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
B. Fristensky Email: email@example.com
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.
Despite the importance of canola (Brassica napus) to agriculture in many countries, few papers have been published regarding the molecular biology of disease resistance in the Brassicas. Rasmussen et al. [ 1992] cloned a basic chitinase that was more strongly induced by L. maculans (Phoma lingam) in a resistant cultivar, compared to a susceptible cultivar. Beta-1,3-glucanase was also induced in B. rapa inoculated with Xanthomonas campestris [Newman et al., 1994]. Pathogenesis-related gene PR1 was found to be expressed in senescent B. napus leaves, but expression in response to pathogens was not studied [Hanfrey et al., 1996].
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  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 materials and pathogen isolates
B. napus seeds and L. maculans (Desm.) Ces. et De Not. isolate PG2 86-12 were provided by Dr. Rachael Scarth and Dr. Roger Rimmer, respectively, Dept. of Plant Science, Univ. of Manitoba. Pycnidiospores were prepared according Mengistu et al. . All plants were grown in Jiffy Pots filled with Metromix (W.R. Grace & Co. Ltd., Ajax, Ontario, Canada) in a growth chamber at 20/16°C day/night temperature and a 16 hr. photoperiod.
cDNA library construction
Amplification of cDNA inserts
cDNA probes and hybridization
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 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.
Sequence analysis and database management
Screening for clones representing mRNAs inducible by fungus, constitutively-expressed, or expressed at low levels
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.
|To obtain a more diverse sampling of the mRNA population additional clones were chosen, 110 showing equal hybridization with both probes, and 37 showing a low level of expression. All clones were screened in two independent hybridizations. It should be noted that using these methods, relative levels of hybridization with both probes were not always consistent between experiments. Therefore, quantitative hybridization results are not presented here. cDNA clones corresponding to the PCR products identified in this manner were grown from the original glycerol stocks. Plasmid DNA was isolated for each clone and single sequencing reactions were performed using the T3 primer. Because the cloning system (Stratagene Uni-ZapII) predetermines cDNA orientation, sequencing with the T3 primer should always provide sequence corresponding to the 5' end of the mRNA. For DH26-2, DH26-20, DH26-23 DH26-24 and MB56-1G sequence was also obtained using the T7 primer. |
Identification of clones by DNA sequencing
| The next most prevalent class (20%) is sequences homologous to defense-related proteins, representing 10 distinct gene families. Because 120 of the cDNAs were selected by screening for genes that preferentially hybridized cDNA probe from blackleg-inoculated leaves, it is likely that this group overestimates the true levels of defense-related transcripts in blackleg-inoculated leaves. By far the most prevalent defense transcript is a homologue of the Arabidopsis Cxc750 gene [Aufsatz and Grimm, 1994], for which 24 clones were sequenced. In Arabidopsis, Cxc750 is expressed constitutively but is strongly induced by incompatible isolates of Xanthomonas campestris. The function of the 10 kDa Cxc750 gene product is unknown, and no homologues of this gene have been found in other species. Nine clones showed homology with non-specific lipid transfer proteins (LTP) which are induced by Rhizobium in Medicago truncatula during root nodule development [Gamas et al. 1996]. LTP proteins from maize and barley also demonstrated antifungal and antibacterial activity [Molina et al. 1993]. Nine clones were also identified for pathogenesis-related protein PR1 and seven for beta-1,3 glucanase (PR2). Single clones were identified for a polygalacturonase inhibitor protein, phenylalanine ammonia lyase, which is involved in both phytoalexin and lignin production, pathogenesis-related protein PvPR3 from common bean (not related to PR3 chitinases) [Sharma et al., 1992], Chia1 and Chia4 (PR3) endochitinases, and hin1, which is activated in the hypersensitive response of tobacco to Pseudomonas [Gopalan et al. 1996]. |
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
| Of the clones in Figure 3, only MB75-2H (25S rRNA) and MB75-4A (18S rRNA) hybridized with both L. maculans and B. napus genomic DNA. |
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 an array of ESTs could be grouped into many alternative functional categories, Table 1 groups them according to the primary goal of this work, which was to identfy a set of genes that would be useful for studying plant/pathogen interactions. Any categorization scheme necessarily oversimplifies the true picture of biochemical pathways. For example, phenylalanine ammonia lyase (PAL) is grouped among the defense genes, because of its role in the production of phenolic phytoalexins and lignins, which are produced in response to pathogenic attack. However, lignins are also produced during the course of development, and flavonoid pigments produced downstream of PAL play no known role in disease resistance. Disease resistance genes have been shown to be components of the signal transduction pathways leading to the activation of defense responses [Dangl et al., 1995]. However, there is no guarantee that a given resistance gene homologue recognizes a pathogen-derived signal. Conversely, it is possible that some of the genes categorized in Table 1 as "regulatory" are actually components of disease resistance signal transduction pathways.
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.  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.  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.  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.  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.  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.
This work was supported by Research Grant OGP0105628 from the Natural Sciences and Engineering Research Council of Canada, the Western Grains Research Foundation, and the NSERC/AAFC Research Partnership Program Grants 695-023-93 and 661-01996. Thanks to David Lonsdale for searching the EST panel against the Mendel database, and to Don Palmer for advice on assigning ESTs to biochemical or functional groups.
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