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First published online May 1, 2003; 10.1104/pp.103.021683 Plant Physiology 132:999-1010 (2003) © 2003 American Society of Plant Biologists Systemic Gene Expression in Arabidopsis during an Incompatible Interaction with Alternaria brassicicola1,[w]Cooperative Research Centre for Tropical Plant Protection (P.M.S., K.K., J.M.M., J.P.A., D.J.M.), and Department of Biochemistry and Molecular Biology (P.M.S., J.P.A., R.S.S., D.J.M.), The University of Queensland, St. Lucia, Queensland 4072, Australia; and Commonwealth Scientific and Industrial Research Organization Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia, Queensland 4067, Australia (K.K., J.M.M.); and Department of Plant Biology, Carnegie Institution of Washington, 260 Panama Street, Stanford, California 94305 (I.W.W., S.C.S.)
Pathogen challenge can trigger an integrated set of signal transduction pathways, which ultimately leads to a state of "high alert," otherwise known as systemic or induced resistance in tissue remote to the initial infection. Although large-scale gene expression during systemic acquired resistance, which is induced by salicylic acid or necrotizing pathogens has been previously reported using a bacterial pathogen, the nature of systemic defense responses triggered by an incompatible necrotrophic fungal pathogen is not known. We examined transcriptional changes that occur during systemic defense responses in Arabidopsis plants inoculated with the incompatible fungal pathogen Alternaria brassicicola. Substantial changes (2.00-fold and statistically significant) were demonstrated in distal tissue of inoculated plants for 35 genes (25 up-regulated and 10 down-regulated), and expression of a selected subset of systemically expressed genes was confirmed using real-time quantitative polymerase chain reaction. Genes with altered expression in distal tissue included those with putative functions in cellular housekeeping, indicating that plants modify these vital processes to facilitate a coordinated response to pathogen attack. Transcriptional up-regulation of genes encoding enzymes functioning in the  -oxidation pathway of fatty acids was particularly interesting. Transcriptional up-regulation was also observed for genes involved in cell wall synthesis and modification and genes putatively involved in signal transduction. The results of this study, therefore, confirm the notion that distal tissue of a pathogen-challenged plant has a heightened preparedness for subsequent pathogen attacks.
Plants have evolved a number of defense strategies to protect themselves from pathogen invasion. The hypersensitive response at the site of attempted infection is one of the most common features of disease resistance response in incompatible plant-microbe interactions. In many instances, the onset of this initial response also activates a signaling process that makes the plant not only locally, but also systemically, more refractory to subsequent infections by a broad spectrum of pathogens. This response is known as systemic acquired resistance (SAR) and is accompanied with activation of many plant genes (Ryals et al., 1996  ). The defense signal, salicylic acid (SA) is one key modulator of SAR. Although the specific role of SA as a systemic signal is still not clear, its accumulation in local and distal (uninoculated) tissues is accompanied with coordinated expression of a specific subset of defense genes (Malamy et al., 1990; Métraux et al., 1990; Maleck et al., 2000). Pathogen challenge also leads to the synthesis of other signaling molecules such as jasmonates and ethylene, which along with SA regulate distinct but overlapping patterns of defense gene expression (Reymond and Farmer, 1998; Feys and Parker, 2000; Schenk et al., 2000; Devadas et al., 2002). Recent evidence also suggests that there is an extensive overlap between the gene expression triggered by pathogen inoculation, defense-signaling compounds, and other physiological processes such as senescence (Quirino et al., 1999; Morris et al., 2000; He et al., 2002).
It has been hypothesized that systemic resistance may result from induced tissues being "primed" to provide a rapid and strong defense response following subsequent pathogen challenge (Conrath et al., 2002
We previously reported gene induction in Arabidopsis leaves locally challenged by an incompatible isolate of A. brassicicola and after treatment with defense regulators (Schenk et al., 2000
Time Course of PDF1.2 Expression in Distal and Local Tissue The aim of this study was to gain insights into transcriptional changes that occur in distal tissue during defense responses triggered by an incompatible fungal pathogen. Because this type of systemic response can potentially last several days, we first set out to identify a representative time point where full spectrum of systemic gene expression could be studied ("systemic maintenance period"). We hypothesized that the expression from known defense genes should be relatively high during this period to provide the plant a long-lasting and a broad spectrum of resistance. A well-established marker gene for the defense response in Arabidopsis is the PDF1.2 gene that encodes an antimicrobial peptide. A time-course study after inoculation with A. brassicicola was performed for this gene using real-time quantitative reverse transcriptase-PCR (RT-qPCR) and showed that induction occurred as early as 6 to 12 h after inoculation (Fig. 1A). Interestingly, no difference in the timing of the first induction of PDF1.2 was observed between local and distal tissue, suggesting that the signal for systemic activation of this gene travels quite rapidly. However, accumulation of the PDF1.2 transcript in time points after 12 h was much higher in local tissue (118-fold induction at 24 h) than that in distal tissue (4.3-fold induction at 24 h). The highest induction ratios of PDF1.2 compared with uninoculated control plants were found at 72 h for both local (202-fold induction) and distal (7.8-fold induction) tissue. Northern-blot analysis of the PDF1.2 gene confirmed significant expression at 72 h in locally inoculated and in distal leaf tissue of two independent inoculation experiments (Fig. 1B). Thus, a time point corresponding to 72 h after fungal inoculation was chosen to study the systemic responses during the maintenance period. It is important to note that genes that show specific induction during the early stages of systemic defense responses in this interaction may not be represented in our analysis.
Three independent inoculation experiments were performed, and total RNA was isolated from locally inoculated and uninoculated (distal) leaves as well as from uninoculated control plants, converted to cDNA, labeled with separate fluorescence dyes, and hybridized onto cDNA microarrays in binary comparisons of samples to equivalent controls in each replicate experiment. The cDNA microarray we used was previously described and contains 2,375 Arabidopsis expressed sequence tags (ESTs) with a bias toward putative defense-related and regulatory genes (Schenk et al., 2000 A total of 100 genes that showed differential expression with a mean change in expression greater than 2-fold in distal tissue as compared with equivalent tissue from uninoculated control plants were identified after analysis of microarray data, and also passed all other selection criteria for spot quantifications (see "Materials and Methods"). These included 83 induced and 17 repressed genes (Table I; supplementary table, which can be viewed at www.plantphysiol.org). Statistical analyses of microarray data across the three independent experiments showed that 35 of these genes had t test probability values higher than 95% (P < 0.05), suggesting that the expression values observed for these particular genes in different biological replicates were the most robust. Of these genes, 25 were up-regulated and 10 were down-regulated in the distal leaves (Table I). The PR1 and the PDF1.2 marker genes were among these 35 significantly altered genes and showed 2.55- and 2.86-fold increases in expression, respectively, with high significance values (P < 0.01). The induction ratios and other data for the remaining identified genes that showed probability values below 95% (P > 0.05) but otherwise met all other microarray data analysis criteria are shown in the supplementary table.
Twenty-seven of the genes with significantly altered systemic expression have been previously shown to be affected by SA and/or jasmonate treatment, and 15 of the significantly altered systemic genes were also altered in expression in the locally inoculated tissues at 72 h after inoculation (Schenk et al., 2000 ; see also Table I). This further suggested that the genes identified in this analysis were relevant to plant defense responses. Interestingly, there was a strong relationship between the direction of up- or down-regulation by these signals and the direction of change in the distal tissue. This is consistent with the results of Kazan et al. (2001) and Chapman et al. (2002), who have used cluster and principal component analysis, respectively, to examine the extent of overlap in pathogen- and chemically (SA, MJ, and ethylene) induced gene expression. These analyses have shown that there was a close relationship between jasmonate and ethylene responses. In addition, the systemic response showed a strong correlation with both SA- and MJ-induced responses (Kazan et al., 2001; Chapman et al., 2002), suggesting that both pathways play significant roles during the systemic response triggered by A. brassicicola inoculation.
We also compared the genes induced in Arabidopsis-A. brassicicola interaction with those reported by Maleck et al. (2000
To test whether induction of gene expression in distal tissue could be confirmed by another method, we performed RT-qPCR assays on a subset of 22 induced and 1 repressed genes selected from each putative functional group given in Table I. Cross-hybridization from closely related members of the gene families may be problematic in microarray experiments, whereas because of the use of specific primers, expression detected from qPCR experiments should be a true reflection of gene expression measured. These RT-qPCR experiments used RNA from three biological replicates that were previously used for microarray hybridizations and additional independent biological experiments (Table I). To allow a direct comparison of the response in inoculated and distal leaf tissue, RNA obtained from inoculated tissue was used in addition to the RNA from distal tissue of the same plants and the equivalent uninoculated control plants grown in parallel. Results from these RT-qPCR experiments generally confirmed the induction patterns of genes in distal tissue, which was usually accompanied by a similar response in locally inoculated tissue (Table I). Altered expression of three genes (At1g66140, At1g04680, and At1g06620) was not confirmed at 72 h after inoculation using RNA previously used for microarray analysis. However, further analysis of independent replicates by RT-qPCR confirmed altered expression of these genes in distal tissue. In addition, differences in the induction values obtained by these two methods of gene expression analysis were observed. In general, -fold induction values measured by RT-qPCR were lower than those measured by cDNA microarray hybridization. However, five genes, including three highly expressed genes (encoding PDF1.2, ATMPK3, and a putative cold acclimation protein), showed lower induction ratios in microarray experiments, possibly due to saturation of the hybridization signals measured. To obtain further expression data for individual members of gene families, three additional genes were selected that had close DNA sequence homology to the ESTs used in microarray hybridization experiments. These included three pairs of tandemly linked genes encoding putative disease resistance proteins (At1g33590 and At1g33600), polygalacturonase inhibitors (At5g06860 and At5g06870), and cold stress-associated proteins (At1g20440 and At1g20450). These experiments showed induction of all three gene pairs in both local and distal tissues (Table I). However, there were substantial differences in the levels of induction between different family members. For example, At1g33600 showed significantly higher induction (4.35-fold) in distal tissue than the gene with closest DNA sequence homology (84%) to the EST used in microarray hybridizations (At1g33590; 1.93-fold). It appears likely, therefore, that transcripts of At1g33600 cross-hybridized to the cDNA of this EST on the microarrays, contributing to the overall induction ratio of 4.00 (Table I).
Although it was not possible to test the expression patterns of all genes identified by RT-qPCR due to time and cost involved, some genes from each functional group were chosen for further expression profiling. For this purpose, two additional inoculation experiments were carried out (independent from previous microarray experiments). In these experiments, samples of locally inoculated and distal leaf tissue together with tissue from control plants were collected at 48 and 72 h after inoculation with A. brassicicola. Additional samples were collected for one replicate at 1, 3, 6, 12, and 24 h to allow for further characterization of selected genes which also included three genes (encoding putative pectate lyase [At1g04680], acyl-CoA synthetase [At4g23850], and a multifunctional protein [At5g06860]) that were of particular interest but that did not pass the initial microarray data analysis criteria. The results obtained by RT-qPCR from these independent replicates confirmed the induction patterns in systemic tissue of the genes tested at either 48 or 72 h after inoculation (Table I). However, expression for some genes was higher at 48 h than at 72 h (e.g. genes encoding a basic helix-loop-helix protein or desacetoxyvindoline 4-hydroxylase), suggesting that some variability of gene expression occurred for these independent experiments despite careful control of the experimental procedures, including the plant growth and incubation environment. Variability observed in gene induction values between the replicates may have also contributed to the finding that only 35 of 100 genes that showed a mean of 2-fold alteration in gene expression were significant at 95% probability in statistical analysis. This indicates the importance of substantial experimental replication to obtain reliable results, an issue that has been overlooked in some studies.
Putative Disease Resistance and Regulatory Genes
Strong induction in distal tissue was also observed for several putative regulatory genes, e.g. encoding a putative Arabidopsis response regulator (ARR1-like) and a basic helix-loop-helix protein. Recently, we have undertaken functional studies on the latter gene showing that this gene is likely to play a role in the regulation of the jasmonate-dependent defense responses (J.P. Anderson, P.M. Schenk, K. Kazan, and J.M. Manners, unpublished data). We also identified two genes encoding putative mitogen-activated protein (MAP) kinases (AtMPK3 and AtMEKK1) induced in distal tissue but at relatively low significance (P = 0.06 and P = 0.12, respectively; Table I; supplementary table). MAP kinases are known to be involved in transducing extracellular stimuli into intercellular responses and confer resistance to both bacterial and fungal pathogens via a signaling cascade that has recently been found to be remarkably conserved among plants, insects, and mammals (Kovtun et al., 2000
Cell Wall Modifications
Our microarray analysis also identified a gene (AtPGIP2, At5g06870; Table I) encoding a polygalacturonase inhibitor, an enzyme specifically inhibiting fungal endogalactorunases and located within the Arabidopsis cell wall (Ferrari et al., 2003
Fatty Acid Metabolism
We further examined the activation of the genes encoding enzymes involved in the
Heightened fatty acid metabolism may be significant in priming the synthesis of plant defense-signaling molecules such as jasmonic acid (JA) and other oxylipins (Graham and Eastmond, 2002
To further explore the relationship between
Genes Involved in Secondary Metabolism, Cell Maintenance, and Development
Interestingly, a gene putatively involved in indole metabolism (desacetoxyvindoline 4-hydroxylase) was induced significantly. Additionally, at a lower significance (P = 0.07), Trp decarboxylase was also induced. Although involvement of Trp decarboxylase in the synthesis of the Arabidopsis phytoalexin (camalexin) is not yet known, this enzyme is involved in the biosynthesis of pharmaceutically important monoterpenoid indole alkaloids in opium poppy (Papaver somniferum) and Madagascar periwinkle (Catharanthus roseus; Verpoorte and Memelink, 2002
A gene encoding a malic enzyme isoform was among the housekeeping genes that were induced after inoculation with A. brassicicola. NADP-malic enzymes from maize (Zea mays) and cucumber (Cucumis sativus) have recently been found to accompany other plant defense responses (Havelda and Maule, 2000
A gene showing homology the rice (Oryza sativa) OsNAC6 gene, which is thought to play a role in plant development (Kikuchi et al., 2002 In conclusion, the results of the microarray experiments described here supports the notion that plant defense is a complex physiological event with potential involvement of many genes. This complexity is further enhanced by the fact that resistance against most necrotrophic fungal pathogens does not follow a gene for gene-type interaction but rather is influenced by many genes with relatively smaller effects. In this context, some of the genes reported here might have more direct or specific effects on the final outcome of the interaction between Arabidopsis and A. brassicicola, whereas induction of other genes may be as a result of an overall stress response triggered by the pathogen inoculation. Our results suggest that alterations that occur in the transcription of genes involved in pathogen perception, signal transduction, cell wall modification, fatty acid metabolism, and secondary metabolism may be key processes associated with the systemically activated primed state.
Inoculation Experiments Arabidopsis plants were grown to eight- to 12-leaf stage in controlled environment rooms (24°C–20°C day and night temperature) and a photo-period of 8 h light (170 µE m-2 s-1) under transparent covers (Yates, Brisbane, Australia) and were treated with either defense-inducing chemical signal compounds or fungal pathogens. All treatments were carried out at 1 h after the start of the illumination period. To identify genes whose expression changes during plant defense responses in uninoculated (distal) tissue, we inoculated three to four leaves on one side of the rosette of 6-week-old Arabidopsis cv Columbia plants (200 plants replicate-1) with 5-µL drops of a freshly prepared spore suspension of (105 spores mL-1) Alternaria brassicicola (isolate UQ4273; freshly grown on agar plates containing clarified V8 vegetable juice (Campbell Soup Company, Camden, NJ). The remaining leaves of the rosette were left uninoculated, and plants were incubated under a transparent dome to provide high humidity. Uninoculated distal leaves of the same age as the locally inoculated leaves were collected opposite of the inoculation side. This inoculation system ensured that no accidental cross-contamination of distal uninoculated leaves with A. brassicicola spores occurred. Uninoculated control plants were grown in parallel under the same conditions, and leaf tissue was collected at the same time as for the inoculated plants. A total of five completely independent experiments were performed on separate occasions to provide replicate RNA samples for either statistical analysis of microarray data or RT-qPCR assays.
The methods of total RNA isolation, northern-blot analysis, preparation of probes, hybridizations, and scanning of slides have been described previously (Schenk et al., 2000 To monitor and assess differential gene expression, normalized signal intensities were used to calculate induction or repression ratios as well as normalized differences using Perl scripts. Genes that showed induction or repression ratios of at least 2.00, normalized differences of at least 0.500, and t test probabilities of at least 80% are reported (Table I; supplementary data). The genes that did not meet these criteria have been removed from the data set (except for a gene encoding a multifunctional protein [T43247] whose induction ratio [1.69] was below 2.00 but was highly significant [P < 0.01]). It is, therefore, probable that due to the stringent criteria used in data analysis, our results may underestimate the extent of altered gene expression in distal tissue.
Results from microarray experiments were also validated for a subset of selected genes by RT-qPCR experiments using either the same RNA samples used for microarray experiments or RNA isolated from two additional biological replicates (e.g. plants grown and inoculated separately from microarray experiments). In these experiments, we collected Arabidopsis leaf tissue samples for RNA isolations at 48 and 72 h after inoculation. Additional samples were taken for one biological replicate at 1, 3, 6, 12, and 24 h after inoculation. Leaf tissue from 50 plants each of Arabidopsis cpr5 mutant and wild-type cv Columbia (grown in parallel) plants was collected at the eight- to 12-leaf stage for RNA preparation.
For RT-qPCR experiments, 5 µg of total RNA was denatured at 70°C for 5 min followed by quick chill on ice in a 13-µL reaction containing 10 ng of anchored oligo(dT) 23-mers, 4.5 ng of random hexamer primers (Invitrogen, Carlsbad, CA), and 0.5 µL of 20 mM dNTPs. After the addition of 4 µL of 5x reaction buffer (Invitrogen) and 2 µL of 0.1 M dithiothreitol, the reaction was preheated to 42°C for 2 min before adding 1 µL (200 units) of Superscript II reverse transcriptase (Invitrogen) followed by incubation at 42°C for another 50 min. After terminating the reaction at 70°C for 15 min, the resulting cDNA was subsequently taken up in a volume of 500 µL, and SYBR green-labeled PCR fragments were amplified by using gene-specific primers designed from the coding sequence and over an RNA splice junction (if available) of each gene using the Primer Express 1.5 software (Applied Biosystems, Foster City, CA). RT-qPCR using the ABI PRISM 7700 sequence detector and SYBR Green Master mix (Applied Biosystems) was carried out using primers (listed in Table II) at a final concentration of 0.28 µM each and 1 µL (the equivalent of 10 ng total RNA) of cDNA as template. PCR-cycling conditions comprised an initial polymerase activation step at 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 59°C for 1 min. Real-time DNA amplification was monitored and analyzed using the Sequence Detector 1.7 program (Applied Biosystems). Differences in cycle numbers during the linear amplification phase between samples containing cDNA from treated and untreated plants were used to determine differential gene expression, each cycle representing a 2-fold change in template abundance. SEs of the means were calculated using the JMP In statistics software (SAS Institute Inc.). Expression detected from three
The sensitivity and accuracy of transcript abundance detection was examined by using a dilution series of 1-, 1.5-, 2-, and 3-fold template, set up as triplicates to detect transcript levels of At2g28040 encoding a receptor protein kinase (primers 5'-CTGCTCTTCTCTTAAATTGTTAGTTTGTCTC-3' and 5'-GCTTATTAGCCTCATGCTTTAAAATCTTGA-3') Template changes of 1.33-, 1.50-, 2.00-, and 3.00-fold were detectable as 1.36 ± 0.13, 1.53 ± 0.08, 2.07 ± 0.09, and 3.34 ± 0.09, respectively (shown as average ± SDs, assuming that each amplification cycle difference corresponded to a 2-fold change).
We thank Dr. Chris Somerville for kind provision of the Arabidopsis EST collection, Dr. Todd Richmond for use of the Perl scripts, and the Arabidopsis Biological Resource Center for supplying seeds of cpr5 mutant. We are grateful to Drs. Paul Ebert and Luis Oñate-Sanchez for critical reading of the manuscript. Received February 4, 2003; returned for revision February 26, 2003; accepted March 13, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.021683.
1 This research was partly supported by the Grains Research and Development Corporation of Australia (to J.M.M.) and by the U.S. Department of Energy and the Carnegie Institution of Washington (to S.C.S.).
[w] The online version of this article contains Web-only data. The supplemental material is available at http://www.plantphysiol.org.
2 Commonwealth Scientific and Industrial Research Organization Plant Industry, G.P.O. Box 1600, Canberra, ACT 2601, Australia. * Corresponding author; e-mail p.schenk{at}tpp.uq.edu.au; fax 61–7–3365–4771.
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ABSTRACT
RESULTS AND DISCUSSION
