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1 Department of Plant Pathology, University of Minnesota, St Paul, MN 55108, USA
2 Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA
3 Department of Plant Biology and Department of Plant Pathology, Michigan State University, East Lansing, MI 48824, USA
4 USDA ARS Cereal Disease Laboratory, University of Minnesota, St Paul, MN 55108, USA
Correspondence
H. Corby Kistler
hckist{at}umn.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are DV998659–DV998726 and DW005257–DW005273.
Three supplementary tables are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), has now become a devastating disease in many parts of the world. The major causal organisms of this disease are fungal pathgens that are members of the recently described Fusarium graminearum (Fg) species complex (O'Donnell et al., 2004). The Fg species complex comprises strains belonging to at least nine biogeographically structured cryptic species that may differ significantly in aggressiveness on wheat and in mycotoxin production (Goswami & Kistler, 2005). The species Fusarium graminearum (O'Donnell et al., 2004) [teleomorph Gibberella zeae (Schweinitz) Petch] is the most common causal agent of FHB in North America and many other regions of the world. FHB commonly affects wheat, barley and other small grains in both temperate and semi-tropical areas and has the capacity to destroy a potentially high-yielding crop within a few weeks of harvest (McMullen et al., 1997). Additionally, infected grains often contain significant levels of trichothecene and oestrogenic mycotoxins, making the grain unfit for food or feed (McMullen et al., 1997).
The current status of genomic research on F. graminearum recently has been reviewed (Goswami & Kistler, 2004). Initial studies on gene expression in F. graminearum involved analysis of expressed sequence tag (EST) libraries to study the expression patterns in F. graminearum grown under different culture conditions (carbon and nitrogen starvation) and at the reproductive phase in maturing perithecia (Trail et al., 2003). In planta gene expression was studied using a similar approach (Kruger et al., 2002). In the latter study, an EST database containing 4838 sequences was created using the inflorescence of the partially resistant wheat cultivar Sumai 3 infected with F. graminearum. However, the focus of that study was primarily on plant genes expressed during this interaction, and only 2 % of the non-redundant sequence set was attributable to the fungus.
Functional genomic studies in F. graminearum have mostly targeted genes potentially involved in pathogenicity and mycotoxin production. Mutants for eight such genes have been characterized using various techniques. The genes identified and verified by gene disruption or replacement include the Tri5 gene of the trichothecene gene cluster encoding trichodiene synthase (Proctor et al., 1995), two MAP kinase genes, Mgv1 (Hou et al., 2002) and Map1, also called gpmk1 (Jenczmionka et al., 2003; Urban et al., 2003), a secreted lipase gene, Fgl1 (Voigt et al., 2005), a gene similar to Cps1 from Cochliobolus heterostrophus (Lu et al., 2003) and two polyketide synthase genes, Zea1 (PKS4) and Zea2 (PKS13), involved in the synthesis of the mycotoxin zearalenone (Gaffoor et al., 2005; Kim et al., 2005). Apart from these, genes encoding a predicted NADH : ubiquinone oxidoreductase, a putative b-ZIP transcription factor, a transducin 
-subunit-like protein, as well as hydroxymethylglutaryl-CoA reductase (HMR1) and cystathionine beta-lyase (CBL1) have also been identified to be involved in pathogenicity through restriction-enzyme-mediated insertion (REMI) and directed mutagenesis (Seong et al., 2005, 2006).
The release of the draft genome sequence assembly of this pathogen (http://www.broad.mit.edu/annotation/fungi/fusarium/) has allowed new approaches for genomic studies. However, there is still very limited information regarding pathogen genes expressed early in the development of disease within the host. This information is vital for understanding the host–pathogen interaction and characterizing genes involved in pathogenesis. To address this issue, we chose the technique of suppression subtractive hybridization (Diatchenko et al., 1996). This technique has been used successfully to identify plant and fungal genes up-regulated in various pathosystems (Beyer et al., 2002; Bittner-Eddy et al., 2003; Cramer & Lawrence, 2004; Guilleroux & Osbourn, 2004; Lu et al., 2004; Xiong et al., 2001).
The objective of this study was to establish a comprehensive method for identification and functional analysis of novel genes potentially involved in the pathogenicity of the fungus. We specifically chose to identify pathogen genes that are expressed at early stages of the infection process by F. graminearum on wheat; the time point of 48 h after inoculation was chosen based on previous microscopic studies on infection (Pritsch et al., 2000; Skadsen & Hohn, 2004) and reports on mycotoxin production (Evans et al., 2000). Overall fungal gene expression was studied by preparing a subtraction library using the susceptible wheat cultivar Norm, inoculated with a highly aggressive F. graminearum strain or mock-inoculated with water. Considering the large variation in pathogenicity observed among members of the Fg species complex (Goswami & Kistler, 2005; O'Donnell et al., 2000), an attempt also was made to identify fungal genes differentially expressed in planta during interactions between wheat and a highly aggressive or less aggressive strain. Four fungal genes found to be expressed early during infection were subsequently targeted for gene deletion in order to determine their effect on pathogenicity.
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METHODS |
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, 2004). The strains were stored and macroconidia prepared as previously described (Goswami & Kistler, 2005; O'Donnell et al., 1998). Comparisons of spore production between wild-type and transformants were made by counting the number of macroconidia 8 days after plating on mung-bean agar. Mean numbers of spores per ml from five replicates were compared using a two-tailed t-test (two-sample unequal variance).
Plant growth, inoculation, disease assessment and mycotoxin analysis.
Wheat plants, cultivar Norm, were grown as previously described (Goswami & Kistler, 2005). For RNA extraction, wheat spikelets, at early-to-mid anthesis, were inoculated with approximately 104 conidia of strain NRRL 31084 or NRRL 28303 in 10 µl 0.01 % Triton 60. Mock inoculations were conducted in a similar manner with 0.01 % Triton 60 solution alone. After inoculation, wheat plants were placed in a humidity chamber for 48 h, after which inoculated wheat heads were collected and immediately frozen at –80 °C for RNA extraction. For disease assessment, inoculum was prepared and applied using the same procedure except that only a single central spikelet within each head was inoculated. After inoculation, the plants were placed in a humidity chamber for 72 h, and then transferred to a greenhouse maintained at approximately 27 °C. The wild-type strain NRRL 31084 was used as a positive control. Wheat heads inoculated with sterile 0.01 % Triton were used as negative controls. Inoculation treatments consisted of a total of ten plants, with five plants per pot and two pots per treatment. Each strain was tested three times. Head blight was rated 14 days after inoculation (dai) by counting the number of spikelets showing disease symptoms (necrosis and/or bleaching of palea/lemma) as described previously (Goswami & Kistler, 2005). A total of ten spikelets on each wheat head were evaluated for the presence of disease symptoms by scoring five spikelets above and four spikelets below the point of inoculation. Mean number of symptomatic spikelets on inoculated plants were compared using a two-tailed t-test (two-sample unequal variance). Inoculated spikelets also were collected, weighed and stored for mycotoxin analysis. Mycotoxin determination was conducted as previously described (Goswami & Kistler, 2005).
Nucleic acid extraction.
A revised version of the hot phenol method (Verwoerd et al., 1989) was used for extraction of total RNA from infected wheat heads from each of the three treatments (inoculation with NRRL 31084, NRRL 28303 or water). All of the solutions and water used in the extraction procedure were rendered RNase free by mixing them with 0.1 % diethyl pyrocarbonate, incubating at 37 °C overnight and then autoclaving at 121 °C, 15 p.s.i. pressure for 30 min. Infected tissue (3–4 g) was ground in liquid nitrogen. A preheated 1 : 1 mixture of extraction buffer (0.1 M Tris/HCl pH 8; 0.1 M LiCl; 10 mM EDTA; 1 % SDS) and Tris-saturated phenol at 80 °C was added to the frozen powder followed by vigorous vortexing. Subsequently, half the buffer/phenol volume of choloroform was added and the mixture vortexed again. This was then centrifuged at 2500 g at room temperature for 30 min and the supernatant transferred to a 50 ml Teflon tube. One-third of the volume of 8 M LiCl was added to the supernatant and precipitated on ice for more than 2 h. The solution was centrifuged at 12 000 g for 30 min at 4 °C. The pellet was then subjected to washes with 3 ml 2 M LiCl and 70 % ethanol at room temperature, each followed by 5 min centrifugation at 12 000 g. The pellet was air-dried and dissolved in 2 ml RNase free water. Any undissolved debris was removed by another round of centrifugation, followed by precipitation using 0.1 vol. 3 M sodium acetate (pH 5.2) and 2.5 vols room temperature 100 % ethanol and incubation at –80 °C for 15 min. The clean pellet was then washed in 70 % ethanol, air-dried and dissolved in 100 µl RNase-free water. mRNA was prepared using the polyA Spin mRNA Isolation kit (New England Biolabs) according to the manufacturer's instructions. For RT-PCR, total RNA was isolated from infected wheat heads (cv. Norm) inoculated with NRRL 31084 or water at 0, 24, 48 and 72 h after inoculation using the RNeasy Plant Mini kit (Qiagen), following the manufacturer's instructions, and subjected to DNase (Roche Applied Science) treatment. DNA was extracted from lypholized mycelium of the two strains mentioned above using a protocol previously described (Rosewich et al., 1999).
Library preparation.
For the Fgr-S3/S4 library, suppression subtractive hybridization (SSH) was performed between the driver sequences derived from mock-inoculated wheat heads and tester sequences from wheat heads inoculated with NRRL 31084. The Fgr-S subtraction library was constructed using material from NRRL 31084-infected wheat as the tester and from NRRL 28303-infected wheat as the driver; the Fgr-S2 library was constructed using material from NRRL 28303-infected wheat as the tester and from NRRL 31084-infected wheat as the driver. The SSH procedure was performed using the PCR-Select cDNA Subtraction kit according to the manufacturer's directions (BD Biosciences-Clontech). The PCR product, enriched in differentially expressed genes from the Fgr-S3/S4 library, was cloned into the pGEM-T Easy vector (Promega) and the FgrS and Fgr-S2 libraries were cloned into pT-Adv vector using the AdvanTAge PCR Cloning kit (BD Biosciences-Clontech). After transforming DH5
MCR competent cells (Invitrogen), individual white colonies were picked into 96-well plates either manually or using a QBot robotic colony manipulator (Genetix).
DNA sequencing and bioinformatic analysis.
Sequencing reactions for individual cDNA clones were performed using ABI BigDye version 3.0 chemistry (Applied Biosystems). Reaction products were ethanol-precipitated and run on an ABI3100 genetic analyser (Applied Biosystems). The CAP3 sequence assembly program based on a multiple sequence alignment method (Huang & Madan, 1999) was used to align the ESTs in each library and generate consensus sequences for contigs using the default parameters. Sequences have been deposited in the GenBank database under accession numbers DV998659–DV998726 and DW005257–DW005273.
The fungal origin of the contigs and singletons was determined based on BLASTN searches against the F. graminearum whole-genome sequence available at the Broad Institute website http://www.broad.mit.edu/annotation/fungi/fusarium/ (F. graminearum sequencing project). Sequences with E-values <10–10 were considered to be derived from the pathogen unless they had better matches, according to BLASTX, with other sequences in GenBank. Comparisons also were made by conducting BLASTN searches against EST sequences from F. graminearum libraries created using the fungus grown on various culture media. This included EST sequences available in GenBank for libraries grown on a trichothecene induction medium, complex plant medium, cornmeal and simple substrates, and on nitrogen- and carbon-limited media (believed to provide conditions similar to those on the host). BLASTX comparisons were also made with Magnaporthe grisea (Dean et al., 2005) and Neurospora crassa assembly version 3 (Galagan et al., 2003), whose draft genome sequence assemblies are available. Putative functions of genes corresponding to the ESTs were ascertained by BLASTX matches to non-redundant protein sequences in GenBank (E-value <10–5). In cases where the EST matched an ORF in the F. graminearum genome, the sequence of the entire ORF was used for the searches in GenBank, in order to better predict function of the gene. Gene predictions by the Broad Institute were primarily used for identification of ORFs corresponding to the ESTs. However, in cases where the ESTs matched regions with no predicted genes in the Broad Insititute database, predictions from the Munich Information Center for Protein Sequences F. graminearum genome database (Güldener et al., 2006a) were considered.
Verifying fungal origin of genes and gene expression in planta by PCR and RT-PCR.
Ten ESTs were selected for verification of their fungal origin; six ESTs (Fgr-S4_2_M02_T7, Fgr-S4_3_G23_T7, Fgr-S4_3_H08_T7, Fgr-S4_3_I05_T7, Fgr-S4_2_K04_T7 and Fgr-S4_3_G14_T7) matched the assembled F. graminearum genome sequence and four ESTs (Fgr-S4_1_J09_T7, Fgr-S4_1_K24_T7, Fgr-S4_1_D04_T7 and Fgr-S3_1_L11_T7) matched the excluded read files of the Fusarium sequencing project. Primers specific to each EST were designed using the WebPrimer available at http://seq.yeastgenome.org/cgi-bin/web-primer. Primers for a F. graminearum translation elongation factor 1 (EF1 and EF2; O'Donnell et al., 2000) were used to generate positive controls. The primer sequences (see Supplementary Table S1, available with the online version of this paper) were used for amplification of the DNA sequences corresponding to the above-mentioned ESTs from fungal (NRRL 31084) and wheat (cv. Norm) DNA using the following cycling conditions: 94 °C for 2 min followed by 30 cycles of 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min and a final extension for 10 min at 72 °C.
RT-PCR reactions were conducted for the genes FG08308.1 (Abc2), FG03405.1 (Lyp1), FG00215.1 (Rrr1) and FG02874.1 (Zbc1), corresponding to the ESTs Fgr-S4_3_G23, Fgr-S4_2_M02, Fgr-S4_3_I05 and Fgr-S4_3_G14 selected for targeted gene replacement. The EST/gene-specific primers mentioned above were also used for RT-PCR. The cycling parameters were as follows: one cycle of 94 °C for 2 min followed by 30 cycles of 94 °C for 1 min, 62 °C for 1 min, 68 °C for 3 min and one cycle at 68 °C for 10 min. The transformants generated after replacement of each of the genes were also initially tested using the primers in supplementary Table S1 and primers for the hph gene (HYG/F and HYG/R) (Supplementary Table S2). The same PCR conditions as described above were used for all the genes except the annealing temperature was lowered to 55 °C using the hph primers.
Identifying chromosomal positions of ESTs/contigs.
The ESTs/contigs were located on the physical map by identifying the position of the contig that they matched according to BLASTN searches against the F. graminearum genome. Chi-squared analysis was used to analyse the distribution of these sequences on the chromosomes. The whole-genome size of 36 002 487 bp and projected size of each chromosome (Gale et al., 2005) were used for calculating the expected number of genes on each chromosome.
Protoplast isolation and transformation.
F. graminearum strain NRRL 31084 was maintained in 15 % glycerol at a spore density of 108 macroconidia ml–1 and stored at –80 °C. For protoplast preparation, 1 ml of the stock spore suspension was inoculated into 100 ml YEPD medium (yeast extract, 3.0 g; Bactopeptone, 10.0 g; glucose, 20.0 g; and distilled water to 1 l) and incubated for 12–14 h at room temperature with shaking at 175 r.p.m. Fungal protoplasts were prepared essentially as described previously (Hou et al., 2002). Pure cultures were prepared by single-spore isolation from the putative transformant colonies growing on V8 juice medium and cultured on mung-bean agar (Evans et al., 2000) medium containing 250 mg hygromycin l–1. Macroconidia were harvested from these plates as mentioned above and used for pathogenicity tests.
Targeted gene replacement.
The ligation PCR approach (Zhao et al., 2004) developed for M. grisea was used for replacing Abc2 and Lyp1 in F. graminearum strain NRRL 31084. Primers used for each gene are presented in Supplementary Table S2. The split marker recombination procedure (Catlett et al., 2003a) was used for gene replacement of Rrr1 and Zbc1 with modifications. The modifications included the use of the entire hph gene fragment amplified with the HYG/F and HYG/R primers for the fusion PCR step with the flanking regions using primers HY/R and YG/F located inside the hph gene along with the 1F and 2R primers for this step. Changes also were made to the HYG/F and HYG/R primers to fit regions flanking the gene in the plasmid pCX62 from which we amplified the gene (Supplementary Table S2). An annealing temperature of 60 °C was used for the first PCR cycle. An extra purification step of the first PCR product was added using a QIAquick PCR Purification kit (Qiagen). The cycling conditions for the second round of PCR were also modified from the original protocol and involved one cycle of 94 °C for 2 min followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 68 °C for 2.5 min and one cycle at 68 °C for 10 min.
Southern hybridization.
Samples (5 µg) of DNA from each of the mutants and the wild-type were digested with restriction enzymes as follows: Abc2 mutants, HindIII and AflII; Lyp1 mutants, HpaI and SacII; Rrr1 mutants, HindIII; and Zbc1 mutants, BlpI and SpeI. Southern hybridization for the Abc2 mutants and wild-type was performed as described by Rosewich et al. (1999) except that the Decaprime DNA II Random Priming DNA Labelling kit from Ambion was used for labelling the probes. The AlkPhos Direct Labelling and Detection System with CDP-Star from Amersham Bioscience was used for Southern hybridization using the mutants for the other three genes, following the manufacturer's instructions with minor modification. Gene-specific fragments amplified from NRRL 31084 genomic DNA with the primers in Supplementary Table S2 were used as probes to ascertain replacement of the gene and identify those mutants where the hph gene had integrated at locations other than the target gene in the genome (ectopic mutants). The hph gene fragment used in the split marker protocol was used as a probe to verify integration of the hph gene.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The second and third subtraction libraries, named Fgr-S and Fgr-S2 were generated by using cDNA synthesized with RNA from wheat heads inoculated with a highly aggressive strain NRRL 31084 or RNA derived from wheat inoculated with the less aggressive strain NRRL 28303. The Fgr-S subtraction library was constructed using RNA from NRRL 31084-infected wheat as the tester and RNA from NRRL 28303-infected wheat as the driver; the Fgr-S2 library was constructed using RNA from NRRL 28303-infected wheat as the tester and RNA from NRRL 31084-infected wheat as the driver. The relative aggressiveness of strains had been determined by our initial studies (Goswami & Kistler, 2005). A total of 1361 clones from these libraries were sequenced: 588 from the Fgr-S library and 773 from the Fgr-S2 library. The mean EST size was 0.35 kb. Marked differences in genes represented in Fgr-S and Fgr-S2 were observed, as only 6.4 % of the ESTs were found in both the forward and reverse subtraction libraries. However, the number of fungal sequences found in each library was quite low. Only seven ESTs of fungal origin were identified from these libraries: three ESTs from the forward subtraction library and four from the reverse subtraction library.
Distribution of ESTs into contigs and singlets and their comparisons to ESTs from F. graminearum in culture and to the M. grisea and N. crassa genomes
The CAP3 sequence assembly program was used to group together redundant ESTs from each library (Fgr-S3/S4, Fgr-S and Fgr-S2) which had overlapping sequences. A consensus sequence was obtained for each contig, and every EST in the contig was considered to be a copy of the transcript from the same gene. The 1236 ESTs from the Fgr-S3/S4 library could be assembled into 182 contigs and 630 singlets. Of these, 22 contigs and 56 singlets matched the whole-genome assembly, and 45 contigs and 30 singlets matched the excluded reads of the F. graminearum genome sequence. Remaining contigs and singlets are presumed to correspond to wheat genes. The 78 contigs and singlets matching the Fusarium genome assembly were chosen for further analysis. In addition to searching against GenBank, these EST contigs and singlets were compared to the predicted proteins from the rice blast fungus M. grisea and the saprophyte N. crassa. Twenty contigs and 38 singlets that had no significant homologue in either of the two genomes initially were considered to be specific to F. graminearum. However, among these, 12 contigs and 13 singlets matched predicted proteins from other organisms (Table 1). The EST contigs and singlets that also did not have any matches in GenBank are presumed to be specific to F. graminearum (Table 2). Those ESTs and contigs that had matches to proteins coded by M. grisea and N. crassa genomes are listed in Table 3. Comparisons also were made to EST sequences available in GenBank for libraries grown on trichothecene induction medium (Tag et al., 2001), complex plant medium, cornmeal and simple substrates, and on nitrogen- and carbon-limited media. The ESTs/contigs that were identified that match EST sequences from the fungus grown in culture also are noted in Tables 1, 2 and 3.
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Gene replacement
Genes corresponding to four ESTs (Fgr-S4_3_G23, Fgr-S4_2_M02, Fgr-S4_3_I05 and Fgr-S4_3_G14) were selected for targeted gene replacement. These genes, FG08308.1 (Abc2 – ATP binding cassette transporter 2), FG03405.1 (Lyp1 – Lysine permease 1), FG00215.1 (Rrr1 – Receiver response regulator 1) and FG02874.1 (Zbc1 – Zinc finger binuclear cluster regulator 1), had BLASTX matches to an ABC transporter, a lysine permease, a putative two-component response regulator and the alcR regulatory gene respectively. Two genes, Abc2 and Rrr1, had BLASTX matches with predicted proteins from both M. grisea and N. crassa. Based on BLASTX comparisons, the gene Zbc1 appeared to encode a protein related to a zinc binuclear cluster regulatory protein similar to alcR from Aspergillus nidulans with sequence similarity to the pathogen M. grisea, but not to the non-pathogen N. crassa. The Lyp1 gene, homologue of a lysine permease gene from Saccharomyces cerevisiae, did not have matches to either M. grisea or N. crassa. Based on these matches and their expression in planta it was suspected that these genes could be involved in pathogenicity or development of the fungus on the host; they were therefore selected for targeted gene replacement studies to test their influence on pathogenicity.
To confirm that these genes were indeed expressed in planta, RT-PCR was conducted using RNA collected from NRRL 31084 inoculated and mock (water)-inoculated wheat heads. The amplified products demonstrated that the four selected genes were expressed in planta on wheat heads inoculated with NRRL 31084 only. The expression of these genes was detected 24 h after inoculation and was observed at both time points, 48 and 72 h, thereafter. These results were consistent among all four genes and the gene for translation elongation factor 1 (EF-1), used as a positive control (Fig. 1).
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) are missing in four of the five transformants (labelled T1, T2, T4 and T5), indicating that for these strains, the Rrr1 gene has been deleted. Strain T3 maintains the 1.0 and 0.4 kb internal fragments of the Rrr1 gene, although it appears to be altered at the 2.8 kb flanking region. The 4.8 kb HindIII fragment corresponding to the intact gene for hygromycin resistance is found in all transformants but additional copies of the gene are also found in T3 and T4. A summary of the numbers of hygromycin-resistant colonies, deletion mutants and integration events not resulting in deletion for each of the four targeted genes is provided in Supplementary Table S3, available with the online version of this paper. Five strains with mutations in each of the four genes studied were tested for pathogenicity. For the genes Lyp1, Rrr1 and Zbc1, pathogenicity tests involved at least one successful gene deletion mutant and one transformant for which the targeted gene was not deleted (called ectopic mutants). Ectopic mutants were not obtained for the Abc2 gene and so could not be included in the pathogenicity tests. All four Rrr1 deletion strains exhibited reduced sporulation (P<0.05) as demonstrated by spore counts taken 8 dai (Fig. 3a) and also produced fewer symptomatic spikelets per wheat head (P<0.05), 7 dai (Fig. 3b), thereby indicating a delay in spread of the fungus. Nevertheless, the percentage of symptomatic spikelets observed 14 dai with Rrr1 mutants was found not to be significantly different from the wild-type. The ectopic mutant Rrr1T3 was similar to the wild-type in both spore production (P=0.92) and number of symptomatic spikelets (P=0.63). Mutants of each of the other three genes did not differ from wild-type with respect to pathogenicity or sporulation under the conditions tested (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Moreover, studies on barley have revealed that while extensive colonization of brush hairs may occur by 48 h, colonization of glumes, awns and the outer lemma surface is still sparse at this timepoint (Skadsen & Hohn, 2004). The trichothecene mycotoxin DON accumulates in detectable amounts only 48 h after inoculation of barley (Evans et al., 2000). Therefore, 48 h was considered an appropriate time point for investigating early stages of the host–pathogen interaction. Eighty-four putative fungal genes expressed during host–pathogen interaction between F. graminearum and wheat were identified. A predicted function for 49 of these genes was inferred by bioinformatic analysis.
Fifty-eight per cent of the fungal ESTs from the Fgr-S3/S4 library were found to be of mitochondrial origin, a much higher percentage than that from the fungus grown in culture (Trail et al., 2003). This could indicate a general increase in mitochondrial RNA, which reflects increased mitochondrial activity and/or increased abundance of these organelles in the active disease state. Increase in levels of fungal mitochondrial RNA during plant infection has been reported previously for M. grisea on rice (Talbot & Tongue, 1998). The F. graminearum mitochondrial ESTs most frequently observed were related to either the NAD reductase or cytochrome c oxidase (COX) complexes. These results suggest that respiratory competence is likely to be critical for development of the fungus in the host tissues. This is further supported by the recent finding that reduced virulence in a REMI mutant of F. graminearum was caused by an insertion in a nuclear gene predicted to encode the NADH : ubiquinone oxidoreductase complex I (Seong et al., 2005). The fungus may need enhanced mitochondrial activities to metabolize some plant molecules (for energy or detoxification) or simply to provide the energy for infectious growth. The mitochondrial antioxidant defence system also may be important for a necrotrophic pathogen like F. graminearum. Thus, while perhaps not specifically and uniquely involved in pathogenicity, these basic metabolic systems could prove to be effective targets for control of this pathogen.
Several ESTs from our libraries match scaffold 32 of the F. graminearum genome assembly, which has not been assigned a chromosomal location (Gale et al., 2005). Interestingly, the FgrS3S4Contig120 matching a region on scaffold 32 is homologous to a DNA polymerase from the mitochondrial plasmid pCry1 from Cryphonectria parasitica (Monteiro-Vitorello et al., 2000). This result suggests that this scaffold may be associated with a mitochondrial plasmid previously not identified in F. graminearum, and warrants further investigation.
Within the Fgr-S3/S4 EST libraries were several predicted protein-coding sequences corresponding to genes that may be associated with pathogenicity. Among these is a gene encoding a polyketide synthase (PKS14 – FG03964.1). Polyketide synthases (PKSs) are large multifunctional enzymes involved in production of polyketide secondary metabolites, including toxins such as aflatoxin, T-toxin, fumonisin, patulin and ochratoxin (Kroken et al., 2003). The F. graminearum gene identified as an EST during infection corresponds to a previously undescribed PKS orthologue of unknown function currently found only in F. graminearum. Expression of this gene has recently been described as ‘grain-specific’ since it has been found by RT-PCR to be expressed by the fungus during growth on rice and corn meal, but seemingly not during infection of wheat (Gaffoor et al., 2005). Our results indicate that the gene may indeed be expressed during plant infection, and microarray data of the gene during infection of barley indicates this is the case (Güldener et al., 2006b). The potential significance of the gene to pathogenicity remains to be determined.
An EST corresponding to a gene for an ABC transporter (FG08308.1) of unknown function also is represented in the Fgr-S3/S4 libraries. ABC transporters are membrane-associated proteins involved in transport of small molecules across membranes against a concentration gradient, coupled to the hydrolysis of ATP. Some fungal ABC transporters have been associated with plant pathogenicity (Fleissner et al., 2002; Stergiopoulos et al., 2003; Urban et al., 1999), including a gene in the closely related species F. culmorum (Skov et al., 2004); ABC transporters also have been shown to confer resistance to plant phytoalexins (Schoonbeek et al., 2001). However, deletion of FG08308.1 in this study resulted in no loss of pathogenicity to wheat under the conditions tested.
EST sequence Fgr-S4_2_M02 corresponds to predicted gene FG03405.1 (Lyp1) that has strong sequence similarity to and conserved features of a lysine permease. Fungal nutritional requirements in planta are being increasingly recognized as factors essential for establishing pathogenic interactions with plants (Solomon et al., 2003). Some plant-pathogenic fungi have been shown to be rendered non-pathogenic by mutations leading to deficiency in the biosynthesis of certain amino acids (Balhadère et al., 1999; Namiki et al., 2001). Amino acid transporters, especially lysine transporters, have been previously noted to be strongly up-regulated during plant infection (Hahn et al., 1997). Despite these observations, deletion of Lyp1 alone in F. graminearum resulted in no loss of pathogenicity under the conditions tested here. This could be due to the fact that the F. graminearum genome has many predicted amino acid permeases that perhaps are redundant in function so that deletion of any one of these genes would have no profound effect on pathogencity.
The EST sequence Fgr-S4_3_I05_T7 has a significant match to a gene encoding REC1, a putative two-component response regulator from Gibberella moniliformis. Two-component signal transduction pathways are phosphorelay mechanisms by which various organisms sense and adapt to their environment. They are generally composed of a histidine kinase (HK) and a response regulator (RR) domain, which are easily identifiable by sequence alignment (Borkovich et al., 2004). They have been found in bacteria, slime moulds, plants and fungi but not yet in animals (Borkovich et al., 2004; Catlett et al., 2003b). These systems have been shown to be involved in virulence responses in fungi. For example, the fos-1 gene from Aspergillus fumigatus (Clemons et al., 2002) and the cos-1 gene from Candida albicans (Selitrennikoff et al., 2001) are two-component HKs that have been shown to be putative virulence factors. The eukaryotic two-component phosphorelay systems mostly contain hybrid HKs where both the HK and RR domains are contained in a single polypeptide (Catlett et al., 2003b). These hybrid HKs are believed to function in multi-step phosphorelays where the phosphate is transferred from an RR domain of the hybrid HK to a second histidine residue and then to a second RR domain. The REC1-encoding gene was identified while searching for such potential downstream response regulators. It could not be related to other known fungal RRs or RR domains in hybrid HKs. Since G. moniliformis is taxonomically closer to G. zeae than the other fungi to which it has been previously compared (Catlett et al., 2003b), we propose that this gene could potentially be a novel RR or part of a previously uncharacterized two-component phosphorelay mechanism.
Among the four genes selected for our mutation studies, Rrr1 was found to play a role in sporulation and disease expression. Mutants for this gene exhibited significantly reduced ability to produce macroconidia on mung-bean agar medium as compared to the wild-type strain. They also produced a significantly lower percentage of symptomatic spikelets as compared to the wild-type 7 dai on the wheat head. The ectopic mutant retained its ability to produce conidia and spread to generate symptomatic spikelets at levels similar to the wild-type (Fig. 3
). This suggests that the Rrr1 gene is likely to play a role in pathogenicity and development of the fungus. As signal transduction pathways controlled by genes such as Rrr1 are phosphorelay mechanisms by which organisms sense and adapt to their environment (Borkovich et al., 2004), reduction in this ability due to deletion of the gene could account for the delay in spread of the mutants on the wheat heads as reflected by our results. The observation that the total number of symptomatic spikelets 14 dai was similar to that produced by the wild-type strain leads us to infer that this gene is likely to be involved in the initial response of the fungus to the host plant.
We have been successful in identifying several fungal genes expressed in the early stages of infection on wheat that have the potential to be involved in host–pathogen interaction. This information will help not only in understanding changes in overall gene expression in the pathogen during infection of the host, but also in identifying potential targets for development of disease control strategies. Surprisingly, a large percentage of sequences expressed in planta were not called genes by the ab initio gene prediction models used by the Broad Institute and MIPS. Forty-five per cent of the ORFs represented in the Fgr-S3/S4 library belong to this category. This suggests that currently available gene prediction models are not sufficient for identifying all F. graminearum genes and highlights the need for developing more appropriate models better suited to this organism. The ESTs from this library have thus been helpful in manual annotation of the F. graminearum genome and have supported the identification of 35 new ORFs. A more comprehensive study of fungal gene expression during plant infection is ongoing using a newly designed F. graminearum DNA microarray.
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Received 7 December 2005;
revised 30 January 2006;
accepted 8 February 2006.
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