation of the tomato expansin gene LeEXP5 in syncytia induced by Globodera rostochiensis. Using microarray technology, quantitative RT-PCR and in situ localization LeEXP5 was recently also found in gall cells adjacent to the feeding site induced by Meloidogyne javanica in the tomato (Gal et al., 2005).
Expansins were first identified more than a decade ago as the key cell wall factors responsible for ‘acid growth’ (McQueen-Mason et al., 1992). Characteristically, expansins induce cell wall extension at an acidic pH optimum in vitro, and enhance stress relaxation of isolated cell walls over a broad time range (Cosgrove, 2000a,b). They comprise two major gene families: α-expansins (EXPA) and β-expansins (EXPB) (Kende et al., 2004). EXPA proteins bind tightly to cellulose and hemicellulose, but they have no hydrolytic activity against these major polysaccharides of the cell wall (McQueen-Mason and Cosgrove, 1995). It has been proposed that expansins disrupt non-covalent bonding between cellulose microfibrils and matrix glucans, thereby allowing turgor-driven slippage of microfibrils relative to one another. Comparable studies of EXPB binding and hydrolytic activity have not yet been published, but their wall-loosening action is similar to that of EXPA (Cosgrove et al., 1997).
In Arabidopsis, EXPA proteins are encoded by a subfamily of 26 genes with 52–99% amino acid sequence identity. The EXPB subfamily is smaller, with five genes (six in some Arabidopsis ecotypes; http://www.bio.psu.edu/expansins). In addition, Arabidopsis has two related groups of genes that have been named expansin-like family A and B (EXPLA and EXPLB, respectively). Their functions, however, have not yet been ascertained.
Recently, it was shown that nematodes secrete proteins with sequence similarity to expansins (Kudla et al., 2005; Qin et al., 2004). Nematode secretions containing these and other cell wall-loosening proteins may assist the rapid penetration of the nematode into the root tissues. However, the highly orchestrated patterns of altered cell growth and syncytium formation would seem to require more subtle spatial and temporal control of cell wall loosening and growth processes that could not be achieved through nematode secretion alone.
In this study, we investigated whether the expression of members of the expansin gene family are specifically and developmentally regulated during syncytium formation in roots of Arabidopsis thaliana. In fact, our results demonstrate highly specific expression and implicate a substantial role of certain expansins in the cell wall re-organization occurring in the host response to cyst-forming nematodes.
Expansins are differentially expressed in shoots and roots of uninfected control plants
In order to get a basic overview of the distribution of expansin gene expression in uninfected plants, shoots were separated from roots, and each sample was taken to perform RT-PCR reactions. Primer pairs were designed for 26 AtEXPA genes (AtEXPA1–26) and three AtEXPB genes (AtEXPB1–3; see Supplementary Material. RT-PCR with total RNA isolated from shoots and roots of 21-day-old A. thaliana plants showed that most isoforms are expressed in both shoots and roots. Only AtEXPA3, AtEXPA5 and AtEXPA16 were found exclusively in the shoot, and AtEXPA18 was found exclusively in the root. For AtEXPA13, AtEXPA14, AtEXPA21, AtEXPA22, AtEXPA23, AtEXPA24, AtEXPA25, AtEXPA26 and AtEXPB2 no products could be detected, suggesting that these isoforms are expressed neither in the shoot nor in the root at the selected plant developmental stage (Table 1).
Table 1. Results obtained for PCR reactions with the syncytium-specific cDNA library and for RT-PCR reactions with total RNA isolated from Arabidopsis shoots and roots
cDNA library of syncytial cytoplasm
+, PCR product was detected; −, no PCR product detected.
Expansin genes are expressed in nematode-induced syncytia
Expansin gene expression in syncytia was determined using a syncytium-specific cDNA library from 5–7-day-old syncytia induced by H. schachtii. This library was made from micro-aspirated syncytial cytoplasm. The quality and specificity of this library has been evaluated with different genes (e.g. Atpyk20) known to be expressed specifically in syncytia (Jürgensen et al., 2003; Puzio et al., 2000). In PCR reactions with primers differentiating between the 26 EXPA and three EXPB genes, transcripts of nine different AtEXPA genes (AtEXPA1, AtEXPA3, AtEXPA4, AtEXPA6, AtEXPA8, AtEXPA10, AtEXPA15, AtEXPA16 and AtEXPA20) and AtEXPB3 were amplified (Table 1). Compared with the expression pattern in the uninfected plants, the expansin genes detected in the syncytium can be divided into two groups: AtEXPA1, AtEXPA4, AtEXPA6, AtEXPA8, AtEXPA10, AtEXPA15, AtEXPA20 and AtEXPB3 were found in syncytia, shoots and roots, whereas AtEXPA3 and AtEXPA16 were found in syncytia and shoots. Results obtained for uninfected plants are supported by Genechip data collected at Genevestigator (http://www.genevestigator.ethz.ch; Zimmermann et al., 2004).
Several expansin genes are specifically expressed during syncytium formation
After having identified which expansin genes are expressed in syncytia, we compared the expression of these genes in root segments containing syncytia versus segments of coeval uninfected roots. The samples contained neither root tips nor primordia of secondary roots. Semi-quantitative RT-PCR (sqRT-PCR) was performed for all the expansin genes that had been detected in the syncytium-specific cDNA library and for 18S rRNA and UBQ1 as controls (Figure 1). According to the results the expressed expansin genes can be divided into two groups. Group one comprises AtEXPA3, AtEXPA6, AtEXPA8, AtEXPA10 and AtEXPA16, which gave positive signals with syncytium material collected at 5, 10 and 15 days after infection (dai), whereas no products could be amplified from control root segments. Except for AtEXPA16 the group-one signals were weak at 5 dai, strongest at 10 dai and slightly reduced at 15 dai. AtEXPA16 gave the weakest signal at 5 dai, but gave a strong signal at both 10 and 15 dai. Group two consists of AtEXPA1, AtEXPA4, AtEXPA15, AtEXPA20 and AtEXPB3, which could be detected in samples with and without syncytia. However, they are strongly upregulated in root segments with syncytia. AtEXPA1 is generally expressed in all samples with syncytia, whereas its expression in uninfected roots is increased in older samples. Expression of AtEXPA4 became weaker in older segments with syncytia, whereas it was detectable in control roots coeval to 5 dai, but reduced at the age coeval to 10 dai and no longer detectable in control roots corresponding to 15 dai. AtEXPA15 is expressed at the same high level at all time points in root segments with syncytia. In the control roots a weak signal was detected only at the age corresponding to 5 dai, whereas at later time points no signal could be detected. Maximum expression of AtEXPA20 and AtEXPB3 occurred in syncytium material collected at 10 dai. The signal of AtEXPA20 was slightly stronger at 5 dai than at 15 dai, whereas for AtEXPB3 it was slightly reduced at 5 dai compared with 15 dai. Signals for both expansin genes were weaker in uninfected root samples than in samples with syncytia.
The results clearly show that AtEXPA3, AtEXPA6, AtEXPA8, AtEXPA10 and AtEXPA16 are specifically expressed during syncytium formation, and are not transcribed in the corresponding parts of healthy roots. AtEXPA1, AtEXPA4, AtEXPA15, AtEXPA20 and AtEXPB3 are also upregulated during syncytium formation but are also expressed in control roots.
Analyses of expansin expression profiles in syncytia and uninfected roots were also made using Affymetrix Genechips. The basis material was again micro-aspirated syncytial cytoplasm sampled at 5 and 15 dai compared with corresponding uninfected root segments. These data confirmed the results of all other experiments (Table 2). Significant upregulation was detected for AtEXPA1, AtEXPA3, AtEXPA5, AtEXPA6, AtEXPA8, AtEXPA10, AtEXPA16 and AtEXPB3. Upregulation of AtEXPA3, AtEXPA6, AtEXPA8, AtEXPA16 and AtEXPB3 was found in syncytia both at 5 and 15 dai, with either a similar level (AtEXPB3) or a higher level observed in the older samples. Increased expression of AtEXPA1, AtEXPA5 and AtEXPA10 was only detected at 15 dai. Upregulation of AtEXPA4, AtEXPA15 and AtEXPA20 was not detected with significance in this assay.
Table 2. Genechip expression profiles of Arabidopsis expansin genes during the development of syncytia induced by Heterodera schachtii. Changes in gene expression were obtained in comparison between micro-aspirated syncytia content at 5 dai (5d) and 15 dai (15d) and coeval root fragments from the elongation zone without root tips and lateral root primordia (Ctl). Values displayed have been normalized and are on a log2 scale (see Experimental procedures for details). The differences shown are consequently log2 ratios, with values of ± 1 corresponding to either a twofold up- or downregulation
Ctl versus 5d
Ctl versus 15d
5d versus 15d
In the table, significance in a regularized Benjamini–Hochberg corrected test is indicated by asterisks (*q < 25%; **q < 10%; ***q < 5%; see Experimental procedures for details).
EXPA22 & 26
EXPA23 & 25
EXPB2 & 4
Raw group means
Pairwise contrasts in a batch detrending linear model
A highly significant reduction of expression was found for AtEXPA7 and AtEXPA18, both at 5 and 15 dai. For all other expansin genes no significant changes in expression during syncytium development were detected.
Promoter::GUS studies support differential regulation of expansin genes in syncytia
For some expansin isoforms, promoter::GUS lines were studied (AtEXPA1, AtEXPA3, AtEXPA4, AtEXPA6, AtEXPA10, AtEXPA15 and AtEXPA16). In order to analyse the specificity of expression of these genes during syncytium formation, plants were infected with nematodes and stained for GUS activity. In the context of this work we focused our attention to the roots, whereas a general expression analysis of uninfected plants will be presented elsewhere (Cosgrove and Durachko, in preparation).
GUS assays with uninfected roots gave the following results: in AtEXPA1, AtEXPA4 and AtEXPA15::GUS plants, a generally strong staining was found mainly in the vascular cylinder of the primary and lateral roots, as well as in the root tips (Figure 2b,c,h,i,q,r). In AtEXPA6::GUS plants staining was restricted to root tips (Figure 2k), emerging primordia of lateral roots (Figure 2l) and young lateral roots. In the roots of AtEXPA3::GUS plants, no GUS activity could be observed (Figure 2e,f). The AtEXPA10::GUS line showed a faint expression in the root tips (Figure 2n). In AtEXPA16::GUS plants gus expression was detected at a very low level in lateral roots and root tips (Figure 2t,u).
Blue staining in syncytia was found in AtEXPA1, AtEXPA3, AtEXPA4, AtEXPA6, AtEXPA10, AtEXPA15 and AtEXPA16::GUS plants (Figure 2). Syncytia in the lines with promoters of AtEXPA1, AtEXPA4 and AtEXPA15 showed a remarkably strong staining (Figure 2a,g,p). In the lines AtEXPA3::GUS, AtEXPA10::GUS and AtEXPA16::GUS activity was restricted to the syncytium, and was not observed in the root tissue above and below the feeding site (Figure 2d,m,s).
A time course analysis was performed with the AtEXPA6:GUS line because of its gus expression in syncytia and the background expression in both root tips and lateral root primordia (Figure 3j,k,l). Samples were taken at 3, 4, 5, 7, 12, 15 and 18 dai. At 3 and 4 dai GUS activity was located in a diffuse zone within and around the young syncytia (Figure 3a,b). It became stronger and more focused to syncytia at 5 dai (Figure 3c). The strongest gus expression was found in syncytia at 7 and 10 dai. At this stage GUS staining was restricted to the feeding site (Figure 3d,e). In syncytia at 12 and 15 dai expression decreased (Figure 3f,g) and was no longer detectable at 18 dai (Figure 3h), which is in contrast to our Genechip data showing a strong expression in syncytia at 15 dai. However, this phenomenon is explained by a general decrease of GUS staining in older syncytia (see Discussion).
In situ analysis: expression of AtEXPA3, AtEXPA6, AtEXPA8, AtEXPA10 and AtEXPA16 is restricted to syncytia
AtEXPA3, AtEXPA6, AtEXPA8, AtEXPA10 and AtEXPA16 mRNA localization within 7-dai syncytia was also investigated using in situ RT-PCR. Transcripts of AtEXPA3 were mainly found in young and relatively small cells either freshly incorporated or being prepared for fusion with syncytium (Figure 4a). Results with specific primers for AtEXPA6 on sections of syncytia showed that AtEXPA6 transcripts occurred in syncytial elements, but not in the surrounding tissue (Figure 4d). Reactions with primers for AtEXPA8, AtEXPA10 and AtEXPA16 showed specific accumulation of transcripts of these expansin genes in syncytia. Low background staining occurs in the surrounding root tissue (Figure 4g,j,m). No products of all these genes were detected with the control reactions without Taq Polymerase (Figure 4b,e,h,k,n). Control reactions on root sections above the syncytium gave no staining in the vascular cylinder and the surrounding root cell layers (Figure 4c,f,i,l,o).
Expansins are known to play an important role in cell wall formation and modification. Therefore it can be anticipated that they are involved in plant–pathogen interactions that go along with major structural changes in cell wall architecture, such as the formation of hypertrophic and hyperplastic tissues.
In this paper we studied the expression of the expansin gene family in roots of Arabidopsis that were infected with the beet cyst nematode H. schachtii. A group of ten expansin genes were found to be expressed in the syncytia induced by the nematode. In uninfected plants the majority of these genes are expressed in roots, whereas two genes, AtEXPA3 and AtEXPA16, are expressed mainly in the shoot. As far as uninfected control plants are concerned, these data match with gene expression profiles that are available in the Genevestigator database (http://www.genevestigator.ethz.ch; Table 3). For this comprehensive analysis we applied several methods that differ in their potential to detect, quantify and localize gene expression. Complementing each other, these methods gave a clear picture of expansin genes expression during syncytium development.
All listed genes are specifically either up- (↑) or downregulated (↓) in syncytia.
Specific expression of expansin genes in nematode-infected root tissue
Arabidopsis contains 26 AtEXPA and six AtEXPB expansin genes, and so far the advantage of this high number of isomers is not known. In general, there is not much information on the specific functions of single members of the expansin gene family. One may speculate that there is either a variability in function or a variability in regulation, but so far there is no experimental evidence in either the one or the other direction. However, as different functions have not yet been found, it is highly probable that variability is an approach to specify expression during either different developmental stages or under different environmental influences.
With the aid of the specific cDNA library and the Genechip, expression of ten expansin genes within syncytia could be clearly shown. However, as these analyses were based on micro-aspirated syncytium samples, they do not indicate whether or not there is additional expression in other areas of the root. This information was obtained by promoter::GUS lines, in-situ RT-PCR and sqRT-PCR with samples of infected and uninfected root segments. The analysed genes can be divided into four different categories according to their expression pattern (Figure 5).
Category I comprises five genes that are expressed in syncytia and the entire root system, including the tissue surrounding the syncytia. It comprises AtEXPA1, AtEXPA4, AtEXPA15, AtEXPA20 and AtEXPB3. Durachko and Cosgrove (unpublished results) found that AtEXPA1 is mainly expressed in the stomatal guard cells and very young vascular bundles, whereas the AtEXPA4 promoter directs expression in the vascular bundles throughout the plant. For AtEXPA1, AtEXPA4 and AtEXPA15 promoter::GUS lines were available and expression in the whole root including the area adjacent to syncytia could be shown clearly. Slight differences between the Genechip data and the results of the sqRT-PCR experiments (Figure 2) in the case of AtEXPA4 and AtEXPB3 can be explained with the different origin of the starting material. For sqRT-PCR, root segments containing syncytia were taken, whereas micro-aspirated syncytial cytoplasm without surrounding tissue was used for Genechip analysis.
Category II contains three genes that are expressed in syncytia and in other parts of the roots, but not in the surrounding root tissue. It includes AtEXPA6, AtEXPA8 and AtEXPA10. Expression analyses of promoter::GUS lines indicate that AtEXPA8 is expressed in specific cells in the root, whereas AtEXPA10 occurred in leaf petioles and midribs and at the base of the pedicels (Cosgrove, 1998).
Category III consists of AtEXPA3 and AtEXPA16, which are upregulated in syncytia and are otherwise expressed only in shoot tissue. Expression of AtEXPA3 was found in the shoot apical meristem (Cosgrove and Durachko, unpublished results). Comparing Genechip data and the results of sqRT-PCR with AtEXPA3 the same phenomenon occurred as explained with AtEXPA4 and AtEXPB3.
Category IV contains two expansin genes, AtEXPA7 and AtEXPA18, which are downregulated in syncytia. Expression data from Genevestigator for expansin genes belonging to all four categories are shown in Table 3.
Expression of expansin genes changes during the development of nematode feeding sites
Using GUS assays we performed a time-course analysis of the expression pattern of AtEXPA6 during syncytium development. GUS activity was observed already in syncytia at 3 dai and reached its maximum in syncytia at 7–10 dai. These data were supported by results of the Genechip analysis at two time-points, where a significant increase of expression in syncytia at 5 and 15 dai was measured (Table 2). No blue staining was found in syncytia at 18 dai. This can be explained by earlier studies with this host-pathogen system, which revealed that GUS activity generally decreases in the syncytia of older plants independent of the used construct (Barthels et al., 1997; Puzio et al., 1998).
Jammes et al. (2005) performed similar gene expression profiling during the formation of galls induced by the root-knot nematode Meloidogyne javanica in roots of Arabidopsis. They identified seven AtEXPA and two AtEXPB genes upregulated in galls. Expression of six genes (AtEXPA1, AtEXPA6, AtEXPA10, AtEXPA15, AtEXPB1 and AtEXPB3) continuously increased in galls from 7 to 14 dai. AtEXPA7 is more strongly expressed in galls at 7 dai than in galls at 14 dai. For two genes (AtEXPA11 and AtEXPA16) no changes in expression levels between 7 and 14 dai were measured. The observed signal intensities were generally much weaker than those reported here. However this is not surprising because they dissected the galls, so that mRNA from giant cells was diluted in the samples by contamination from other tissues, whereas either micro-aspirated or laser-captured cell contents (Ramsay et al., 2004) are specifically sampled and therefore much less contaminated. Nevertheless, there are some clear differences in the expression dynamics of expansin genes in galls obtained by these authors (Jammes et al., 2005), and in cyst nematode-induced syncytia as described in this paper (Table 2). Although the expression of AtEXPA7 is strongly downregulated in syncytia, it is slightly upregulated in galls. In the case of AtEXPA15 no difference was observed in younger and older syncytia, whereas in galls an increase of expression was observed. AtEXPA16 is specifically upregulated in syncytia at 5 and 15 dai. Interestingly, there is no change in its expression at 5 dai, but a slight increase at 14 dai in galls. There are also differences in the expression of AtEXPB genes between syncytia and galls. In syncytia the expression of AtEXPB1 is not changed significantly, whereas in galls this expansin gene is upregulated. AtEXPB3 has its strongest expression in syncytia at 5 dai, whereas in galls the maximum of its expression occurs at 14 dai. Data for AtEXPA3 were not provided by Jammes et al. (2005).
This comparison shows that root-knot and cyst nematodes differ in their influence on the expression of expansin genes (either activation or reduction) during feeding site development in Arabidopsis.
Another study with root-knot nematodes was recently performed in the tomato. Gal et al. (2005) described the expression of the tomato expansin gene LeEXP5, which was observed at a very low expression level in the uninfected root, but was upregulated in gall cells adjacent to the giant cells induced by the root-knot nematode M. javanica. Using in situ RT-PCR they could not detect the transcripts in giant cells. Furthermore, these authors have generated LeEXP5-antisense transgenic roots using Agrobacterium rhizogenes transformation. After nematode infection they observed a decrease of the egg mass per gall, the number of eggs per gall mass and the giant cell diameter in LeEXP5-antisense transgenic lines in comparison with the control plants. They concluded that expression of LeEXP5 is required for gall cell expansion, and thus gall formation, and that a decrease of its transcription caused a reduced parasitism by the nematodes. However, there are several possible problems associated with the presented data. It is known that the use of rhizogenic roots can be problematic for studies of nematode infections because of their artificial hormone status (Plovie et al., 2003). Furthermore, a conserved region of the LeEXP5 gene was used for the antisense construct. Considering the high sequence homology among members of this large gene family, this means that also other expansin genes will be downregulated. To characterize a specific function of the LeEXP5 gene it would be necessary to use more specific parts of the sequence for RNAi constructs. Such experiments are underway in our laboratory for both Arabidopsis and tomato expansin genes.
Expansins may have specific functions in plant-microbe interactions
To date there are only a few reports showing plant expansin gene expression in plant–microbe interactions. Balestrini et al. (2004) found the cucumber expansins CsEXPA1 and CsEXPA2 to be more abundant in cell walls upon mycorrhizal infection. They proposed that these expansins are directly involved in the accumulation of Glomus veriforme in infected cortical cells, and may be cell wall-loosening agents that facilitate the penetration of the hyphae through the cell wall. Giordano and Hirsch (2004) studied the expression of expansin genes during nodule development induced by Sinorhizobium meliloti in the roots of Melilotus alba and found MaEXP1 to be upregulated.
In other cases, expansin-like proteins of unknown function were found in plant-associated bacteria and fungi (Laine et al., 2000; Saloheimo et al., 2002). A gene with structural and putative functional similarities to plant expansins has recently been found in juveniles of the cyst nematode G. rostochiensis, which is related to H. schachtii (Kudla et al., 2005; Qin et al., 2004). The authors suggested that the protein is produced and secreted by the juvenile during the invasion through the root where it could help to soften cell walls and thus facilitate nematode migration through the root tissue.
On the plant side, future analyses will have to focus on the function of the different described expansins as well as on their regulation. The cell walls of nematode-induced syncytia undergo highly specific modifications that are necessary to meet the specific demands of the cell complex and the associated parasite. Therefore, it is essential to understand how these modifications are formed and controlled. Here we describe the expression pattern of an entire gene family in response to a nematode infection. We show that the different members of the expansin family are regulated in a highly specific manner that includes upregulation as well as downregulation of the single members. The type of expression pattern, in which shoot-specific genes are especially activated in roots during the formation of syncytia, is highly remarkable. Further studies have to be performed in order to clarify the basis of this expression pattern. A detailed promoter analysis and comparison with related genes might reveal specific regulatory elements leading to transcription in shoot organs and syncytia. On the other hand, the specific function of these genes in the shoot has to be studied in order to find out whether this relates to processes in syncytium development.
Seeds of A. thaliana were surface-sterilised for 10 min in 5% (w/v) calcium hypochlorite, submerged for 5 min in 70% (v/v) ethanol and subsequently three times in sterile dH20 (Sijmons et al., 1991). The sterilized seeds were placed into sterile Petri dishes (9 cm in diameter) on a modified 0.2 concentrated Knop medium supplemented with 2% sucrose (Sijmons et al., 1991). Seeds were kept at 4°C for 3 days prior to incubation in a growth chamber at 25°C with a 16-h light and 8-h dark cycle.
DNA and RNA isolation from A. thaliana
Genomic DNA and total RNA were extracted from various organs of A. thaliana (ecotype Columbia) following the method of Gustincich et al. (1991) as modified by Clark et al. (1997). Genomic DNA was isolated from young leaves of A. thaliana. RNA was isolated from complete shoots and roots of 21-day-old A. thaliana plants.
DNA manipulation, including enzymatic digestions, agarose gel electrophoresis, ligation and transformation to Escherichia coli DH5α were performed according to Sambrook et al. (1989). Promoters of various Arabidopsis expansin genes were cloned into the binary vector pGPTV-HPT (Becker, 1992) in order to drive the expression of the β-glucuronidase reporter gene (gus). The promoters were from AtEXPA1 (from −1610 to −70 bp before the ATG start codon), AtEXPA4 (from −2299 to −82 bp), AtEXPA10 (from −1561 to −70 bp) and AtEXPA15 (−1635 to −38 bp). In most cases, genomic fragments containing whole promoter regions were first subcloned from appropriate BAC clones (Arabidopsis Stock Centre, Ohio State University, Columbus, OH, USA) by restriction and ligation into either pUC118 or pBSK plasmids. For AtEXPA10 and AtEXPA15, promoter regions were first amplified by PCR using primers engineered with suitable restriction sites, then cloned into either pUC18 or pUC118. Promoters were then excised with appropriate restriction enzymes and ligated into the polylinker site of pGPTV-HPT (Cosgrove and Durachko, unpublished results). The pGPTV-HPT vectors were amplified in E. coli DH5α and then transformed into Agrobacterium tumefaciens strain C58C1.
A 1704-bp AtEXPA6 promoter fragment was produced and cloned into the binary pMOG819 vector that contains gus and nptII, flanked by the T-DNA border sequences. This AtEXPA6 promoter::GUS construct was transformed from E. coli DH5α into A. tumefaciens strain MOG101 (Goddijn et al., 1993) by triparental mating, using the helper plasmid pRK2013 in E. coli DH5α.
The 953-bp AtEXPA3 and the 577-bp AtEXPA16 promoter fragments were amplified by PCR and cloned into pCambia 1304 vector. The vectors were amplified in E. coli DH10β and then electroporated into A. tumefaciens LBA 4404.
In most cases, the AtEXPA::GUS chimeric constructs were inserted into the genome of A. thaliana, ecotype Columbia, by Agrobacterium-mediated transformation using the floral-dip method (Bechtold et al., 1993; Bent et al., 1994). For AtEXPA10, ecotype C24 was used. Transformants were identified by hygromycin selection, selfed, and homozygous lines were characterized (Cosgrove and Durachko, unpublished results). AtEXPA3::GUS and AtEXPA16::GUS transformants were selected on kanamycin. In the case of the AtEXPA6::GUS construct, 51 transformants were regenerated from A. thaliana (ecotype C-24) root explants according to Valvekens et al. (1988).
Cysts of H. schachtii cultures were harvested from in vitro stock cultures on mustard (Sinapsis alba cv. Albatros) roots growing on 0.2 concentrated Knop medium supplemented with 2% sucrose (Sijmons et al., 1991). Hatching of juveniles was stimulated by soaking cysts in 3 mm ZnCl2. The larvae were then washed four times in sterile H2O and resuspended in 0.5% (w/v) Gelrite (Duchefa, Haarlem, The Netherlands) before inoculation. Twelve-day-old roots of A. thaliana plants were inoculated under axenic conditions with about 30 juveniles.
Histochemical localization of GUS activity
Histochemical detection of GUS activity was performed by staining, according to the method of Schrammeijer et al. (1990), using a solution of 2 mm 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-gluc; Biomol, Hamburg, Germany) in 0.1 m sodium phosphate buffer pH 7.0, 0.1% Triton-X 100, 0.5 mm K3[Fe(CN)6], 0.5 mm K4[Fe(CN)6] and 10 mm Na2EDTA incubated overnight at 37°C. After staining, chlorophyll was extracted from photosynthetic tissues with 70% (v/v) ethanol. The gus expression was detected microscopically by the distinct blue colouration resulting from the enzymatic cleavage of X-gluc. The GUS staining of roots of AtEXPA1::GUS, AtEXPA4::GUS, AtEXPA6::GUS, AtEXPA10::GUS and AtEXPA15::GUS lines containing syncytia was examined at 5 dai. Plants of line AtEXPA6::GUS used for the histochemical localization of GUS activity were additionally examined at 3, 4, 5, 7, 12, 15 and 18 dai.
Oligonucleotide primers flanking the protein coding sequences of A. thaliana expansin genes (see Supplementary Material) were used for first-strand synthesis and amplification of mRNA templates. Control reactions were performed using the 5′ primer (5′-GGTGGTAACGGGTGACGGAGAAT-3′) and 3′ primer (5′-CGCCGACCGAAGGGACAAGCCGA-3′) designed from the sequence of A. thaliana 18S ribosomal cDNA. Total RNA (50 ng) was denatured for 3 min at 65°C and added to the RT reaction mix (final concentrations: 1 × RT buffer; 0.5 mm of each dNTP; 1 μm gene-specific 3′ primer; 10 U Rnasin, Promega, Mannheim, Germany; 1.0 μl Sensiscript Reverse Transcriptase, Qiagen, Helden, Germany; in a total volume of 20 μl). Samples were incubated at 37°C for 1 h, heated to 95°C for 5 min, and cooled to 10°C for 15 min. The cDNA was amplified by PCR using a PCR mix containing 1 × PCR buffer (Qiagen), 1.5 mm MgCl2, 200 μm of each dNTP, 1.0 μm each of the gene specific primers, 2.5 U HotStarTaq DNA Polymerase (Qiagen). Two PCR protocols were used, PCR1 and PCR2. The cycle order for PCR1 was as follows: denaturation for 2 min at 94°C; cycles 1–20, 15 sec at 94°C, 0.7°C sec−1 to 65°C, 30 sec at 65°C, 1.5°C sec−1 to 72°C and 2 min at 72°C; cycles 21–40, 15 sec at 94°C, slope 0.7°C sec−1 to 45°C, 30 sec at 45°C, 1.5°C sec−1 to 72°C, 2 min at 72°C; 5 min at 72°C. The cycle order for PCR2 was as follows: denaturation for 2 min at 94°C; cycles 1–40, 40 sec at 94°C, 0.7°C sec−1 to 60°C, 1 min at 65°C, 1.5°C sec−1 to 72°C; 5 min 72°C. RT-PCR products (18 μl) were separated on a 1.0% agarose gel. The specificity of each primer pair was established by RT-PCR reactions from A. thaliana shoot RNA of a predicted unique fragment, the identity of which was confirmed by DNA sequencing.
Syncytia and corresponding uninfected root fragments without root tips and lateral root primordia were collected, and total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions, including DNA digestion with DNaseI. Syncytia were dissected at 5, 10 and 15 dai. cDNA was amplified using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random primer [oligo(dN)6]. For PCR experiments a 1:10 cDNA dilution and specific primer pairs were used(see Supplementary Material). PCR conditions were as described above. Because of the very limited quantity of the RNA isolated from syncytia samples, the measurement of the RNA concentration was not possible. Therefore, PCR conditions (number of cycles) were established for 18S rRNA and UBQ1 to give bands of similar intensity with each RNA sample. Because of the lower level of expansin transcripts, the number of cycles was increased for PCR reactions with expansin primers (Table 4). In this way the bands for all RNA samples can be compared directly. The PCR cycles for 18S rRNA and UBQ1 controls were: 20 cycles for syncytia at 5 and 10 dai, 22 cycles for control roots at the age corresponding to 5 dai, 24 cycles for syncytia at 15 dai and 26 cycles for control roots at the age corresponding to 10 and 15 dai. For expansin genes, 39 cycles for syncytia at 5 and 10 dai, 41 cycles for control roots at the age corresponding to 5 dai, 43 cycles for syncytia at 15 dai and 45 cycles for control roots at the age corresponding to 10 and 15 dai, were performed.
Table 4. Number of cycles performed in semi-quantitative RT-PCR for 18S rRNA, UBQ1, and expansin genes
RNA isolation from syncytia and cDNA library construction
The cytoplasm of syncytia was extracted with a microcapillary and a micromanipulator (Eppendorf AG, Hamburg, Germany) without contamination from either uninfected root cells or nematodes (Jürgensen et al., 2003). Samples of cytoplasm were collected between 5 and 7 dai. Total RNA was isolated from 100 micro-aspirated syncytia by using the RNeasy Plant Mini Kit (Qiagen). An aliquot of approximately 15 syncytia was used for cDNA library construction. A syncytium-specific cDNA library, containing approximately 2.5 × 106 primary recombinants, was produced with the SMARTTM cDNA library construction kit (Clontech Laboratories, Palo Alto, CA, USA), according to the manufacturer's instructions. The quality of the cDNA library was determined by PCR amplification of known nematode-responsive plant genes (P.S. Puzio, P. Voss, F.M.W. Grundler, Institut für phytopathologie, Universität Kiel, Germany unpublished results). A sample of the primary non-amplified cDNA library (2 μl) was used as the template for PCR reactions.
PCR reactions with A. thaliana gDNA and syncytium-specific cDNA library
For PCR reactions 1 μg μl−1 gDNA from A. thaliana and 2 μl of syncytium-specific cDNA were used, respectively. The reactions were performed with HotStarTaq DNA Polymerase (Qiagen) as described above.
In situ RT-PCR
The in situ RT-PCR was performed according to the method described by Koltai and Bird (2000) and Urbanczyk-Wochniak et al. (2001). Infected and non-infected A. thaliana control roots were cut into small pieces and fixed at 4°C for 24 h in fixation solution (63% ethanol, v/v; 2% formalin v/v) at 5 dai. Fixed samples were washed three times for 10 min each in 63% (v/v) ethanol and once in phosphate-buffered saline (PBS; 10 mm Na3PO4 and 130 mm NaCl, pH 7.5). Samples were embedded into 5 % (w/v) low-melting point agarose in PBS. Small blocks of agarose containing root samples were attached to the block of a Vibratom (VT 1000, Leica, Wetzlar, Germany), sections (20–30 μm thick) were cut and then digested overnight at 37°C with 8 U of DNase (Fluka, Sigma-Aldrich, Seelze, Germany). Washing steps were always performed for 10 min at 37°C: once with 0.5 m EDTA, twice with 2 × SSC, once with 1 × SSC and 0.5 × SSC and finally with RNase-free water. Afterwards, about ten agarose-free root sections were transferred into 10 μl of RT mix per reaction tube. For in-well RT amplification the same conditions as described for normal RT-PCR were used. PCR was performed in a 50 μl reaction volume containing 0.25 μl of Taq polymerase (5 U μl−1; BioTherm, GeneGraft, Lüdinghausen, Germany) and the appropriate 10 × buffer, 1 μl primer (10 μm), 1 μl each of dCTP, dGTP and dATP (10 mm), 2.36 μl dTTP (2 mm) and 0.5 μl digoxigenin-11-dUTP (DIG; 1 mm; Roche Diagnostics, Indianapolis, IN, USA). For PCR profiles see RT-PCR and Supplementary Material. Positive control reactions were performed using the 5′ and 3′ primers designed from the sequence of A. thaliana 18S ribosomal cDNA, as describe above. Three different negative controls reactions were performed, by omitting primers, Taq DNA polymerase or digoxigenin-11-dUTP, respectively. Afterwards cross sections were washed twice with 1 × PBS for 5 min, once with 0.1% (v/w) BSA (Roth, Karlsruhe, Germany) in PBS for 30 min and finally with anti-DIG antibodies (1:500; 150 U; Roche Diagnostics) in PBS containing BSA for 1 h at room temperature (25°C). Root sections were then washed twice for 15 min with washing buffer (0.1 m Tris-HCl, 0.15 m NaCl, pH 9.5). Staining reactions (5–10 min) with NBT/BCIP (Roche Diagnostics, Mannheim, Germany) were performed according to the manufacturer's recommendations. Sections with satisfactory signals were photographed under an inverse microscope (Axiovert 200M; Zeiss, Hallerbergmoos, Germany) containing an integrated camera (AxioCam MRc5; Zeiss).
Affymetrix Genechip analysis
Syncytia were aspirated and RNA isolated as described above. Root segments cut from the elongation zone were used as controls. Care was taken to avoid any either root tips or lateral root primordia. Biotin-labelled probes were synthesized according to the Affymetrix protocol with some modifications. Details will be published elsewhere. ATH1 genechips were hybridized by German Resource Centre for Genome Research GmbH (Berlin, Germany) according to the manufacturer's protocols.
Affymetrix CEL files were read into the R statistical analysis environment (http://www.r-project.org) using the affy package of the Bioconductor suite (http://www.bioconductor.org). Probe sequence-specific ‘background correction’ (Wu et al., 2004) was performed using routines available in the Bioconductor gcrma package. Both ‘PM’ and ‘MM’ probes were employed for this correction. A heuristic estimate for optical instrument background as offered in gcrma, however, was not subtracted. An inspection of exploratory pairwise scatter and ‘MA’ plots confirmed the necessity for inter-chip normalization. As an examination of pairwise quantile–quantile plots showed only random fluctuations, inter-chip normalization could be achieved using quantile–quantile normalization (Bolstad et al., 2003). See Supplementary Material.
After normalization, robust summaries of probe set signals were obtained for each gene using an iterative weighted least-squares fit of a linear probe level model (Bolstad, 2004) through the fitPLM function of the Bioconductor package affyPLM. This process automatically identifies unreliable chip areas and correspondingly downweights outlier probes. See Supplementary Material.
The normalized data on a log2 scale was then fitted gene by gene with a linear model, including hybridization batch effects, using the lmFit function (Smyth, 2004) of the Bioconductor package limma. The pairwise contrasts from this fit shown in Table 2 also include q-values as indicators of significance after the correction for multiple-testing controlling the False Discovery Rate (Benjamini and Hochberg, 1995). For the statistical tests, individual gene variances have been moderated using an Empirical Bayes approach that draws strength from transferring variance characteristics from the set of all genes to the test for each individual gene (Smyth, 2004).
We would like to thank Krzysztof Jeziorny for technical support in preparing the figures. This research was supported by grant QLK-CT-1999-01501 (‘NONEMA’) from the European Union within the 5th Framework, FWF grant P16296-B06, and by grant IBN-9874432 from the US National Science Foundation. DPK acknowledges funding by the Vienna Science and Technology Fund (WWTF), the Austrian Centre of Biopharmaceutical Technology (ACBT), Austrian Research Centres (ARC) Seibersdorf, and Baxter AG.