Journal of Plant Interactions Vol. 4, No. 4, December 2009, 261269
ORIGINAL ARTICLE Differential gene expression during hypersensitive response in Phylloxera-resistant rootstock ‘Bo¨rner’ using custom oligonucleotide arrays Livia Blank, Tatjana Wolf, Klaus Eimert* and Max-Bernhard Schro¨der Department of Botany, Geisenheim Research Center, Geisenheim, Germany
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(Received 29 June 2009; final version received 20 July 2009) The aim of the present study was to identify genes involved in the hypersensitive response (HR) in ‘Bo¨rner’, a grapevine rootstock cultivar resistant to grape phylloxera. The HR was chemically induced in roots by an application of indol-3-acetic-acid (IAA). Comparisons between treated and untreated roots after different times of HR induction by IAA were realized by advanced custom DNA microarrays using ‘Riesling’, a phylloxerasensitive scion, as a control. IAA induction resulted in higher numbers of differentially expressed genes in ‘Bo¨rner’ than in ‘Riesling’. In total, 27 putative HR-related genes were identified in ‘Bo¨rner’. These genes are presumably involved in the production of phytoalexins, ethylene-associated gene products, cell wall proteins and transcription factors. Thus, the present study is contributing to a better understanding of the signal transduction pathways involved in the hypersensitive reaction underlying the necrosis formation after phylloxera attack in the rootstock cultivar ‘Bo¨rner’.
Keywords: Vitis; grape phylloxera; hypersensitive response (HR); gene expression; DNA oligonucleotide array; indol-3-acetic-acid (IAA)
Introduction Grape phylloxera (Daktulosphaira vitifoliae Fitch, Hemiptera: Phylloxeridae), an aphid-like vine pest, was introduced to Europe in the 19th century. Since that time it has become a considerable threat to viticulture. Grape phylloxera infestation leads to tissue proliferations in susceptible vines in the form of leaf galls and root nodosities or tuberosities, respectively. The latter two lead to substantial loss of vitality which can result in the death of the vine (Sterling 1952; Niklowitz 1955; Anders 1957). Introgression of American wild grapevines into European breeding programs for phylloxera resistance in the beginning of the 20th century led to tolerant rootstock cultivars used in viticulture until today. Resulting hybrids often displayed less or no visible damage upon grape phylloxera attack. They were generally thought to be resistant and widely used as rootstocks for the European varieties. In the last couple of decades, increasing damage was reported from vineyards, only hesitantly at first attributed to a grape phylloxera ‘come-back’. From this development, we know that the majority of rootstock varieties currently in use are only phylloxera tolerant, rather than resistant. Usually, the vigorous root system of tolerant rootstock varieties can compensate for the damage caused by grape phylloxera feeding on their roots. Nevertheless, in the presence of concomitant adverse conditions, such as negative weather conditions or increasing damage by mechanization, the *Corresponding author. Email:
[email protected] ISSN 1742-9145 print/ISSN 1742-9153 online # 2009 Taylor & Francis DOI: 10.1080/17429140903254697 http://www.informaworld.com
tolerant rootstocks can collapse under a severe phylloxera attack. However, these crosses also yielded few hybrids showing a qualitative difference. For instance, one of these crosses (Vitis cinerea Arnold and V. riparia 183G) yielded the only cultivar completely resistant to grape phylloxera (Bo¨rner 1943) which was subsequently named after the breeder and is, up to now, one of the only three commercially available, truly resistant rootstocks, the other two being its siblings. The ‘Bo¨rner’ rootstock reacts to grape phylloxera infestation with a hypersensitive reaction (HR) leading to local necroses on leaves and roots and, thus, preventing feeding and breeding of the insect (Bo¨rner 1943; Niklowitz 1955; Anders 1958). Although a rather high genotypic diversity has been reported for phylloxera (Forneck et al. 2000; Corrie et al. 2002) which can lead to reduced ‘resistance’ in tolerant varieties (Forneck et al. 1998; Corrie et al. 2003), no breach of resistance has yet been reported in ‘Bo¨rner’ from any area it is used in. That does not exclude the possible adaptation of a grape phylloxera biotype to ‘Bo¨rner’ in the future, but we feel it emphasizes the qualitative differences between the quantitative nature of a tolerance (which often has been overcome) in comparison to the HR-like resistance, yet to be broken. Since the cultivar ‘Bo¨rner’ is not equally well suited for all soil conditions, the development of additional phylloxera-resistant rootstocks is of large interest. As breeding is very time-consuming, especially in woody
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species, studies are under way to characterize the molecular and cell biological pathogen-induced hypersensitivity reaction in grapevine rootstocks in an attempt to accelerate breeding programs and to provide the basis for biotechnological approaches. Therefore, the present study was initiated to identify and characterize genes differentially expressed during the early stages of the HR in ‘Bo¨rner’. So far, little is known about the triggering factor(s) of the HR in ‘Bo¨rner’ upon grape phylloxera infestation. However, it is assumed that the plant hormone indol-3-acetic-acid (IAA) present in phylloxera saliva at high concentrations is the component triggering the resistance mechanism (Anders 1958; Scha¨ller 1960, 1965, 1968a, 1968b). Previous studies demonstrated that IAA-treatment of roots results in necrosis formation in resistant cultivars, such as ‘Bo¨rner’, and in gall formation in susceptible ones, such as the scion (Vitis vinifera L.) ‘Riesling’ (ElNady and Schro¨der 2003). For other rootstock varieties than ‘Bo¨rner’, it has been reported that the swellings caused by IAA and phylloxera are not identical (Granett 1990). This is not in conflict with our observations, since the varieties used as resistant controls in those experiments are now only considered tolerant (Granett, personal communication). Thus, the described differences seem to display quantitative changes between sensitive and (moderately) tolerant varieties rather than qualitative ones between sensitive and truly resistant (HR) varieties. Induction of HR in ‘Bo¨rner’ by IAA has successfully been used in earlier studies utilizing heterologous DNA microarrays (Dietrich et al. 2009). While it was possible to identify genes up-regulated during the HR, down-regulation could not be analyzed using that system. Also, the question of missing the regulation of Vitis genes not present in the Arabidopsis microarray remained open. Therefore, we decided to use a custom oligonucleotide array based on Vitis sequences available in the NCBI GenBank. The experimental system used the advanced Geniom one† microarray technology (febit biomed GmbH, Heidelberg, Germany) for comparison of gene expression in induced and non-induced ‘Bo¨rner’ roots. Furthermore, we introduced the susceptible scion ‘Riesling’ for comparison and as an additional control to identify gene regulation unrelated to the HR.
Materials and methods Plant material and experimental HR induction Adventitious roots were obtained from internodes of in vitro cultures of ‘Bo¨rner’ (Vitis cinerea Arnold and V. riparia 183G) and ‘Riesling’ (V. vinifera L.) using a temporary immersion system (RITA, VITROPIC, France) as described by Dietrich et al. (2009). A root induction phase in ½ strength MS salts medium
(pH 5.6) complemented with 2% sucrose and 10 mM indol-butyric acid (IBA) for two days in the dark was followed by about seven days in the same medium lacking IBA. Developed adventitious roots of 35 mm length were induced with a 5.7 mM IAA solution as described by El-Nady and Schro¨der (2003). The treated roots were harvested and frozen in liquid nitrogen after incubation times of 7, 15, 30, 60 and 90 min. In addition, non-induced control material was harvested and frozen directly without IAAtreatment. Root material of incubation and control experiments was collected for each species over time and pooled (150200 rootlets of each treatment) to have enough base material for RNA-extraction. RNA extraction and cDNA synthesis Total RNA from frozen root tissues was extracted following the method described by Baiges et al. (2003). Starting material for each extraction ranged between 0.8 and 1.5 g (from 150200 roots). RNA was dissolved in 50 ml DEPC-water. RNA concentration was determined by UV absorption. Single strand (ss) cDNA was produced from 0.21 mg total RNA using the iScriptTM cDNA synthesis kit (BioRad, Munich, Germany) according to the manufacturer’s instructions. Oligonucleotide arrays Oligo design, array production, hybridization, washing and detection were carried out by febit biomed GmbH (Heidelberg, Germany) using the Geniom one† benchtop unit. The Geniom chip features eight channels with 6776 spots each. The independent design of each channel allows hybridizing eight different samples on one chip at the same time. For each sequence to be analyzed, 10 oligonucleotides were designed and spotted onto the chip using a micromirror device. Sources for the sequences used in the present study were the NCBI and UniGene Vitis genebanks. Vitis sequences (altogether 552), presumably belonging to five functional groups (auxin responsive genes, genes involved in biotic and abiotic stress, HR-associated genes and transcription factors), were chosen for spotting along with ‘Bo¨rner’ sequences identified in earlier works (Dietrich et al. 2009) and controls. In this study, one chip for each IAA incubation time was analyzed. On every chip, one IAA treated sample of each, ‘Bo¨rner’ and ‘Riesling’, and one untreated control sample from each species were hybridized. Every sample was hybridized in two technical repeats. No biological repeats were conducted as each sample already constituted a pool of 150200 roots (from 30150 internodes of 1030 plants). Array hybridization, detection and part of the data analysis were performed at the febit biomed GmbH. In short, RNA (1 mg) labeling was conducted
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using MessageAmp II Biotin Enhanced Kits (Ambion, Darmstadt, Germany) according to manufacturer’s instructions. Hybridizations were carried out at 458C overnight. Washing, labeling and detection were also carried out in a Geniom one† benchtop unit. Microarray data-analysis Raw data were directly imported in the GeneSpring GX software (Agilent Technologies, USA), which was used for all post-processing steps. Values below 0.01 were set to 0.01. In order to compare the expression values for each chip, per chip normalizations were arranged by dividing each measurement by the 50.0th percentile of all measurements. Furthermore, the value for each gene was divided by the median of its measurements in all samples. Lists of differentially expressed genes were created by using a parametric Student’s t-test with a p-value cut-off of 0.05 and a false discovery rate correction of 2% according to Benjamini and Hochberg (1995). Finally, the fold changes for the listed genes were calculated and only values greater than 1.5 were analyzed. Significantly regulated genes were sequenced from ‘Bo¨rner’ cDNA, annotated and deposited in the NCBI database.
Results Plant material and RNA extraction First adventitious roots could be harvested after the explants had been in the expression medium for six days. Sporadically, explants did not show adventitious root growth until two weeks later. The number of lateral roots per internode varied between one and four. Starting from 0.81.5 g root material, about 1.0 mg RNA could be isolated. The RNA was of high quality as determined by UV absorption (OD260/ 2801.6 and OD260/230 ] 2.0). The amount of cDNA yielded by first strand synthesis ranged between 1.0 and 1.5 mg.
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Oligonucleotide arrays This study compared the genetic reaction of two vine cultivars to treatment with IAA. Therefore, a custom oligonucleotide array was designed using the Geniom one† system (febit biomed GmbH, Heidelberg, Germany). Each chip contained two replicates of four probes: Treated and untreated ‘Bo¨rner’ and ‘Riesling’ roots. Roots were treated with IAA and incubated for five different durations. Accordingly, five chips were generated and analyzed. Technical replicates were performed to determine the reproducibility of array synthesis, hybridization and technical readout. Aliquots of the same cRNA sample were hybridized to two identical arrays. The Pearson correlation of raw fluorescence intensities for each individual feature was performed for the analyses. It varied from 0.930 and 0.994 certifying a high reproducibility of the technical replicates and high quality of hybridizations. Significantly differential regulated genes In order to compare treated and untreated samples, the significance level of p 50.05 was used as a threshold for regulation. Based on this cut-off value, genes were detected as regulated with fold changes greater than 1.5 between two samples. The amount of up- and down-regulated genes in ‘Bo¨rner’ and ‘Riesling’ after different IAA incubation times is shown in Figure 1. Generally, in our experimental settings, a higher number of genes seem significantly regulated after IAA induction in ‘Bo¨rner’ than in ‘Riesling’. Furthermore, in ‘Bo¨rner’ most genes were regulated already after 7 min after IAA induction, in ‘Riesling’ most regulations occurred after 90 min, only. In ‘Bo¨rner’ most genes were down-regulated after IAA treatment. It was only after 90 min that there were more genes up-regulated than down-regulated, following the pattern of ‘Riesling’. In ‘Riesling’ there was approximately the same number of genes up- and down-regulated at any analyzed time point. The oligonucleotide arrays identified a total of 72 genes significantly regulated after IAA treatment in
Figure 1. Distribution of the quantity of differential regulated genes after five different IAA incubation times generated by Geniom one† analyses. (a) ‘Bo¨rner’ roots; (b) ‘Riesling’ roots.
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‘Bo¨rner’. Only 30 of these 72 genes also showed a significant regulation in ‘Riesling’. The detailed gene names, Gen-IDs and fold-change values of gene regulation are provided in the supplement (online only). The detected genes can be classified into six categories according to their assumed functions. Table 1 shows two meaningful examples for each category along with the respective NCBI-IDs of sequenced ‘Bo¨rner’ ESTs listed in the third column.
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Discussion The intention of this study was to identify genes differentially expressed during the hypersensitive response (HR) in ‘Bo¨rner’ compared to ‘Riesling’ and non-induced controls. Such a HR occurs in response to phylloxera attack in the phylloxeraresistant ‘Bo¨rner’ but the sensitive ‘Riesling’. During phylloxera infestation, the insects forage on grapevine roots probing for the optimal feeding and breeding position by repeatedly inserting their stylet-like proboscis. During their search for that position they also often rest on the surface not inserting their stylus at all. On ‘Bo¨rner’, necroses develop around the insertion areas earliest six hours after insertion depending on the developmental age of the tissue (El-Nady and Schro¨der 2003). In the experimental set-up, it proved impossible to determine whether an insect had penetrated the root tissue at the place it was resting upon until the actual necrosis appeared 612 h later. This unpredictable feeding pattern, uncertain insertion time and also the miniscule size of the necroses made it practically impossible to use the insects themselves for controlled induction of the HR in order to obtain sufficient quantities of samples. Hence, we had to rely on the chemical induction of the HR by auxin. Auxin, a major component of phylloxera saliva (Scha¨ller 1965, 1968b) has been shown to be sufficient to cause leaf galls and root nodosities/tuberosities in susceptible cultivars and necroses in resistant ones, which were
virtually indistinguishable from those caused by phylloxera (Scha¨ller 1968b; El-Nady and Schro¨der 2003; Dietrich et al. 2009). For that reason we chose to use auxin for HR induction despite the obvious shortcomings. An earlier work using a subtractive hybridization approach identified only few genes differentially expressed during HR in ‘Bo¨rner’ (Dietrich et al. 2009). Also, that analysis was limited to overexpressed sequences, only. Here, we report on the identification of genes differentially expressed at certain stages of the auxininduced HR in the phylloxera-resistant grapevine rootstock cultivar ‘Bo¨rner’. Furthermore, differences in the response to the stimulus were found in the susceptible non-necrotic cultivar ‘Riesling’. For this analysis we used an advanced custom oligonucleotide DNA microarray (Geniom one† ) allowing for the integration of all steps, from synthesis of the array to hybridization and detection to data analysis, in a compact benchtop unit. Automation in this system allowed us to design and perform microarray experiments using self-selected and, if necessary, modified sequences derived from public databases. Accordingly, for hybridization, we chose ESTs of Vitis genes from public databases associated with pathogen defence, HR and auxin transport. However, in doing so, we have to consider the possibility of missing genes not being among the selected groups. The Geniom one† technology offers another advantage in that manual gridding, necessary after common microarray analyses, is not required. Here, oligonucleotide synthesis is limited to the area which is illuminated by the micromirror device. Accordingly, there is a clearly determined square spot geometry allowing for a high reproduction of data (Baum et al. 2003) as could be verified in this study by the consistency of technical replicates on each chip. The results of the microarray analyses showed differences in the amount of significantly regulated genes in treated roots of ‘Bo¨rner’ and ‘Riesling’. On average, there is a three times higher number of genes
Table 1. Examples of genes significantly regulated in roots of ‘Bo¨rner’ and ‘Riesling’ after different times of IAA incubation and NCBI-IDs of sequenced ‘Bo¨rner’ ESTs. Category Ethylene-associated genes Phenylpropanoids and enzymes of their biosynthesis Transcription factors Pathogen-related genes Cell wall proteins Universal stress-associated genes
Example: Vitis gene ID and denotation
NCBI-ID of ‘Bo¨rner’ EST
AY484580, Ethylene response factor 4 AY159556, Lipoxygenase DV466767, Stilbene synthase AB015871, Phenylalanine ammonia-lyase AF281656, transcription factor AY484579, WRKY4 transcription factor AF061329, PR-4 type protein (PR-4a) AF532965, Thaumatin-like protein AF305093, Polygalacturonase inhibitor AY046416, Proline-rich protein 1 (PRP1) AF194174, Alcohol dehydrogenase 2 AJ237991, Ripening-related protein (grip32)
FK938954 FK938960 FK938964 FK938956 FK938961 FK938972 FK938962 FK938970 FK938968 FK938975 FK938973 FK938959
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Journal of Plant Interactions regulated during the HR in ‘Bo¨rner’ than in ‘Riesling’. This seems to be in accordance with the different type of response of those two cultivars to phylloxera attack. In total, 27 putative HR-related genes were identified in ‘Bo¨rner’. Also, it should be noted that a large number of this set of genes was regulated already after 7 min after induction of the HR. The Geniom one† technique used also allowed good quantification of gene expressions, permitting us to hypothesize on the potential function of these genes in the auxin-induced resistance mechanism in ‘Bo¨rner’. Doing so, one has to keep in mind that, due to induction of the HR by IAA, some of the identified genes may be regulated by IAA solely in its function as a growth hormone and may play no role in the resistance mechanism. Assuming that this regulation is similar in resistant ‘Bo¨rner’ and susceptible ‘Riesling’ we should be able to identify those. The same holds true for universally stress-associated genes which are regulated in IAA-treated roots of ‘Bo¨rner’ and ‘Riesling’. These genes can rather be seen as a common component of many defence reactions than are an important participator or elicitor in this specific HR. Based on the assumed gene function, we clustered the genes identified in this work into different functional groups. In the following, we exemplarily discuss the potential role of some of those genes for each cluster. Ethylene-associated genes Seven identified genes are putatively associated with the plant hormone ethylene (see supplementary material, online version only). These genes were mostly up-regulated in IAA-treated ‘Bo¨rner’ roots but hardly regulated in treated ‘Riesling’ roots. Examples are ethylene response factor (ERF) and lipoxygenase. Ethylene biosynthesis is significantly influenced by auxin. Several groups demonstrated the dependence of the synthesis and accumulation of 1-aminocyclopropane-1-carboxylate synthase (ACC) on IAA concentrations in plant cells (Yu and Yang 1979; Yoshii and Imaseki 1981). In turn, the ACC amount regulates the rate of ethylene production. While this could also account for the up-regulation of ethylene production via IAA independent of a HR in our experiments, we do not think this to be the case for two reasons. First, two ACC genes on the microarray were not regulated differentially in any of the samples, treated or untreated. Second, there was hardly any regulation of most of the identified ethylene associated genes in ‘Riesling’. Thus, we rather assume that the ethylene-associated regulation is part of the specific defence response in ‘Bo¨rner’. Ethylene is of particular importance for plant pathogen defence as it plays a role in the activation of defence genes and as a signal component in wound response (O’Donnell et al. 1996; Penninckx et al.
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1996). Furthermore, it has been shown to be involved in the activation of programmed cell death (PCD) (Young et al. 1997; De Jong et al. 2002). De Jong et al. (2002) demonstrated that ethylene is a potentiator of the PCD and that blocking ethylene synthesis results in reduced PCD symptoms. PCD has been described as a typical characteristic of HR in general (reviewed in Greenberg 1997) and also for the local necroses of ‘Bo¨rner’ in response to phylloxera attack or IAA treatment (El-Nady and Schro¨der 2003). The ethylene-associated gene ERF, up-regulated in IAA-treated ‘Bo¨rner’ roots, belongs to a family of plant-specific transcription factors. It is known that ERF activation increases the resistance of plants to pathogens (Berrocal-Lobo et al. 2002). Hence, it is possible that the activation of ERF in IAA-treated ‘Bo¨rner’ roots also leads to the regulation of further defence genes. The characteristic GCC-box, typical for several defence associated and ethylene-regulated genes (Sessa et al. 1995; Hao et al. 1998) was also found in genes identified by the Geniom one† analyses in the present study. Examples, like PRgenes, PAL and WRKY are discussed below. Phenylpropanoids and enzymes of their biosynthesis Within this study, a set of genes were identified in IAA-treated ‘Bo¨rner’ roots which belong to the group of phenylpropanoids or enzymes of their biosynthesis, such as chalcone synthase (CHS), phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS). Most of them were significantly up-regulated in ‘Bo¨rner’ roots 90 min after IAA treatment after an initial slump in expression. No significant regulations could be detected in treated ‘Riesling’ roots. For of this reason, we assume a certain role of phenylpropanoids in the chemically-induced HR in ‘Bo¨rner’. While phenylpropanoids are known to play different roles in plant growth and development (Sukrasno and Yeoman 1993), they are also accumulated after diverse stresses, such as high UV radiation (Reuber et al. 1996), in wound response and upon pathogen attack (reviewed in Dixon and Paiva 1995). In our study, two PAL genes were shown to be activated within 90 min after induction by IAA. While one of them is similarly up-regulated in both induced ‘Bo¨rner’ and induced ‘Riesling’, the second PAL gene is regulated in ‘Bo¨rner’ only. Thus, it is possible that for the first gene the increased PAL expression could be due to IAA-mediated ethylene induction rather than to a defence-specific reaction, as we discussed above. Such PAL accumulation by ethylene is a widespread phenomenon in plants and has been demonstrated repeatedly (Riov et al. 1969; Ecker and Davis 1987; Xu and Heath 1998). It is likely that this PAL expression is a downstream event in the IAA signal transduction, but not the elicitor of the resistance reaction. As the second gene is differentially regulated only in ‘Bo¨rner’, it may well be a
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specific element involved in the induced HR. Several studies have shown that PAL, a key enzyme in the phenylpropanoid biosynthetic pathway, is linked to HR in plants (Davies et al. 1991; Dong et al. 1991; Wanner et al. 1993). It is thought that nitric oxide (NO) which accumulates during a HR (Durner et al. 1998; Delledonne et al. 2001) activates the production of PAL (Durner et al. 1998). NO itself acts as a toxic compound against pathogens as well as an element in the chain of signal transduction (Lamb and Dixon 1997; Delledonne et al. 2001; Mittler 2002), while PAL is the first enzyme in the phenylpropanoid synthesis pathway. Such phenylpropanoids are, for instance, stilbene synthases (STS), which synthesize resveratrol, a phytoalexin which is activated during HR (Tsuji et al. 1992). Other studies demonstrated the relation between phytoalexin production and resistance of grapevine against Boytritis cinerea or Plasmopara viticola (Adrian et al. 1997; Bais et al. 2000; Be´zier et al. 2002). While it has been shown that, apart from jasmonic acid (JA) and salicylic acid (SA), ethylene can also lead to the regulation of phytoalexin (Chung et al. 2003) we do not think the reason for the STS regulation observed during our experiments is due to IAA-mediated ethylene production, but is rather HRspecific. Several stilbene synthases (STS) have been identified as differentially regulated upon IAA-induced HR in ‘Bo¨rner’. Hardly any of those genes showed any regulation at all in ‘Riesling’. Even for the few only slightly regulated stilbene synthases in ‘Riesling’, no regulation pattern similar to that in ‘Bo¨rner’ could be observed. That leads us to believe that we observed a pattern of gene regulation attributable to plant defence rather than to unspecific regulation by IAA via the ethylene pathway, as discussed above. Transcription factors Defence reactions are linked to a transcriptional reprogramming which is regulated by transcription factors (TFs). Our microarray analyses revealed the regulation of five TFs in IAA-treated roots of ‘Bo¨rner’ and ‘Riesling’, including the WRKY transcription factor. The activation of WRKY has been linked to the induction and regulation of a HR in several plant species (Yoda et al. 2002; Park et al. 2006). Transcription factors of the WRKY class recognize various W-box elements [TGAC(C/T)] which are found within promoters of many defence-associated genes, such as PR (pathogen-related) genes, ATPbinding cassette-transporters and genes involved in the systemic acquired resistance (Dong et al. 2003; Yamamoto et al. 2004). Such W-box elements are also present in sequences of genes which were regulated in the IAA-treated roots of ‘Bo¨rner’, such as polyphenol oxidase (PPO), polygalacturonase inhibiting protein (PGIP) and an ethylene receptor.
Thus, it seems possible and likely that there is an overlapping in the signal transduction networks of the defence reactions and the IAA signalling. Pathogen-related genes A set of genes, regulated in ‘Bo¨rner’ and ‘Riesling’ roots after IAA treatment, belongs to the group of pathogen-related (PR) genes. PR genes are usually defined as host specific genes which are induced in plants during a pathogen infestation. The activation of PR genes is controlled by a complex interaction of signal components such as ethylene, JA or SA (reviewed in van Loon and van Strien 1999). Our analyses revealed no activation of PR genes after IAA treatment but rather a clear down-regulation of these genes in nearly all IAA-treated samples from ‘Bo¨rner’ and in some of ‘Riesling’. Here, we suspect that this down-regulation of PR genes after IAA treatment is the result of a negative effect of auxin to PR genes in general as has been described before (Shinshi et al. 1987; Harpster et al. 1998). These authors reported on the inhibition of the PR genes b-1,3-glucanase and chitinase after IAA treatment in strawberries and tobacco. The relevance of the down-regulation of PR genes during a HR in ‘Bo¨rner’ is not very apparent. On the other hand one has to keep in mind that IAA is a major component of the phylloxera saliva and that this down-regulation could well be a part of the evolved attack strategy of the insect, reducing defence reactions in the host. Cell wall proteins One of the cell wall proteins, which were included in our analyses, is xyloglucan transglycosylase (XT). This protein showed an up-regulation in treated ‘Bo¨rner’, but no regulation in treated ‘Riesling’ roots. XT has been shown to be activated upon application of the phytohormons ethylene and auxin (Saab and Sachs 1996; Catala´ et al. 1997). Thus, it could be that the observed XT induction in ‘Bo¨rner’ was caused by the IAA treatment. However, since no such induction occurred in ‘Riesling’, we rather assume it is part of a specific defence reaction. Such a role has previously been described for XT (Fry et al. 1992, 2008). XT is accountable for the cleavage of xyloglucan compounds, which are essential for the remodelling of the cell wall and responsible for the cell wall architecture. In other cases, a direct involvement in the pathogen response could be demonstrated (Divol et al. 2007). It was shown that XT was induced in different plants upon aphid infestation, and its role in cell wall modification as part of the plant defence was established. During the HR in ‘Bo¨rner’ upon phylloxera attack we also observed cell wall modifications resulting in the isolation of the affected tissues (El-Nady and Schro¨der 2003). Such modifications prevent feeding by phylloxera as well as secondary infections in the penetration area. We assume that the
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Figure 2. Possible interactions between IAA-regulated genes and their potential involvement in the HR in ‘Bo¨rner’; 0 activation; l interaction; I inhibition.
specific XT up-regulation in ‘Bo¨rner’ observed during an auxin-induced HR is part of such cell wall modifications leading to the isolation of the affected tissues. Conclusion Using an advanced microarray technique we could identify a set of genes regulated during an auxininduced HR in the phylloxera resistant rootstock ‘Bo¨rner’. We used the phylloxera susceptible grapevine cultivar ‘Riesling’ as a control to distinguish between auxin-induced gene regulation unrelated to HR and auxin-mediated HR-related gene regulation. Further, we observed the gene regulation at different times after auxin induction to reveal potential time courses of regulation of those genes. Our results are in agreement with many observations in literature, that there seems to be an enormous amount of interaction and cross-talk between signal transduction pathways. We present a schematic illustration attempting to outline (some of) the possibilities of such interaction we can construct from our and other available data (Figure 2). What seems to ensue from our data is that several genes known to be activated during pathogen responses in other plant species are also induced by auxin in Vitis. This is in agreement with the notion of auxin functioning as an activating signal in plant defence. On the other hand, we could also identify a number of genes which have been described to be activated during plant defence in other plants but were suppressed by auxin in our system. This apparently atypical behavior could be an artefact intrinsic to our experimental set-up (auxin concentrations, handling of roots etc.). However, it could also be that auxin is a part of the attack mechanism
used by phylloxera to suppress (parts of) the defence system(s) in Vitis. Interestingly, more allegedly defence-related genes are suppressed by auxin in the phylloxera resistant cultivar ‘Bo¨rner’ than in the sensitive cultivar ‘Riesling’. To unravel these apparent contradictions and to dissect more of the above interactions taking place during the hypersensitive response in the phylloxera resistant cultivar ‘Bo¨rner’, further work is planned on more detailed evaluation of the regulation using qRT-PCR, also including other Vitis species, tolerant to phylloxera.
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Supplementary material Fold changes of expression in Bo¨rner (B) and Riesling (R) samples treated with IAA after five different incubation times for genes with a mean ratio higher than 2 and lower than 0.7 in two replicate hybridizations. Green and red highlighted fields indicate up- or down-regulation, respectively. * and (*) denote genes with similar or slightly similar regulation patterns, respectively, in Bo¨rner and Riesling. Bo¨rner time course
Riesling time course
Category/Gene ID
B 07?
B 15?
B 30?
B 60?
B 90?
Ethylene-associated genes AY395744 * ethylene response factor; ERF3a AY395745 (*) ethylene response factor; ERF3b AY484580 ethylene response factor; ERF4 AY484581 (*) ethylene response factor; ERF5 CK986231 ethylene responsive protein; GH-3 AY159556 lipoxygenase; LOX CK906347 methionine synthase; MS
1.50 0.59
0.66
1.68
1.51 1.85 1.66 0.39
1.69 1.69 2.74 1.76
Phenylpropanoids and enzymes of their biosynthesis AB015870 phenylalanine ammonia-lyase; PAL AB015871* phenylalanine ammonia-lyase; PAL X75969 chalcone synthase; CHS EC907674 glutathione S-transferase T4; GTS CK906348 polyphenol oxidase; PPO AY159559 quinone reductase; QOR AB046373 stilbene synthase; RIPST1 AB046374 stilbene synthase; LABST1 AB046375 stilbene synthase; VINST1 AF418567 stilbene synthase; ST2 S63221 stilbene synthase; STS CK906354 stilbene synthase; RIPST1 X76892 stilbene synthase; StSy AY670188 stilbene synthase; S1 AY670227 stilbene synthase; P1 AY670234 stilbene synthase; H3 AF128861 stilbene synthase; STS DV466766 stilbene synthase; VINST1 DV466767 stilbene synthase; LABST1 DV466768 stilbene synthase; RIPST1 DV466771 stilbene synthase; A1 DQ235274 stilbene synthase; STS
0.50 0.65 0.30 0.34 0.33 0.27 0.33 0.34 0.60 0.54 0.42 0.36 0.37 0.32 0.47 0.33
0.61 0.58 0.33 0.30 0.35 0.42 0.32 0.39 0.64 0.39 0.42 0.33 0.54 0.47 0.34
0.49 0.53 0.39 0.54 0.51 0.57 0.64 0.60 0.50 0.43 0.44 0.49 0.57
0.65 0.32 0.36 0.40 0.52 0.38 0.47 0.43 0.43 0.33 0.26 0.36 0.45 0.36 0.51
1.62 2.07 1.56 1.70 1.68 1.54 3.77 3.77 1.59 1.86
1.57
Transcription factors EC907672 transcription factor; GASR AF281656 transcription factor; GASR
R 07?
R 15?
R 30?
R 60?
R 90?
1.51
1.87
1.94 1.96
1.79
0.59 0.61 0.65
0.49 0.64
1.80
1.55
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Table (Continued) (Continued) Bo¨rner time course Category/Gene ID
Riesling time course
B 07?
B 15?
B 30?
B 60?
B 90?
1.58 1.55
3.16 2.09 2.36 1.62 2.50
Pathogen-related genes U68144 beta-1,3-glucanase class 1; G1 AF239617 beta-1,3-glucanase class 1; cIG AY353062 beta-1,3-glucanase class 1; G1 AJ277900 beta-1,3-glucanase; G1 Z68123 acidic chitinase; ChiFIII U97521 endochitinase class IV; VvChi4A U97522 endochitinase class IV; VvChi4B AF532966 endochitinase class IV; Chi4D AY137377 endochitinase class IV; Chi4C Z54234 chitinase; Chi1 DQ267094 chitinase class I; Chi1 AF532965 thaumatin-like protein; Tl3 AJ237999* thaumatin-like protein; Tl1 AF465408 lipid transfer protein; LTP1 AY395741 lipid transfer protein; LTP1 AF061329 pathogenesis-related protein type 4; PR-4a DQ336289 pathogenesis-related protein 10; PR-10
0.53 0.50 0.36 0.48 0.30 0.31 0.28 0.57 0.22 0.57 0.64 0.21
0.53 0.64 0.45 0.58 0.60 0.43 0.46 0.43 0.65 0.62 0.64 0.28 0.62 0.55 0.33
0.50 0.56 0.53 0.50 0.49
0.49 0.59 0.61 0.50 0.28 0.40 0.27 0.54 0.26 0.58 0.56 0.63 0.30
Cell wall proteins AY046416 PRP1 AY046417 PRP2 AB074999 VXET1 AY043237 XET1 AF305093 PGIP
0.24 0.40 0.65 0.24
0.49 0.29
0.67 0.54
Universal stress-associated genes EC907662* cinnamylalcohol dehydrogenase, CAD AF194173 alcohol dehydrogenase 1; ADH1 AF194174 alcohol dehydrogenase 2; ADH2 CK906352 adventitious rooting related oxygenase; ARRO1 AF220407 dehydrin-like protein; Dhn
0.60 0.28 0.57 1.70
0.35 0.64
AY484579 AY509152 AY706986 AY156051 AY953543 AY538261
* transcription factor; WRKY4 (*) transcription factor; WRKY 30 (*)CBF-like transcription factor; CBF4 alanine acetyl transferase; AAT sucrose responsive element binding protein; SREBP (*) hexose transporter; HT5
R 07?
R 15?
R 30?
R 60?
R 90?
1.77 1.80 2.14
3.49 2.79 1.74 4.50
0.63 0.47 0.43 0.47 0.64 0.58 0.66
0.65 0.53 0.47
1.56
0.56 0.43 0.40 0.49 0.62 0.46 0.47
1.99 0.35
1.61
0.66
0.54 0.53 0.58
1.77
2.52 0.58
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Table (Continued) (Continued) Bo¨rner time course
Riesling time course
Category/Gene ID
B 07?
B 15?
B 30?
B 60?
B 90?
BE846416 ABA stress ripening protein; ASR AJ237988* ripening-related protein; grip21 AJ237991 Grip32 AJ237992 ripening-related bZIP protein; Grip55 AJ237987 ripening-related protein; Grip68 AJ237981 ripening-related protein; Grip3 EL930131 Hsp90
1.56 0.66 0.60 1.54
1.79
1.79
2.58 1.59
R 07?
R 15?
R 30?
R 60?
R 90?
2.36
