Abstract
Introduction
Materials & Methods
Results
Discussion
Acknowledgements
References

Molecular Plant Pathology On-Line [http://www.bspp.org.uk/mppol/1999/0301FRISTENSKY]

Expressed sequence tags from the defense response of Brassica napus to Leptosphaeria maculans

Brian Fristensky¹ Margaret Balcerzak, Daifen He and Peijun Zhang²
Department of Plant Science, University of Manitoba, Winnipeg, R3T 2N2, Canada.

²Current address: AAFC/ECORC, 960 Carling Avenue, Ottawa K1A 0C6 Canada

Despite the value of canola (Brassica napus) as an oilseed crop, little molecular biology has been done on its defense responses to pathogens. To expedite the cloning of an array of defense genes from B. napus, 277 cDNAs were sequenced from a library derived using mRNA from Brassica napus leaves inoculated with an incompatible isolate of the blackleg fungus, Leptosphaeria maculans. The resultant Expressed Sequence Tags (ESTs) include sequences homologous to previously-identified defense genes, disease resistance genes, and stress-related genes, as well as genes involved in photosynthesis, metabolism and other functions not associated with defense. This EST panel will find utility in measuring gene expression in plant/pathogen interactions. Because of the wide range of ESTs identified, it may also be useful in other systems, such as stress or light regulation. Our experience with this strategy has convinced us that sequencing ESTs can be a more efficient method of gene discovery, as compared to repeated library screenings using probes from other species. Details on the EST panel can be found at http://home.cc.umanitoba.ca/~frist.

The strategy of gene discovery by sequencing of large numbers of cDNAs was first used for identifying genes expressed in the human brain [Adams et al. 1991]. In this approach, single sequencing reactions are performed on randomly-chosen cDNAs, and the resultant partial sequences, referred to as Expressed Sequence Tags (ESTs) are compared with sequence databases for identification. In this context, research in the Brassicas is benefiting from the large scale EST projects being undertaken in the closely-related crucifer Arabidopsis thaliana [reviewed in Bouchez and Höfte, 1998, Cooke et al., 1996]. In addition to such broadly based projects, EST projects have focused on specific tissues, developmental or environmental conditions. For example, the EST strategy has been used to identify genes expressed in C₄ photosynthesis in Sorghum [Wyrich et al., 1998], in cultured rice cells under stress from high salt or nitrogen-starvation [Umeda et al., 1994], rice endosperm [Liu et al., 1995], in guard cells [Kwak et al. 1997] and flower buds of Brassica rapa (B. campestris) [Lim et al. 1996] and in leaves of B. napus [Lee et al. 1998].

Few studies have used ESTs to identify genes involved in plant/microbe interactions. Tagu and Martin [1995] sequenced 100 cDNAs from ectomycorrhizal tissues comprising the symbiosis of Eucalyptus globulus and Pisolithus tinctorius. In Arabidopsis, ESTs showing similarity to disease resistance genes were mapped [Botella et al. 1997].

To expand the set of genes available for studying plant/pathogen interactions in Brassica, we have sequenced 277 cDNAs derived from B. napus leaves expressing resistance to the blackleg fungus Leptosphaeria maculans. ESTs were found representing 10 defense gene families, 3 resistance gene families, 16 regulatory gene families, as well as 14 stress-related gene families. Genes associated with other physiological processes, such as photosynthesis and secondary metabolism were also identified. Twenty percent of the ESTs had no similarity to any proteins of known function. Contamination of the EST population by cDNAs of fungal origin was determined to be negligible.

Plant inoculation
Pycnidiospore suspensions (2 x 10⁷ spores/ml) in sterile distilled water were infiltrated into cotyledons or leaves using a 1-cc syringe without a needle. The outlet of the syringe was covered with a section of Tygon tubing. Infiltration was conducted by shooting inocula directly into cotyledons or leaves through the abaxial face while supporting the cotyledons or leaves on the operator's gloved index finger. One or two inoculated plants were kept in the growth chamber for at least two weeks to verify interaction phenotype development.

cDNA library construction
Plant materials were frozen in liquid N₂ immediately at harvest. Frozen tissues were used directly for RNA extraction, or stored at - 70°C. Total RNA was extracted from leaves using the method of Kim et al. [1992]. Total RNA from B. napus cv. Glacier leaves inoculated with L. maculans isolate PG2 for 48 hr. was used for cDNA library construction. mRNA was prepared by hot-KCl oligo-dT cellulose column protocol (Collaborative Research Incorporated, Bedford, USA). cDNA was synthesized using Stratagene cDNA synthesis kit according to the manufacturer's manual, resulting in directional cDNAs with an EcoRI site at the the 5' end, with respect to the mRNA, and an XhoI site at poly-A end. cDNAs were ligated to Strategene Uni-ZAP XR vector and packaged with Strategene Gigpack III. The resultant lambda library was amplified. pBluescript SK- phagemids were excised from phage in vivo to produce ampicillin resistant colonies, using Strategene In Vivo Excision System according to the manual.

Amplification of cDNA inserts
Randomly-chosen bacterial colonies were transferred using sterile toothpicks to microfuge tubes and suspended in 10 µl of sterile ultrapure water. Five µl of bacterial suspensions were used to inoculate 1 ml LB containing 50 µg/ml ampicillin, and cultures were shaken at 37°C overnight, supplemented with 150 µl of glycerol, and stored at -70°C. The remaining 5 µl of cells was added to PCR reactions containing the following components: 2.5 µl of 10 x Taq DNA polymerase reaction buffer (Gibco BRL), 1.5 µl of 25 mM MgCl₂ (final concentration 1.5 mM), 2 µl of dNTP mixture (final concentration of each nucleotide 0.2 mM), 0.25 µl (10 µM each) mixture of primers T₃ and T₇ (Gibco BRL), 0.125 µl of Taq DNA polymerase (0.625 units) (Gibco BRL), 5 µl of bacterial cell suspension, and 13.7 µl of ultrapure water, for a total of 25 µl. Amplification was performed in 96-well microtitre plates in a programmable thermal cycler (Techne, MW-2, Mandel Scientific Co.), using the step cycle program, including complete denaturation of the template at 94°C for 5 min, and 30 cycles of PCR amplification as follows: denaturation at 94°C for 1 min, annealing at 55°C for 1 min., and polymerization at 72°C for 3 min. 5 µl of DNA from amplification reactions was directly electrophoresed on a 1.5% agarose gel. Clones with estimated inserts of 500 bp or larger were chosen for further study.

Filter arrays
Dot blots were prepared by spotting 1.5 µl of denatured (heated at 100 °C for 5 min.) PCR product amplified directly from bacterial cell suspensions, onto Hybond-N+ nylon membranes (Amersham). To ensure that duplicate blots contained the same DNA samples at each position, sample tubes were arrayed in the same order as the filters, and each sample was spotted onto both filters using the same pipette tip, as opposed to pipetting all samples onto one filter, and repeating the process for the second filter. For screening with cDNA probes, control spots included 1.5 µl of PCR products of B. napus cDNAs for PR1 (GenBank U64806), PAL, (AA960723) and Cab (AA960724). As controls for blots using genomic DNA probes, for L. maculans (0.1µg) and B. napus (0.15 µg) genomic DNA, along with 1.5 µl of PCR product from the L. maculans nitrate reductase gene (NR) [GenBank U04445, Williams et al. 1994], were spotted in the right column of the array. DNA was fixed to the membrane by UV cross-linking using a UV Stratalinker 1800 (Stratagene).

cDNA probes and hybridization
Complex cDNA probes were prepared from total RNA by simultaneous reverse transcription and labeling with digoxigenin (DIG)-11-dUTP, alkali-labile, according to the supplier's instructions [Anonymous, 1995]. The labeling reaction included: 2 µl of heat denatured RNA (3 µg), 6 µl of 5 x cDNA reaction buffer, 1 µl RNase inhibitor (40 units), 1.5 µl of dNTP mixture (10mM each), 1 µl of dTTP (4mM), 6 µl of DIG-dUTP (0.35 mM), 3 µl of oligo-p(dT)₁₅ (0.74 µg/µl), 5µl of AMV Reverse Transcriptase (50 units), and 4.5 µl of DEPC-treated water. The reaction was incubated at 42°C for 90 min. In agreement with the manufacturer's instructions [Anonymous, 1995], we found that further purification to remove unincorporated DIG nucleotides was unnecessary.

Membranes were prehybridized at 65°C for 1 hr. in 15 ml of hybridization solution (1% SDS and 1 M NaCl) with constant agitation. The complex cDNA probes were denatured by boiling for 5 min. before adding to the prehybridization solution. Hybridization was performed with gentle agitation overnight at 65°C in a Micro Hybridization Incubator (Model 2000) (Robbins Scientific). Probe concentrations were approximately 1 ng/ml hybridization solution. After hybridization, membranes were washed twice, 10 min. per wash, in 1 x SSC (0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0) and 1% SDS at room temperature, then twice for 30 min. in 1 x SSC and 0.1% SDS at 68°C.

Chemiluminescent detection was performed with anti-digoxigenin antibody conjugated to alkaline phosphatase and CDP-Star (Boehringer Mannheim) according to the manufacturer's instructions as follows. After posthybridization washes, membranes were washed briefly in Buffer 1 (100 mM maleic acid and 150 mM NaCl, pH 7.5) and incubated in Buffer 2 (1% blocking reagent in Buffer 1) for 1 hr. with gentle agitation. Anti-DIG-alkaline phosphatase was added to fresh Buffer 2 to achieve a dilution of 1:10,000, followed by incubation with gentle agitation for 30 min. Membranes were washed twice for 30 min. in Buffer 1 and then equilibrated in Buffer 3 (100 mm Tris-HCl, pH 9.5, 100mM NaCl) for 3 min. Membranes were placed on plastic sheets, and 250 µl of a 1:200 dilution of a 25 mM solution of CDP-Star was spread over each membrane (68 cm²). Membranes were sealed in the plastic sheets and exposed to Kodak X-ray film for 5 to 20 min. to record chemiluminescence. Intensity of hybridization signals were compared visually. Clones which appeared to bind greater amounts of cDNA probe derived from fungal-treated leaves than with probe from untreated leaves, were selected for sequencing.

Genomic DNA probes
Genomic DNA was extracted from B. napus leaves using a CTAB protocol [Ausubel et al. 1994]. DNA extraction from freeze-dried mycelia of L. maculans used the method of Dellaporta, et al. [1983] with an additional two phenol (pH 8.0) extractions and RNase treatment for 1 hr.

Genomic DNA of PG2 and B. napus cv. Glacier were labeled with DIG-11-dUTP using the random primed method (Boehringer Mannheim BmbH, Germany). Genomic DNA was restriction-digested with EcoRI prior to labeling. The labeling reaction included: 15 µl of heat denatured DNA (10 µg), 2 µl of hexanucleotide mixture, 1 µl of dNTP labeling mixture (2mM dATP, 2mM dCTP, 2mM dGTP, 1.3 mM dTTP), 0.7 µl of 1 mM alkali-labile DIG-dUTP, and 1 µl of DNA polymerase I Klenow fragment (2 units). The reaction was incubated at 37° C overnight [Anonymous, 1995]. Probe concentrations were approximately 10 ng/ml of hybridization solution.

Sequencing reactions
Double stranded plasmid DNA was prepared using the alkaline lysis method [Maniatis et al. 1982]. Single DNA sequencing reactions were performed using the T3 or T7 primers derived from the pBluescript SK- vector (Stratagene, GenBank X52330). Sequencing reactions were carried out on a Perkin Elmer Cetus DNA Thermal Cycler using the Applied Biosystems Prism Ready Reaction Dyedeoxy Terminator Cycle Sequencing Kit and electrophoresed on an ABI Prism sequencer (Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, SK).

Sequence analysis and database management
Raw sequence files generated by the ABI sequencer were uploaded to a Sun Ultrasparc Unix server and read into GDE [Smith et al., 1994] from which all sequence analysis programs were run. After deleting vector sequence, cDNA sequences were compared with GenBank 106.0 using FASTA [Pearson 1990], and FASTY3 [Pearson et al., 1997] was used for comparison of cDNAs with amino acids in the GenPept translation of GenBank [NCBI, 1998]. Translated cDNA sequences were compared with GenBank 106.0 using TFASTA [Pearson 1990]. In Table 1, sequences for which no match was found with a probability of lower than 0.01 were classified as unknown. Annotation of EST data was managed using ACeDB 4.5 [Durbin and Thierry-Meig, 1991] database software. To ensure that all putative members of a given gene family followed a consistent nomenclature, all sequences were sent to the John Innes Centre, UK for comparison with sequences catalogued by the Mendel database [Lonsdale et al., 1999]. To automate submission to GenBank, files generated by ACeDB were converted into GenBank EST submission format by makeEST, a Java program [Fristensky, unpublished].

The cDNA library used in this study is derived from mRNA isolated from B. napus cv. Glacier leaves, inoculated for 48 hr. with L. maculans isolate PG2. Cultivar Glacier expresses resistance to PG2, mediated by a single resistance gene [Rimmer and van den Berg, 1992]. By 48 hr. postinoculation, a strong induction of pathogenesis-related protein PR1 is seen [Zhang and Fristensky, in preparation]. Therefore, this library represents transcripts active during a resistance response to the blackleg fungus.

cDNA inserts from approximately 1000 randomly-chosen colonies were amplified by PCR (See Methods for details.) PCR products were sized on agarose gels, and products measuring 500 bp or larger were spotted in equal amounts onto duplicate filters. Filters were probed with digoxygenin-labeled cDNA produced using either mRNA isolated from B. napus leaves inoculated with L. maculans PG2 or mRNA from uninoculated leaves ( Figure 1). 130 clones were identified whose PCR products hybridized more strongly with probe derived from inoculated leaves versus probe from uninoculated leaves.

Figure 1

Identification of clones by DNA sequencing
Table 1 lists the cDNAs sequenced and their closest homologues based on database searches. As summarized in Figure 2, the largest single group of ESTs (23%) showed similarity to photosynthesis-related genes, apparently reflecting the fact that the library was derived from mature leaf mRNA.

Figure 2

Several disease resistance gene homologues were also identified, including homologues of downy mildew resistance protein RPP5 [Parker et al. 1997], wheat leaf rust resistance gene homologue Lr10 [Feuillet et al. 1997] and the Colletotrichum-inducible SLRR protein from sorghum [Hipskind et al. 1996]. As well, clones were identified from 17 other gene families associated with gene regulation, such as calmodulin, zinc-finger proteins, GTPases and receptor-like kinases.

Another large group of ESTs (9%) showed similarity to other stress-related genes, encompassing 14 distinct gene families. These include proteins associated with heat shock, water stress, cold stress, heavy metal stress, ethylene-inducible proteins, and proteins involved in detoxification.

The remaining ESTs for which homologues could be identified could be broadly categorized as housekeeping genes, associated with diverse cellular functions.

Finally, 20% of the ESTs were categorized as unknown. Eighteen of these ESTs showed similarity to proteins of unknown function, or to hypothetical proteins inferred from genomic open reading frames. For the remaining 33 ESTs, no significant similarity was found with any sequence in GenBank. Neither did these sequences show significant similarity with each other, indicating that they represent 33 distinct gene families.

Screening for clones of fungal origin
Because the cDNA library used in this work was derived using mRNA isolated from fungus-inoculated leaves, it is possibile that some of the clones in the library could be from fungal mRNAs. Three checks were available to detect fungally-derived clones. First, PCR products for each clone were arrayed on filters and hybridized with DIG-labeled genomic DNA from either L. maculans PG2 or B. napus cv. Glacier. As controls, each filter also had spots of B. napus or L. maculans genomic DNA, and B. napus or the L. maculans nitrate reductase cDNA. (Note: As of this writing, no other L. maculans sequences, other than ribosomal or other non-protein coding sequences, have been reported in GenBank.) As illustrated in Figure 3 (bottom), B. napus genomic probe showed detectible hybridization with many clones, as well as with both B. napus and L. maculans genomic DNA. Hybridization with L. maculans genomic DNA was weaker, and probably most of that signal was due to cross hybridization between rRNA genes. Hybridization of an identical filter using L. maculans genomic DNA ( Figure 3, top) resulted in hybridization with only a small number of clones.

Figure 3

In principle, B. napus genomic DNA should hybridize to all clones. However, the haploid genome size of B. napus is 1.2 x 10⁹ bp [Arumaganathan and Earle, 1991]. In comparison, PFGE analysis of highly virulent isolates of L. maculans (which include PG2) give estimates of haploid genome size between 2.3 x 10⁷ and 3.2 x 10⁷ bp [Morales et al., 1993]. Since the B. napus genome is at least 38 times larger than the L. maculans genome, the B. napus probe would be expected to be much less sensitive than the L. maculans probe to single copy sequences, such as those transcribed into mRNA. Therefore, it is not surprising that only a small number of clones hybridized with the B. napus probe.

Sixteen other clones which hybridized with B. napus DNA also showed weak hybridization with L. maculans genomic probe. The L. maculans genomic probe also hybridized with the L. maculans nitrate reductase cDNA, indicating that the sensitivity of the hybridization is adequate for detection of L. maculans genes. However only one clone, MB72-6H, hybridized with the L. maculans probe but not with the B. napus probe ( Table 2). This clone was not among the group that was sequenced, and is not included among the ESTs in Table 1.

As a second check, sequences of fungal origin should show greater sequence similarity to fungal sequences than to plant sequences. Since S. cerivisiae and L. maculans are both ascomycetes, and the entire yeast genome has been sequenced [Mewes et al., 1997], most L. maculans genes should have homologues in the yeast genome, and therefore would be expected to match yeast genes preferentially to plant genes. Only two of the ESTs for which database matches could be found were shown to be most closely-related to genes of fungal origin: MB69-12B and MB75-5A, matching S. cerivisiae putative 60S ribosomal protein and dnaJ, respectively.

As a third check, many of the cDNAs tested in this study showed at least some hybridization with cDNA probe from leaves that were not inoculated with fungus. As shown in Table 2, of the 20 clones showing some hybridization with the L. maculans genomic probe, all but MB72-5G and MB72-6H also showed detectible hybridization with cDNA derived from uninoculated leaf mRNAs. Since MB72-5G is a beta-1,3 glucanase homologue, it is not surprising that it would be undetectable in uninoculated tissue. Since MB72-6H hybridizes only with L. maculans genomic DNA, and not with B.napus genomic DNA nor with the cDNA probe from B. napus leaf mRNA, it is possible that that clone represents a fungal transcript. However, MB72-6H was not sequenced, and was not included in the EST array.

While the identification of cDNAs for many genes previously characterized from other species is important, the value of the "unknown" class of ESTs, comprising 20% of our ESTs, should not be underestimated. Most EST projects yeild a substantial percentage of sequences in this category. For example, out of 5000 non-redundant ESTs from Arabidopsis, Cooke et al. [1996] estimated that only about one-third corresponded to known proteins. Aside from the random factors that govern which genes get sequenced in various plant species, there are other reasons why newly-sequenced ESTs may yet be unclassifiable. Goldberg et al. [1989] have shown that in mRNA populations from both dicot and monocot species, typically 20-60% of the mRNA mass and 95% of the mRNA complexity is composed of low abundance messages, which therefore have a lower likelihood of being cloned. It is also important to consider that as plant species diverge, new gene families evolve, and others are lost. In any given species, then, some proportion of the transcripts may be unique to a family or genus. Finally, there is necessarily a bias in sequence databases for genes associated with well-characterized cellular processes. Genes associated with as yet unknown processes are only likely to be sequenced as part of EST or genomic sequenceing projects. In that case, ESTs may be an important means of discovering new biochemical and regulatory pathways.

Out of several hundred cDNAs screened by hybridization with L. maculans genomic DNA, only one appeared to be of fungal origin. While there have been few attempts to measure the proportion of fungal RNA in RNA populations extracted from plant/fungal interactions, microscopic observation of fungi on plant tissue usually reveals that fungal biomass is small compared to plant biomass. In compatible and incompatible interactions between pea and Fusarium solani, Fristensky et al. [1985] labeled RNA with ³²PO₄^-2 in planta. When labeled RNA was hybridized to F. solani genomic DNA, no signal was detected above background. In the present work, RNA was extracted from a resistant interaction, in which fungal proliferation is by definition inhibited. Consequently, it is not surprising that fungal contamination was minimal in the EST population.

Although the primary goal of this work was to clone defense-related genes, genes representing many physiological processes and biochemical pathways were identified. The breadth of this EST collection should prove useful for multiplex analysis of gene expression. For example, Bernard et al. [1996] arrayed 47 cDNAs onto nylon filters, and measured transcript levels for each of the 47 genes in complex cDNA probes derived from resting mouse T-cells, or T-cells activated by anti-CD3 antibodies. The large set of genes analyzed provided a solid basis for comparison, which made it possible to identify genes most strongly-induced in activated T-cells. On a larger scale, Chu et al. [1998] used microarrays of cloned yeast sequences to monitor the expression of 6200 yeast genes (approx. 97% of all known yeast genes) over a timecourse encompassing all stages of sporulation. The use of gene arrays for monitoring gene expression should be of great value in the study of plant/pathogen interactions, because the larger the group of genes studied, the more complete a picture of the interaction will be developed. At the same time, sets of genes whose expression are coordinately-regulated in association with cellular processes such as disease resistance can serve as candidate genes for cloning of entire biochemical pathways. Another advantage of gene arrays is that the large number of genes increases the chance that unexpected phenomena will be detected. For example, through use of controls whose expression was presumed to be constitutive, Hahlbrock and coworkers made the unanticipated finding that cell cycle-related genes such as histones, cdc2 and cyclin are down-regulated in parsley by Phytophthora or elicitor [Logemann et al., 1995]. The diversity of genes in the EST population should therefore make it possible to discover other unexpected processes occurring during a defense response.

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