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First published online May 27, 2005; 10.1104/pp.105.060947 Plant Physiology 138:1027-1036 (2005) © 2005 American Society of Plant Biologists Loss-of-Function Mutations in Chitin Responsive Genes Show Increased Susceptibility to the Powdery Mildew Pathogen Erysiphe cichoracearum1,[w]Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama 35487–0344 (K.R.); Department of Plant Biology, Carnegie Institution, Stanford, California 94305 (M.B.-L., S.K., S.S.); National Center for Soybean Biotechnology, Divisions of Plant Sciences and Biochemistry, University of Missouri, Columbia, Missouri 65211 (J.W., G.S.); and Department of Biological Sciences, Western Illinois University, Macomb, Illinois 61455–1390 (H.E.)
Chitin is a major component of fungal walls and insect exoskeletons. Plants produce chitinases upon pathogen attack and chito-oligomers induce defense responses in plants, though the exact mechanism behind this response is unknown. Using the ATH1 Affymetrix microarrays consisting of about 23,000 genes, we examined the response of Arabidopsis (Arabidopsis thaliana) seedlings to chito-octamers and hydrolyzed chitin after 30 min of treatment. The expression patterns elicited by the chito-octamer and hydrolyzed chitin were similar. Microarray expression profiles for several genes were verified via northern analysis or quantitative reverse transcription-PCR. We characterized T-DNA insertion mutants for nine chito-oligomer responsive genes. Three of the mutants were more susceptible to the fungal pathogen, powdery mildew, than wild type as measured by conidiophore production. These three mutants included mutants of genes for two disease resistance-like proteins and a putative E3 ligase. The isolation of loss-of-function mutants with enhanced disease susceptibility provides direct evidence that the chito-octamer is an important oligosaccharide elicitor of plant defenses. Also, this study demonstrates the value of microarray data for identifying new components of uncharacterized signaling pathways.
Plants in the environment are constantly under siege by a multitude of disease-causing organisms including bacteria, fungi, viruses, and nematodes. Plants may resist pathogen attack using both preformed defenses (e.g. antimicrobial compounds) and inducible defense responses (for review, see Hammond-Kosack and Jones, 2000  ; Heath, 2000). Inducible defenses can be activated upon recognition of general elicitors such as bacterial flagellin (Gomez-Gomez and Boller, 2002), the polypeptide systemin (Ryan and Pearce, 1998), and multiple host or pathogen cell wall fragments released during pathogen attack (Nürnberger et al., 2004). Recently, oligosaccharide elicitors such as chito-oligomers and oligogalacturonides have received renewed attention as important signals in plant defense responses. The activation of defense genes by these elicitors is thought to be receptor-mediated though little is known about the initial perception and consequent signaling pathways involved in plant cells.
Chito-oligosaccharides can be generated from the cell walls of pathogenic fungi by the action of endochitinases and were shown to elicit strong defense responses in many plant species (Stacey and Shibuya, 1997
In previous experiments, we used a complex mixture of chito-oligomers derived from hydrolyzed crab-shell chitin (CSC) to induce defense responses in Arabidopsis (Ramonell et al., 2002
Chitin Is Present at the Tips of Growing Hyphae and in Conidia of E. cichoracearum during Infection of Arabidopsis
Chitin is one of the major cell wall components in many fungi. Fungal cell walls change during growth and development and the deposition and subsequent removal of chitin is particularly important during septation (Smits et al., 2001
Chito-Oligomers Elicit Distinct Gene Expression Profiles in Arabidopsis
To better understand the responses of Arabidopsis to chito-oligomers, we compared the gene expression of seedlings treated with chito-octamer, CSC, or water as a control using Affymetrix ATH1 full-genome microarrays. Of the approximately 23,000 genes represented on the array, 5,012 genes showed altered expression according to the statistical analysis of microarrays (SAM) program with a false discovery rate of
The chito-octamer treatment induced genes for several defense-related proteins including pathogenesis-related protein 5, two WRKY transcription factors, and four disease resistance proteins (three of the Toll Interleukin-1 receptor [TIR] or TIR-nucleotide-binding site [NBS]-Leu-rich repeat [LRR] class, and the other an LRR class protein). The largest class of genes repressed in the chito-octamer treatment were unknown or hypothetical proteins, but several interesting genes were repressed 1.5-fold including genes encoding ETR2, an LRR transmembrane protein kinase, the plant defensin protein PDF 2.5, a jasmonate-inducible protein, and several zinc-finger proteins potentially involved in signaling. Treatment with chito-octamer was shown previously to elicit strong defense responses in numerous plant species (Yamada et al., 1993 ; Zhang et al., 2002). These data indicate that the chitin octamer is inducing a strong defense response in Arabidopsis that is consistent with data previously observed. It is interesting to note that the chito-octamer induced and repressed several genes involved in plant defense. The plant defensin protein PDF 2.5 was down-regulated, while several disease resistance-like proteins and a WRKY transcription factor were stimulated. It is unclear why defense-related genes were repressed by the chitin octamer. One explanation may be that other signals in addition to the presence of chitin may be necessary to elicit an appropriate defense response to a specific pathogen. Large numbers of genes were either induced or repressed (1,002 and 312, respectively) by both CSC and the chito-octamer. Many of the genes induced by CSC and the chito-octamer were defense related, which is expected in light of data from previous studies (Yamada et al., 1993; Ramonell et al., 2002; Zhang et al., 2002). However, there was no clear trend in the distribution of genes that were repressed by both treatments. Clearly, a combination of chito-oligomer fragments elicits a strong defense response, but a clearer understanding of the corresponding repression of genes will require more study.
In the CSC treatment alone, a large number of genes coding for defense-related proteins were induced 1.5-fold including numerous disease resistance-like proteins, WRKY family transcription factors, and an LRR protein kinase. Several cell wall-related transcripts including a subunit of cellulose synthase and a xyloglucan endotransglycosylase were also induced. Genes for several transport proteins (ATP-binding cassette transporter family, integral membrane protein) and some cell wall extensin-related proteins were repressed 1.5-fold in the CSC treatment, as well as genes encoding two disease-resistance proteins, RAR1 (Required for Mla Resistance 1), an LRR protein kinase, and a geranyl-geranyl phosphate synthase involved in the biosynthesis of defensive terpene compounds. Several classes of signaling molecules, protein kinases, and zinc-finger proteins included both induced and repressed genes in the CSC treatment. In several experiments, CSC was shown to induce the strongest changes in gene expression (Zhang et al., 2002
To validate the changes in gene expression revealed by the microarray experiments, the following categories of genes were selected based on the microarray data for verification tests in northern-blotting experiments (Fig. 3A): (1) four genes up-regulated by CSC and chito-octamer: AtMPK3 (MITOGEN-ACTIVATED PROTEIN KINASE 3 gene, At3g45640), a lectin-like protein gene (At3g16530), ZAT12 (zinc finger protein 12 gene, At5g59820), and a salt tolerance zinc finger protein gene (At1g27730); (2) a gene down-regulated by CSC and the chitin octamer: a putative auxin-regulated protein gene (At2g21200); and (3) a gene not regulated by these treatments: AtERF3 (ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 3 gene, At1g50640). The same RNA samples used in the microarray experiments were used in the northern-blotting analyses, and a gene-specific probe was used for each hybridization. To make a direct comparison between the microarray data and the corresponding northern results, the image was quantified and the fold-change relative to a water control for each gene was calculated with the ACTIN2 gene serving as a loading control. The fold-change values determined from the microarray data (Fig. 3A, white bars) and the northern-blot experiments (Fig. 3A, black bars) correlated well, suggesting that our current microarray data is of good quality.
In addition, the transcript levels of two genes, At2g35000, encoding an E3 ligase-like protein, and At5g25910, encoding a putative disease resistance protein, were determined in independent RNA samples by reverse transcription (RT)-PCR methods. The induction of both genes after treatment with CSC for 30 min was confirmed by RT-PCR (Fig. 3B). Little or no induction of either gene was observed after 15 min of treatment. Quantitative RT-PCR was used to confirm that these two genes were induced by 30 min of treatment with CSC as was observed in the microarray experiments, although the fold-induction values observed in the two experiments differed somewhat (Fig. 3C). The mock value observed for At5g25910 was unusually variable, likely due to the very low expression levels of this gene in mock treated seedlings and the difficulty of reliably measuring these low expression levels.
From the microarray data, nine genes (At2g28200, At2g02950, At2g34930, At2g35000, At5g25910, At4g11830, At2g41940, At4g16820, and At3g57640) were selected on the basis of their expression profiles for further analysis. A search of the insertion sequence database of the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu) was performed to identify T-DNA insertional mutants with insertions in the nine genes of interest. All lines were screened to identify homozygous mutants. One backcross was performed on all T-DNA insertional mutants with wild-type Columbia (Col-0).
If we assume that chito-oligomer fragments are one of the nonspecific signals or pathogen-associated molecular patterns, exchanged between host and pathogen during fungal and insect attack (Nürnberger et al., 2004
At low-density inoculations, two mutants in genes At2g35000 and At5g25910 had more severe macroscopic symptoms compared with Col-0 wild-type plants (Fig. 4A). In contrast, the other seven insertion mutants exhibited a wild-type (Col-0) disease phenotype when challenged by low-density inoculum (data not shown). In addition, there was a clear increase in the growth of fungal hyphae on both mutants (Fig. 4B) compared to growth on Col-0 wild-type plants, though the fungal growth was not as advanced as that on transgenic NahG plants (Fig. 4B). Independent SALK insertion mutants for each of At2g35000 (Salk_066755, Salk_036065, Salk_036066) and At5g25910 (Salk_125444, Salk_107922) showed enhanced disease susceptibility, confirming that the altered disease phenotype can be attributed to the loss of function of At2g35000 and At5g25910 and not to a secondary mutation in the SALK lines (Supplemental Fig. 1). To measure disease development more precisely, conidiophores per colony (c/c) were counted 4 d postinoculation (dpi) and conidiophores per mm–2 (c/mm2) leaf surface were counted at 5 dpi for each of the mutants (Table I). Three of the T-DNA insertion lines, At2g34930, At2g35000, and At5g25910, were significantly more susceptible to powdery mildew than Col-0, Ler, and Kas-1 accessions. However, these three mutants were not as susceptible as transgenic NahG plants, which are impaired in the salicylic acid defense signaling and are known to be hypersusceptible to powdery mildew (Reuber et al., 1998
Chitin fragments may be generated by the action of plant chitinases on fungal or insect cell walls (Boller, 1995 ) and are known to elicit responses associated with plant defense (Stacey and Shibuya, 1997; Shibuya and Minami, 2001). In a previous study, we demonstrated that Arabidopsis responded to treatment with chito-oligomers by initiating a defense response similar to that of other plant species (Ramonell et al., 2002) and that the chitin response pathway did not appear to be operating through any of the well-characterized defense pathways (i.e. salicylic acid, jasmonate, or ethylene; Zhang et al., 2002). To better understand the chitin response, we treated Arabidopsis seedlings with chito-octamer or CSC and examined expression profiles using full-genome microarrays. The pattern of gene expression elicited by the chito-octamer and the CSC mixture was very similar and included a number of up-regulated defense associated genes. In rice and soybean, longer chitin oligomers, such as the chito-octamer, elicit a variety of defense-related cellular responses (Stacey and Shibuya, 1997; Shibuya and Minami, 2001). Both the chito-octamer and CSC repressed the expression of genes for several defense-related proteins (e.g. RAR1, LRR kinases, PDF 2.5, jasmonate-inducible protein). It is unclear why some defense-associated genes are induced and other genes are repressed by CSC or chito-octamer treatment. CSC consists of an undefined mixture of chito-oligomers of varying lengths up to about chito-octamer (oligomers with a higher dp are thought to be insoluble in water) and chito-oligomers of different sizes may induce or repress distinct sets of genes. Analogous to the negative cross-talk between the salicylic acid and jasmonate pathways (Dong, 2004), the purified chito-octamer may induce a specific set of defense-related genes and repress other defense-related genes under the regulation of a competing pathway. A subset of the genes responding to the two chito-oligomer treatments were identified, T-DNA insertional mutations in nine of these genes were obtained and the response of the mutants to the powdery mildew pathogen E. cichoracearum was examined. Of the nine, three mutants were isolated that were more susceptible to powdery mildew: At2g35000 (a putative RING zinc-finger protein), At2g34930, and At5g25910 (putative disease resistance-like proteins). The enhanced disease susceptibility of the mutants in At2g35000 and At5g25910, but not in the remaining seven genes, was also apparent visually following low-density inoculations with the powdery mildew pathogen, suggesting that either these two genes play a larger role in defense against powdery mildew or they more directly influence chitin-activated defense signaling.
The At2g35000 gene product corresponds to a putative RING zinc-finger protein belonging to the Arabidopsis Tóxicos en Levadura (ATL) gene family. Several important Arabidopsis genes with RING domains have been identified including COP1, a photomorphogenesis regulator (Torii et al., 1999
Both At2g34930 and At5g25910 correspond to two uncharacterized disease resistance-like proteins containing LRR regions. The most widely studied LRR-containing genes involved in plant defense are the plant disease resistance (R) proteins (Martin et al., 2003
Using genomics techniques, we identified several genes that may play a role in the chitin-mediated defense pathway in Arabidopsis. Our results showed that T-DNA insertions in three genes, At2g35000 (a putative RING zinc-finger protein), At2g34930, and At5g25910 (putative disease resistance-like proteins) resulted in plants that were more susceptible to powdery mildew than wild type, indicating that these genes may play a role in the defense response of Arabidopsis to powdery mildew. Thus, the general elicitor, chitin, has been linked to a defense response in plants indicating that it plays a role in plant responses to fungal pathogens. In addition, this study demonstrates the power of microarray data for identifying potential targets for mutation in uncharacterized signaling pathways. Additional experiments will now be necessary to elucidate the precise functions of these genes in defense responses and chito-oligomer recognition in Arabidopsis.
Plant Growth Conditions and Chitin Treatments
For microarray analysis and northern hybridization, Arabidopsis (Arabidopsis thaliana) L. Col-0 seeds were sterilized and grown hydroponically according to Zhang et al. (2002)
Seeds of T-DNA insertion lines, Col-0, Kas-1 (Wilson et al., 2001 Chitin treatments of seedlings for the RT-PCR analysis were as follows: seeds were surface sterilized using 75% ethanol followed by 50% bleach and a water rinse. Approximately 500 seeds (10 mg) were grown per Erlenmeyer flask in 125 mL of Murashige Skoog medium (Sigma), at 2 g/L, pH 5.7 supplemented with Gambourg vitamins (Sigma). Erlenmeyer flasks with the seeds were incubated in the cold for 6 d and then were placed in a shaking incubator at 150 rpm for 3 weeks under constant illumination (125 µmol m–2 s–1) at 23°C. After 14 d, hydrolyzed CSC (Sigma) was added to the medium containing the seedlings at a final concentration of 100 µg/mL. Whole seedlings were collected 30 min after treatment, immediately frozen in liquid N2, and stored at –80°C for later use.
Three-week-old Arabidopsis plants were inoculated with the powdery mildew pathogen, Erysiphe cichoracearum UCSC1 (Adam and Somerville, 1996
To visualize fungal hyphae, infected leaves were cleared in ethanol for 2 h and then stained with 250 µg/mL trypan blue in a solution of lactic acid, glycerol, and water (1:1:1) for 15 min and mounted with 60% glycerol solution (v/v) on microscope slides. Individual colonies were photographed using a Nikon Eclipse E600 camera and images were analyzed with the Spot Advance 32 program attached to a Leica microscope. The size of the microscopic image was calculated using a microscopic ruler in order to quantify the numbers of conidiophores per millimeter squared. A Student's t test was performed using Excel (Microsoft, Seattle) to identify ratios that were significantly different from those in wild-type plants. Both conidiophores/colony and conidiophores per millimeter squared were measured. Photographs of whole plants and infected leaves were taken using a Nikon Coolpix E995 digital camera.
For experiments performed using confocal microscopy, 3-week-old Arabidopsis (Col-0) was infected with E. cichoracearum UCSC1 and cut rosette leaves were stained with WGA-AlexaFluor488 conjugate (Molecular Probes, Eugene, OR) at a concentration of 10 µg/mL (w/v) in 1x phosphate-buffered saline (PBS), pH 7.4, and 0.1% silwet for an hour (up to 24 h) at room temperature. Leaves were then washed twice in a solution of 1x PBS, pH 7.4, and 0.01% silwet. The WGA stained and washed leaves were counterstained with propidium iodide (PI; 10 µg/mL) in the same buffer used for both WGA stain and wash. PI stained fungal structures and WGA-AlexaFluor488 stained chitin were visualized simultaneously by Bio-Rad MRC 1024 laser scanning confocal microscope at 568 nm and 488 nm (excitation by Ar/Kr laser) and 598 nm and 522 nm (emission), respectively (Bio-Rad, Hercules, CA). Z-series images were acquired by Lasersharp (Bio-Rad) and merged in Confocal Assistant (version 4.02, Bio-Rad). For electron micrographs, Arabidopsis leaves infected for 24 h with E. cichoracearum UCSC1 were cut into small pieces and fixed in 3% glutaraldehyde (0.05 M phosphate buffer, pH 6.8) for 1.5 h. Tissue was rinsed in buffer and post fixed in 1% OsO4 in buffer for 2 h, rinsed in buffer, dehydrated in an ethanol series, and embedded in Spurr's medium. Thin sections were cut on a Reichert ultramicrotome, picked up on gold-gilded grids and floated on 0.01 M potassium phosphate buffered saline (0.15 M NaCl) adjusted to pH 7.4 containing 0.02 µg/mL of Tween 80 (KPBST) for 5 min. Grids were transferred to KPBST containing WGA-gold (15 nm) complex for 1 h. Control grids were transferred to KPBST containing WGA-gold complex containing N-acetyl-D-glucosamine (0.09 M). Grids were rinsed in a steady stream of buffer followed by a similar rinse in distilled water. After drying, grids were stained with uranyl acetate and lead citrate and viewed in a Philips 201 TEM.
Total RNA was isolated from each sample using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and was further purified using the Qiagen RNeasy Mini Kit (QIAGEN, Valencia, CA).
For northern hybridizations, 10 µg of total RNA was separated on a denaturing, formaldehyde, 1% agarose gel and vacuum blotted onto Zeta-Probe GT blotting membrane (Bio-Rad Laboratories, Hercules, CA). Gene-specific probes were made with a Prime-a-Gene Labeling System (Promega, Madison, WI) from the genes listed below. Mitogen-activated protein kinase 3 (AtMPK3) gene (At3g45640), a lectin-like protein gene (At3g16530), and ACTIN-2 (At3g18780) were from Zhang et al. (2002)
Genomic DNA for PCR reactions was extracted using the method described by Dellaporta et al. (1983)
Quantitative RT-PCR experiments were done using the SYBR Green qPCR kit (Finnzymes, Espoo, Finland) at a final volume of 20 µL with the cycle protocol recommended by the manufacturer. Samples were run using a DNA Engine Opticon 2 system with a PTC-200 DNA Engine Cycler and a CFD-3220 Opticon 2 detector (MJ Research, Waltham, MA). Reactions were carried out using primers at a final concentration of 0.2 µM and 1.5 µL of cDNA as a template. PCR cycling conditions were composed of an initial step at 95°C for 3 min followed by 94°C for 10 s and 62°C for 20 s, 72°C for 24 s before the first plate reading, and a step of 82°C for 1 s, before the second reading, during 40 cycles. All cDNA samples were assayed in triplicate. Data from the quantitative RT-PCR was analyzed using Opticon Monitor Analysis software (version 2.01, MJ Research), and Microsoft Excel (Microsoft). Statistical analysis of data was done as described by Livak and Schmittgen (2001)
The location of the T-DNA insertions within each line was originally identified at the Salk Institute Genomic Analysis (La Jolla, CA) and confirmed using the protocols described by Siebert et al. (1995)
Total RNA samples were processed according to manufacturer's protocols with the following modifications (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix, Santa Clara, CA). Single-stranded, then double-stranded cDNA was synthesized from the poly(A+) mRNA present in the isolated total RNA (20 µg of total RNA starting material for each sample reaction) using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Carlsbad, CA) and custom poly (T)-nucleotide primers that contained a sequence recognized by T7 RNA polymerase. All of the resulting double-stranded cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction, using the Bio-Array High-Yield RNA Transcript Labeling kit (Enzo Diagnostics, Farmingdale, NY). Twenty micrograms of the resulting biotin-tagged cRNA was fragmented to strands of less than 100 bases in length following prescribed protocols (Affymetrix GeneChip Expression Analysis Technical Manual). The fragmented target cRNA (20 µg) was hybridized at 45°C with rotation at 60 rpm for 16 h (Affymetrix GeneChip Hybridization Oven 640) to probe sets present on an Affymetrix ATH1 GeneChip array. The GeneChip arrays were washed and then stained (streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 400, followed by scanning on a Hewlett-Packard GeneArray scanner (Hewlett-Packard, Palo Alto, CA). Image analysis and pixel intensity were quantified using MicroArray Suite 5.0 software (Affymetrix). Text files were then generated and exported into GeneSpring 6.0 software (Silicon Genetics, Redwood City, CA) for normalization and further analysis. Three biological replicates were performed per treatment.
Text files containing raw data were imported into GeneSpring 6.0 (Silicon Genetics, Redwood City, CA) and were normalized as follows. First, values below 0.01 were set to 0.01. Each measurement was then divided by the 50th percentile of all measurements in that sample. Specific samples were then normalized to one another: samples 1 to 9 (CSC, chito-octamer, and water treatment, three replicates of each treatment) were normalized against the median of the control samples (water treatment). Each measurement for each gene in those specific samples was divided by the median of that gene's measurements in the corresponding control samples.
These data on all genes were then extracted and analyzed for significance using the SAM software (Tusher et al., 2001
We thank D. Alexander (Carnegie Institution) for help with the quantitative RT-PCR reactions and B.-H. Hou and S. Thayer (Carnegie Institution) for assistance with the microarray experiments. We also thank Dr. Shibuya (National Institute of Agrobiological Resources, Tsukuba, Japan) for providing the chito-octamer, and the ABRC for providing seed stocks. Received February 9, 2005; returned for revision March 17, 2005; accepted March 17, 2005.
1 This work was supported by the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (grant no. DE–FG02–02ER15309 to G.S.), by the National Science Foundation (grant no. 0114783 to S.S.), by the Carnegie Institution (to S.S. and M.B.-L.), by the Spanish government (fellowship to M.B.-L.), and by the University of Alabama (to K.M.R.). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.060947. * Corresponding author; e-mail kramonel{at}bama.ua.edu; fax 205–348–1768.
Adam L, Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsis thaliana. Plant J 9: 341–356[CrossRef][ISI][Medline]
Adams DJ (2004) Fungal cell wall chitinases and glucanases. Microbiol 150: 2029–2035
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657 Boller T (1995) Chemoperception of microbial signals in plant cells. Annu Rev Plant Physiol 46: 189–214[CrossRef][ISI] Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19–20
Dixon MS, Hatzixanthis K, Jones DA, Harrison K, Jones JD (1998) The tomato Cf-5 disease resistance gene and six homologs show pronounced allelic variation in leucine-rich repeat copy number. Plant Cell 10: 1915–1925 Dong X (2004) NPR1, all things considered. Curr Opin Plant Biol 7: 547–552[CrossRef][ISI][Medline]
Edgar R, Domrachev M, Lash AE (2002) Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30: 207–210 Galun M, Braun A, Frensdorff A, Galun E (1976) Hyphal walls of isolated lichen fungi: autoradiographic localization of precursor incorporation and binding of fluorescein-conjugated lectins. Acta Microbiol 108: 9–16 Gomez-Gomez L, Boller T (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 6: 1003–1011 Gomez-Gomez L, Boller T (2002) Flagellin perception: a paradigm for innate immunity. Trends Plant Sci 7: 251–256[CrossRef][ISI][Medline] Hammond-Kosack K, Jones JDG (2000) Response to plant pathogens. In B Buchanan, D Gruissem, R Jones, eds, Biochemistry and Molecular Biology of Plants. American Society of Plant Biologists, Rockville, MD, pp 1102–1156 Heath MC (2000) Nonhost resistance and nonspecific plant defenses. Curr Opin Plant Biol 3: 315–319[CrossRef][ISI][Medline] Kinoshita T, Cano-Delgado AC, Seto H, Hiranuma D, Fujioka S, Yoshida S, Chory J (2005) Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1. Nature 433: 167–171[CrossRef][Medline] Lawton K, Weymann K, Friedrich L, Vernooij B, Uknes S, Ryals J (1995) Systemic acquired resistance in Arabidopsis required salicylic acid but not ethylene. Mol Plant Microbe Interact 8: 863–870[ISI][Medline]
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(- Martin GB, Bogdanove AJ, Sessa G (2003) Understanding the functions of plant disease resistance proteins. Annu Rev Plant Biol 54: 23–61[CrossRef][Medline] Martinez-Garcia M, Garcidueñas-Piña C, Guzmán P (1996) Gene isolation in Arabidopsis thaliana by conditional overexpression of cDNAs toxic to Saccharomyces cerevisiae: identification of a novel early response zinc-finger gene. Mol Gen Genet 252: 587–596[Medline]
Nawrath C, Metraux JP (1999) Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant Cell 11: 1393–1404 Nimchuk Z, Eulgem T, Holt BF, Dangl JL (2003) Recognition and response in the plant immune system. Annu Rev Genet 37: 579–609[CrossRef][ISI][Medline] Nürnberger T, Brunner F, Kemmerling B, Piater L (2004) Innate immunity in plants and animals: striking similarities and obvious differences. Immunol Rev 198: 249–266[CrossRef][ISI][Medline] Peters W, Latka I (1986) Electron microscopic localization of chitin using colloidal gold labeled with wheat germ agglutinin. Histochemistry 84: 155–160[CrossRef][ISI][Medline] Ramonell KM, Zhang B, Ewing RM, Chen Y, Xu D, Stacey G, Somerville S (2002) Microarray analysis of chitin elicitation in Arabidopsis thaliana. Mol Plant Pathol 3: 301–311[CrossRef] Reuber TL, Plotnikova JM, Dewdney J, Rogers EE, Wood W, Ausubel FM (1998) Correlation of defense gene induction defects with powdery mildew susceptibility in Arabidopsis enhanced disease susceptibility mutants. Plant J 16: 473–485[CrossRef][ISI][Medline] Ryan CA, Pearce G (1998) Systemin: a polypeptide signal for plant defensive genes. Annu Rev Cell Dev Biol 14: 1–17[CrossRef][ISI][Medline] Salinas-Mondragón RE, Garcidueñas-Piña C, Guzmán P (1999) Early elicitor induction in members of a novel multigene family coding for highly related RING-H2 proteins in Arabidopsis thaliana. Plant Mol Biol 40: 579–590[CrossRef][ISI][Medline] Shibuya N, Minami E (2001) Oligosaccharide signaling for defense responses in plants. Physiol Mol Plant Pathol 59: 223–233[CrossRef]
Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23: 1087–1088
Smits GJ, van den Ende H, Klis FM (2001) Differential regulation of cell wall biogenesis during growth and development in yeast. Microbiol 147: 781–794 Stacey G, Shibuya N (1997) Chitin recognition in rice and legumes. Plant Soil 194: 161–169[CrossRef]
Torii KU, Stoop-Myer CD, Okamoto H, Coleman JE, Matsui M, Dong XW (1999) The RING finger motif of photomorphogenic repressor COP1 specifically interacts with the RING-H2 motif of a novel Arabidopsis protein. J Biol Chem 274: 27674–27681 Tronchin G, Poulain D, Herbaut J, Biguet J (1981) Localization of chitin in the cell wall of Candida albicans by means of wheat germ agglutinin. Fluorescence and ultrastructural studies. Eur J Cell Biol 26: 121–128[Medline]
Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98: 5116–5121 Wan J, Zhang S, Stacey G (2004) Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin. Mol Plant Pathol 5: 125–135
Wilson IW, Schiff CL, Hughes DE, Somerville SC (2001) Quantitative trait loci analysis of powdery mildew disease resistance in the Arabidopsis thaliana accession Kashmir-1. Genetics 158: 1301–1309
Yamada K, Lim J, Dale JM, Chen H, Shinn P, Palm CJ, Southwick AM, Wu HC, Kim C, Nguyen M, et al (2003) Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302: 842–846 Yamada A, Shibuya N, Kodama O, Akatsuka T (1993) Induction of phytoalexin formation in suspension-cultured rice cells by N-acetylchitooligosaccharides. Biosci Biotechnol Biochem 57: 405–409 Zhang B, Ramonell K, Somerville S, Stacey G (2002) Characterization of early, chitin-induced gene expression in Arabidopsis. Mol Plant-Microbe Interact 15: 963–970[ISI][Medline] Zou J, Taylor DC (1997) Cloning and molecular characterization of an Arabidopsis thaliana RING zinc finger gene expressed preferentially during seed development. Gene 196: 291–295[CrossRef][Medline] This article has been cited by other articles:
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ABSTRACT
RESULTS
5% (Tusher et al., 2001
1.5-fold change in expression in at least one of the two chito-oligomer treatments relative to the water control. B1, Distribution of 1,252 genes identified as expressed at 
