Application of cDNA array for studying the gene expression profile of mature appressoria of Magnaporthe grisea

Application of cDNA array for studying the gene expression profile of mature appressoria of Magnaporthe grisea 1 first-author 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:htdong@zjuem.zju.edu.cnLidb@mail.hz.zj.cn †E-mail:htdong@zjuem.zju.edu.cnLidb@mail.hz.zj.cn htdong@zjuem.zju.edu.cn 2 3 1 1 1 1 1 3 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:htdong@zjuem.zju.edu.cnLidb@mail.hz.zj.cn †E-mail:htdong@zjuem.zju.edu.cnLidb@mail.hz.zj.cn htdong@zjuem.zju.edu.cn Bioinformatics and Gene Network Research Group, School of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Bioinformatics and Gene Network Research Group, School of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Key Laboratory of Molecular Plant Pathology, Ministry of Agriculture, China Agricultural University, Beijing 100094, China Key Laboratory of Molecular Plant Pathology, Ministry of Agriculture, China Agricultural University, Beijing 100094, China Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, Nanning 530004, China Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, Nanning 530004, China Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea . Analysis of gene expression profiles of appressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST (expressed sequence tag) database of M . grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M . grisea . A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M . grisea . The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTH11 , beta subunit of G protein and SGT1 involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results. Magnaporthe grisea Mature appressoria cDNA array Gene expression profile INTRODUCTION 1 The rice blast pathosystem has emerged as a model system for studying plant-fungal interactions, because of one of attributes of the pathogen Magnaporthe grisea evolving an elaborate mechanism to penetrate host plant cells through a specialized infection structure known as appressorium, which can form on the hydrophobic surface of the host cells (Dean et al., 2005 2005 ; Valent, 1990 1990 ). Appressorium is also induced by hydrophobic signals in vitro 2 to 8 h after conidium germination, followed by maturation in 16 h (Lee and Dean, 1994 1994 ; Talbot, 1995 1995 ). Researches of individual genes on molecular mechanisms of initiation, formation and maturation of appressorium have been carried out one by one, and more than 30 related genes have been cloned (Talbot, 2003 2003 ). Recently, serial analysis of gene expression (SAGE) (Irie et al., 2003 2003 ), cDNA array (Takano et al., 2003 2003 ), suppression subtractive hybridization (Lu et al., 2005 2005 ), and expressed sequence tag (EST) (Ebbole et al., 2004 2004 ; Jantasuriyarat et al., 2005 2005 ) techniques have been adopted to study the molecular mechanism of appressorium development and pathogenesis at the systemic biology level, resulting in identification of many genes differentially expressed in appressorium development of M . grisea . Maturation of appressorium after its morphogenesis is a long and complex biological process. During this development stage turgor in appressorium generates and accumulates, which affects differentiation of penetration pegs and pathogenicity of the pathogen (Talbot, 2003 2003 ; Thines et al., 2000 2000 ). The molecular mechanism of appressorium maturation during turgor accumulation in the aspect of system biology is not well studied. In this study, we used an M . grisea cDNA array to detect gene expression profiles of mycelia and mature appressoria (20 h after conidium germination). Seventy-seven differentially expressed genes that are related to appressorium maturation were identified. Our data provide insight into appressorium development, as well as the molecular mechanisms mediating M . grisea development and pathogenesis. MATERIALS AND METHODS 1 M . grisea culture and total RNA extraction 2 The strain of M . grisea Y34 used in this study was isolated from Yunnan Province of China (Zheng et al., 1998 1998 ). The fungus was grown in oatmeal agar medium (juice after 50 g oatmeal was boiled, 15 g agar, 1 L water) for 10 d (28 °C, 12/12 h alternating day and night), and conidia suspension filtered once through Miracloth (Calbiochem, USA) was used for the following different purposes. For vegetative mycelia, conidia suspension was cultured in liquid complete media (CM) (Talbot et al., 1993 1993 ) for 72 h at 28 °C with a rotary shaking at 150 r/min for RNA extraction. Mycelia were collected from three independent experiments. For appressorium formation, about 50 µl of conidia suspension (10 5 conidia/ml) was placed on transparent films (Gaoke, China) and kept for induction of appressoria at 28 °C in humid box. Twenty hours after inoculation, fluid droplets on the films were removed, the films adhering to appressoria were quickly soaked in liquid nitrogen, and appressoria were scraped with a blade off for RNA extractions from the frozen films. Appressoria was collected from three independent experiments. The total RNA from mycelia and appressoria of M . grisea was extracted immediately with Trizol™ reagent (Gibco-BRL, USA), according to the manufacturer’s instructions. Contaminating genomic DNA was removed by treatment with RNase-free DNase (Promega, USA). Preparation of cDNA arrays 2 We constructed a cDNA library of M . grisea Y34 prepared from mixed mRNA at mycelia, conidia, and different germlings of appressorium development process. From the cDNA library a total 13057 ESTs were acquired, which assembled 4756 unique ESTs. All EST and unique EST sequences can be found at our laboratory database ( www.estarray.org www.estarray.org ). For the preparation of cDNA array, 4756 unique clones were amplified by PCR using universal M 13 primers (5′-CCCAGTCACGACGTTGTAAAACG-3′ and 5′-AGCGGATAACAATTTCACACAGG-3′). The clones that did not amplify or that produced nonspecific PCR products, as determined by gel electrophoresis were discarded, while other 2927 cDNA samples were selected for array printing. The PCR products precipitated by isopropanol were resuspended in 15 µl of a denatured solution containing 0.4 mol/L NaOH and 10 mmol/L EDTA until they were printed on the arrays. The arrays were printed on Immobilon™-Ny+transfer membranes (Millipore, USA) using a GeneTAC™ G3 arrayer (Genomic Solutions, USA). Every cDNA sample was duplicated twice in each array. Additionally, several control clones from ribosomal RNA were also included. Probe preparation and hybridization 2 Hybridization probes were prepared respectively from total RNA samples isolated from mycelia and mature appressoria. The RNA samples were labelled with α- 33 P-dCTP during the first-strand reverse transcription reactions. A typical labelling reaction contained 20 mmol/L each of dATP, dTTP and dGTP, 10 mmol/L dCTP and 5 µl of α- 33 P-dCTP (10 mCi/ml, Amersham, USA), 10 µl of 5×First-Strand Buffer, 0.5 mmol/L DTT and 400 units SuperScript™ II RNase H − Reverse Transcriptase (Invitrogen, USA), 0.3 µg oligo(dT) 16 primer, 0.1 µg random hexamer primer and 5 µg of total RNA in a reaction volume of 50 µl. The reaction proceeded at 42 °C for 2 h, and the products were purified using a Sephadex G-50 column (Amersham, USA). cDNA array was irradiated (60 mJ/cm 2 ) by UV crosslinker (UVP Inc., USA) before hybridization. The hybridizations were performed overnight at 60 °C in a hybridization oven (ThermoHybaid, UK). Subsequently, the nylon membranes were washed twice at 60 °C in 2×SSC and 0.1% (w/v) SDS for 20 min and once 60 °C in 0.1×SSC and 0.1% (w/v) SDS for 20 min. Three individual hybridization experiments were performed for each sample. cDNA array analysis 2 The signals of the array were absorbed by storage phosphor screen (Kodak, Japan) for 3 d, and signal intensities were measured by scanning the storage phosphor screen with Typhoon 9200 PhosphorImager (Amersham, USA). The signal intensity of each cDNA spot on the array was quantified using software ArrayVision 6.0 (Imaging Research, Canada). With this program, the normalization between different cDNA arrays was performed by using a spot’s signal intensity to divide the average signal intensity of the whole image (global normalization). Total 6 nARVOL (normalized AR volume) values were acquired for each gene per sample. The regularized t -test approach in the Cyber-T program ( http://visitor.ics.uci.edu/genex/cybert/ http://visitor.ics.uci.edu/genex/cybert/ ) was used to determine gene expression changes between two samples. All cDNA expression data are available at http://www.estarray.org/array/default.asp http://www.estarray.org/array/default.asp . Quality control 2 In order to test whether PCR products of the genes on the cDNA array were right, 192 PCR products were ligated into pGEM®-T Easy vector (Promega, USA) and transformed into Escherichia coli DH5α cells by electroporation using a Gene Pulser Xcell™ System (Bio-Rad, USA) according to the manufacturer’s instructions. Individual recombinant colonies were inoculated into 1 ml of 2×YT (yeast extract-tryptone) liquid medium (16 g tryptone, 10 g yeast extract, 5 g NaCl in one liter) in the single wells of 96-well micro plates, and centrifuged for 15 h at 200 r/min. Plasmid DNA was extracted following the protocol of plasmid DNA purification kits (Millipore, USA). We sequenced these 192 cDNA clones from 5′ and 3′ end on a MegaBACE™ 1000 automated sequencer (Molecular Dynamics, USA) using standard M 13 primers (5′-CCCAGTCACGACGTTGTAAAACG-3′ and 5′-AGCGGATAACAATTTCACACAGG-3′) and the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham, USA). Sequence alignment was carried out with standalone BLAST against the above M . grisea EST sequence database. Description of preparation, analysis and quality control of cDNA array can be found in the published papers of our laboratory (Deng et al., 2006 2006 ; Dong et al., 2005 2005 ; Guo et al., 2005 2005 ). Northern blot hybridization 2 About 5 µg of total RNA from the above mycelia and mature appressoria was separated on a 1% (w/v) denaturing formaldehyde gel and then transferred to a nylon membrane (Millipore, USA) according to the method in (Sambrook and Russell, 2001 2001 ). Candidate genes as probes came from differentially expressed genes by cDNA array analysis. Probes were labelled through PCR amplification with α- 33 P-dCTP (Amersham, USA) using Prime-a-gene® Labelling System (Promega, USA). Hybridization methods and corresponding buffer were the same to method in (Sambrook and Russell, 2001 2001 ). The hybridization signals on the membranes were absorbed by storage phosphor screen (Kodak, Japan) for 3 d, and signal intensities were measured by Typhoon 9200 PhosphorImager (Amersham, USA). RESULTS 1 Appressorium induction of M . grisea 2 At 20 h after conidia inoculation on hydrophobic surfaces, more than 95% conidia germinated and formed seal brown mature appressoria, with appressorium pores being seen clearly (Fig. 1 1 ). Total RNA samples from mycelia and mature appressoria were used in probe preparation for hybridization of cDNA array. Analysis of cDNA array hybridization results 2 The cDNA array containing 2927 unique genes of M . grisea was based on a large collection of the fungal ESTs. For cDNA array construction, PCR amplified inserts of the unique genes were spotted. In addition, a collection of control clones from ribosomal RNA were included in the cDNA array. The identity of 192 randomly chosen DNA clones from the unique genes was confirmed by resequencing. In all cases, the sequences of the clones plated on the cDNA array were identical to the original EST sequence. This confirmation increases confidence in the identity of clones spotted on the cDNA array. In order to maximize the statistical reliability of the data, different biological and technical replicates were carried out as follows. Three biological replicates were carried out with new tissues grown under the conditions described above. In addition, for each biological replicate, two technical replicates (each PCR fragment was arrayed twice on each nylon membrane) were used for hybridization. The probe made from mature appressoria was hybridized with the cDNA array for three times and the coefficient of determination ( R 2 ) was 0.96, 0.96 and 0.99. The same procedure was done using a probe made from mycelia, and R 2 was 0.94, 0.94 and 0.99. Above results indicated that repeat experiments on cDNA array hybridization were good and that the array data could be used for further analysis. Fig. 2 2 shows scanning images of the cDNA array after hybridization with isotopic nucleotide labelled probes. Images of the cDNA array were quantified using the software ArrayVision 6.0. Scatter plots of the transcript abundance of 2927 unique genes from mycelia and mature appressoria are shown in Fig. 3 3 . Signal values of most scatter dots were less than 1 [log(transformed value) < 0] in mycelia and mature appressoria, which indicated the transcript abundance of most genes at both stages was low. Most of the gene dots lied close to the diagonal line, showing that the transcript abundance of these genes in mycelia and mature appressoria was similar. Some gene dots lied far from the diagonal line, indicating these genes were differentially expressed between two stages; the farther from the diagonal line the dots were, the bigger the difference in expression level. Differentially expressed genes in mature appressoria 2 The Cyber-T program calculates the fold change and significance of the difference in expression. In order to maximize the screening reliability of differentially expressed genes, genes that met the following three criteria were identified as differentially expressed genes: (1) confidence level of P < 0.01, (2) fold change cutoff of the signal value being more than 3, (3) larger signal value of gene with at least one of any two stages being larger than 0.2 (nARVOL). According to these criteria, 77 differentially expressed genes were identified in mature appressoria compared with mycelia. Of these, 55 genes were up-regulated and 22 genes were down-regulated (Table 1 1 ). indicates that clone showed no similarity to the genome sequences of M . grisea 70-15 ( E ≥10 −3 ) indicates that fold changes reached statistical significance of the difference in expression level between mature appressoria and mycelia (confidence level of P < 0.01) 2 Clone 2 GenBank No. 2 Annotation 2 E value 2 Signal intensity 2 Fold change b b Mycelia Appressoria 7 Up-regulated p3fmgcf_003968 CK916894 Pyruvate carboxylase 2 ( Candida robusta ) 0 3.5 13.8 4.0 p3fmgc_016531 CK925431 Related to coclaurine N-methyltransferase ( Neurospora crassa ) 2E−110 5.0 15.4 3.1 p3fmgc_014963 CK924418 Similar to dTDP-D-glucose 4,6-dehydratase ( Arabidopsis thaliana ) 1E−77 1.6 4.8 3.1 p3fmgc_017013 CK925780 Heterochromatin protein 1 ( Drosophila melangaster ) 7E−10 0.6 4.0 6.3 p3fmgc_015770 CK924917 Cutinase transcription factor 1 beta ( Fusarium solani ) 0 3.1 9.5 3.1 p3fmgcf_004759 CK917547 60S ribosomal protein L27 ( Cryptococcus neoformans ) 2E−14 0.5 1.9 3.8 p3fmgc_014039 CK923805 60S ribosomal protein L20 ( Candida robusta ) 0 2.3 6.9 3.0 p3fmgc_009740 CK920976 60S acidic ribosomal protein P2 ( Fusarium culmorum ) 1E−20 0.4 1.2 3.1 p3fmgcf_003681 CK916638 60S acidic ribosomal protein P0 ( Podospora anserina ) 0 0.9 2.9 3.1 p3fmgcf_003730 CK916679 Protein transport protein sec73 ( Schizosaccharomyces pombe ) 0 0.2 0.9 3.8 p3fmgc_006682 CK918982 H + /nucleoside cotransporter ( Aspergillus fumigatus ) 7E−167 1.4 4.6 3.2 p3fmgc_010096 CK921272 Nucleoporin nup184 ( Schizosaccharomyces pombe ) 8E−44 0.5 2.4 4.5 p3fmgc_015331 CK924608 Tubulin alpha-1 chain ( Aspergillus nidulans ) 0 4.0 13.1 3.3 p3fmgc_007888 CK919831 Hydrophobin-like protein Mpg1 precursor ( Magnaportha grisea ) 0 0.4 1.5 4.0 p3fmgc_007879 CK919824 Putative zinc knuckle domain protein (Byr3) ( Aspergillus fumigatus ) 1E−25 0.4 1.3 3.1 p3fmgc_015536 CK924732 Putative NADH-ubiquinone oxidoreductase B18 subunit ( Aspergillus fumigatus ) 6E−29 3.4 10.8 3.2 p3fmgcf_005058 CK917797 Phospholipid metabolism-related protein ( Cryptococcus neoformans ) 1E−24 0.9 3.3 3.5 p3fmgc_015911 CK924986 Putative transcriptional regulator ( Magnaporthe grisea ) 2E−40 1.9 8.6 4.4 p3fmgc_006559 CK918905 Putative Sgt1 protein ( Aspergillus fumigatus ) 9E−101 6.4 21.7 3.4 p3fmgc_018263 CK926361 G-protein beta like WD repeat protein ( Fusarium oxysporum ) 0 0.9 2.8 3.0 p3fmgc_016443 CK925364 Integral membrane protein ( Aspergillus fumigatus ) 3E−95 0.4 1.3 3.1 p3fmgc_018350 CK926422 UVI-1 gene induced by near-UV light ( Bipolaris oryzae ) 4E−72 0.3 1.7 5.0 p3fmgc_012062 CK922573 Signal recognition particle 14 kD protein ( Aspergillus fumigatus ) 3E−11 0.4 1.6 4.0 p3fmgc_019369 CK927054 Copper metallothionein ( Neurospora crassa ) 7E−4 0.3 0.9 3.2 p3fmgc_010478 CK921529 Hypothetical protein MG03625.4 ( Magnaporthe grisea ) 0 0.2 0.8 3.4 p3fmgc_006276 CK918694 Predicted protein MG04367.4 ( Magnaporthe grisea ) 0 0.5 1.7 3.4 p3fmgc_013687 CK923540 Hypothetical protein MG04654.4 ( Magnaporthe grisea ) 0 1.4 4.8 3.5 p3fmgc_006351 CK918755 Hypothetical protein MG04836.4 ( Magnaporthe grisea ) 0 1.8 5.8 3.2 p3fmgc_016377 CK925316 Hypothetical protein MG09618.4 ( Magnaporthe grisea ) 0 3.1 9.8 3.1 p3fmgc_012651 a a CK923046 Unknown − 3.1 12.9 4.1 p3fmgcf_004538 CK917359 Unknown − 1.8 6.2 3.4 p3fmgc_010655 a a CK921636 Unknown − 2.7 9.2 3.4 p3fmgc_007568 CK919581 Unknown − 1.1 3.3 3.0 p3fmgc_017202 CK925886 Unknown − 1.0 3.2 3.2 p3fmgc_006167 a a CK918615 Unknown − 1.1 3.8 3.5 p3fmgc_005954 CK918534 Unknown − 3.7 13.1 3.6 p3fmgc_016946 CK925761 Unknown − 6.8 25.2 3.7 p3fmgc_018594 CK926628 Unknown − 1.1 5.3 4.9 p3fmgc_009430 a a CK920812 Unknown − 8.2 25.4 3.1 p3fmgc_009675 a a CK920940 Unknown − 1.5 5.0 3.4 p3fmgcf_004668 CK917466 Unknown − 2.3 8.4 3.7 p3fmgc_006635 a a CK918945 Unknown − 1.4 4.3 3.1 p3fmgc_007193 a a CK919313 Unknown − 0.4 2.2 5.2 p3fmgc_015653 a a CK924820 Unknown − 0.4 1.9 4.6 p3fmgc_015571 a a CK924761 Unknown − 0.6 2.1 3.3 p3fmgc_010647 a a CK921629 Unknown − 0.4 1.6 3.9 p3fmgc_016358 CK925300 Unknown − 0.5 1.6 3.2 p3fmgc_012659 a a CK923053 Unknown − 0.3 1.2 3.6 p3fmgcf_004487 CK917315 Unknown − 1.1 3.7 3.3 p3fmgc_012479 a a CK922902 Unknown − 0.4 1.8 4.0 p3fmgc_016180 CK925181 Unknown − 0.3 1.4 4.9 p3fmgcf_004752 CK917541 Unknown − 8.4 25.4 3.0 p3fmgc_008171 a a CK920009 Unknown − 0.4 1.3 3.4 p3fmgc_012910 CK923215 Unknown − 2.4 7.7 3.2 p3fmgc_014484 a a CK924087 Unknown − 0.3 0.8 3.1 7 Down-regulated p3fmgc_018329 CK926411 Glyceraldehyde 3-phosphate dehydrogenase ( Podospora anserina ) 0 3.9 0.6 6.0 p3fmgcf_000490 CK913975 RNA 3′-terminal phosphate cyclase-like protein ( Candida robusta ) 0 3.2 1.0 3.2 p3fmgcf_004048 CK916959 Heat shock protein Sti1 ( Candida robusta ) 7E−16 1.0 0.3 3.7 p3fmgc_013455 CK923388 Peptidyl-prolyl cis-trans isomerase ( Neurospora crassa ) 9E−115 1.6 0.5 3.3 p3fmgc_016518 CK925421 Possible copper transport protein Ctr2 ( Candida robusta ) 1E−19 1.2 0.3 4.4 p3fmgc_017343 CK925929 Related to GTPase activating protein ( Neurospora crassa ) 0 2.0 0.6 3.3 p3fmgc_009930 CK921137 b-ZIP transcription factor IDI-4 ( Podospora anserina ) 3E−15 1.0 0.2 4.9 p3fmgc_010939 CK921810 Reverse transcriptase ( Magnaporthe grisea ) 1E−179 3.5 1.1 3.3 p3fmgcf_000222 CK913775 Elongation factor 1-alpha ( Aspergillus oryzae ) 0 1.7 0.6 3.0 p3fmgc_007834 CK919784 Putative TBC domain protein ( Aspergillus fumigatus ) 7E−100 1.2 0.3 3.6 p3fmgcf_000566 CK914039 Integral membrane protein, Pth11 ( Magnaporthe grisea ) 5E−17 2.8 0.6 4.4 p3fmgc_005845 CK918469 Putative transposase ( Magnaporthe grisea ) 0 1.3 0.3 4.0 p3fmgc_017874 CK926198 Related to spore coat protein sp96 precursor ( Neurospora crassa ) 4E−12 3.5 0.5 7.3 p3fmgc_007636 CK919625 Male sterility protein 2 ( Arabidopsis thaliana ) 4E−15 1.3 0.4 3.3 p3fmgc_006724 CK919007 Hypothetical protein MG00432.4 ( Magnaporthe grisea ) 0 1.9 0.6 3.0 p3fmgc_013422 CK923366 Hypothetical protein MG10198.4 ( Magnaporthe grisea ) 0 3.0 0.9 3.4 p3fmgc_015509 a a CK924710 Unknown − 155.5 48.7 3.2 p3fmgc_017596 CK926044 Unknown − 1.2 0.2 4.9 p3fmgcf_003124 a a CK916195 Unknown − 0.8 0.2 3.9 p3fmgc_007826 CK919778 Unknown − 2.8 0.7 3.8 p3fmgc_005375 CK918157 Unknown − 2.5 0.5 4.7 p3fmgcf_000736 CK914184 Unknown − 1.2 0.4 3.0 The sequences of 77 differentially expressed genes from mature appressoria of M . grisea Y34 were performed BLASTn searches against the draft sequences of the M . grisea 70-15 genome ( http://www.broad.mit.edu/annotation/fungi/magnaporthe/ http://www.broad.mit.edu/annotation/fungi/magnaporthe/ ). A total of 61 genes could be matched with the M . grisea genome database ( E < 10 −3 ), the remaining 16 genes showed no similarity to the genome sequences of M . grisea ( E ≥10 −3 ) (Table 1 1 ). We know that only about 4% genome sequence of M . grisea was not obtained (Dean et al., 2005 2005 ). So the 16 unique transcripts could not match the genome sequences of M . grisea 70-15, probably because they fall within gaps of genome sequences although they may also represent genes unique to M . grisea Y34. In order to annotate the 77 differentially expressed genes, we performed BLASTx searches against the NCBI (the National Center for Biotechnology Information) protein database (Table 1 1 ). A total 32 genes showed no homology with identified proteins deposited in the NCBI database ( E ≥10 −3 ). These genes were considered unknown novel genes differentially expressed in the mature appressoria of M . grisea . Other 45 genes had significant matches ( E < 10 −3 ) and were given functional annotation. Among the 45 annotated genes, only 12 genes were annotated known or hypothetical protein of M . grisea , the remaining 33 genes had hits with proteins of other origins. Novel homologue of peptidyl-prolyl cis-trans isomerase 2 A gene CYP1 encoding peptidyl-prolyl cis-trans isomerase (PPIase) (GenBank accession No. AF293848) can serve as a virulence determinant during plant infection (Viaud et al., 2002 2002 ), another homologue of PPIase (GenBank accession No. AI068531) was found up-regulated in conidia of M . grisea (Takano et al., 2003 2003 ). We found a homologue of PPIase (GenBank accession No. CK923388) down-regulated in mature appressoria. BLASTn searches were performed against M . grisea genome database with the results indicating three clones CK923388, AF293848 and AI068531 could match respectively three predicted genes MG08104.4 ( E value=10 −48 ), MG10447.4 ( E value=0) and MG07389.4 ( E value=10 −174 ). We further compared conserved domains of three predicted genes encoding proteins using NCBI Conserved Domain Search Database ( http://ncbi.nih.gov/Structure/cdd/cdd.shtml http://ncbi.nih.gov/Structure/cdd/cdd.shtml ) (Fig. 4 4 ). The clone AI068531 had PPIC (peptidylprolyl isomerase C)-type PPIase domain (rotamase). Clones CK923388 and AF293848 had a cyclophilin_ABH domain, but the domain laid in different location of open reading frame of two genes, N-terminal extension of the former gene and C-terminal extension of the later gene. So the homologue of PPIase identified from this study was a novel gene expressing weakly in mature appressria of M . grisea . CK923388, cyclophilin_ABH [cyclophilin A, B and H-like cyclophilin-type peptidyl-prolyl cis-trans isomerase (PPIase) domain], E value=10 −70 ; AF293848, cyclophilin_ABH, E value=10 −61 ; AI068531, rotamase (PPIC-type PPIase domain), E value=10 −16 , WW [domain with 2 conserved Trp (W) residues], E value=10 −4 . The number above bold lines indicates the number of amino acids in open reading frames of peptidyl-prolyl cis-trans isomerase homologues Northern blot analysis 2 Northern blot analysis was done to confirm the cDNA array results. Eleven genes for analysis were randomly picked from 77 differentially expressed genes (Fig. 5 5 ). Seven genes (CK918905, CK917797, CK926361, CK919831, CK926422, CK922573 and CK925364) were up-regulated in mature appressoria compared with mycelia. The remaining four genes (CK926411, CK914039, CK923388 and CK921137) were down-regulated in mature appressoria. These results are consistent with the cDNA array results and confirm the cDNA array experiment is reliable. MY: Mycelia; MA: Mature appressoria; N: Northern blot; C: cDNA array nARVOL value DISCUSSION 1 Metabolism changes related to appressorium turgor 2 Strong turgor in appressorium allows a penetration peg to invade the host tissue. Accumulation of high concentration glycerol in appressorium was considered to be the main cause of turgor (de Jong et al., 1997 1997 ). Glycerol is a degradation product of triacyglycerol, and can also come from gluconeogenesis demonstrated by a mutant research of gene ICL1 encoding isocitrate lyase in M . grisea (Thines et al., 2000 2000 ; Wang et al., 2003 2003 ). We also found a homologue of pyruvate carboxylase involved in gluconeogenesis up-regulated in mature appressoria. The gene might be related to glycerol production involved in turgor accumulation and still remained to verify its function in appressoria. Some carbohydrates such as glycogen also participate in turgor generation in appressoria (Talbot, 2003 2003 ). We found up-regulation of phospholipid metabolism-related gene involved in lipid catabolism and down-regulation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) homolog involved in glycolysis in mature appressoria. Takano et al.( 2003 2003 ) also showed the down-regulation of GAPDH in developing appressoria using cDNA array analysis. All the above results might be cues of induced lipid catabolism and reduced glycolysis in appressorium development. Reduction in glycolysis in appressoria is unlikely to ensure macromolecules or carbohydrates accumulation (Talbot, 2003 2003 ). It more likely reflected that the carbohydrates such as glycogen are not degraded in appressorium development and that lipid catabolism is the main source of energy and glycerol. Genes related to cell signaling of appressorium 2 Cell signaling pathways regulate appressorium formation and development (Dean, 1997 1997 ). PTH11 gene encoding a plasma membrane protein mediates appressorium formation of M . grisea (DeZwaan et al., 1999 1999 ). Our data showed that a homologue of PTH11 had lower expression in mature appressoria. It might imply that PTH11 had no significant relation to appressorium maturation. The heterotrimetric G proteins, which are composed of α, β and γ subunits, are essential in signal transduction and regulation of fungal growth and development. We found a homologue of FGB1 encoding β subunit of G protein highly expressed in mature appressoria. Gene FGB1 regulates Fusarium development and virulence through multiple signaling pathways (Delgado-Jarana et al., 2005 2005 ). In this study the homologue of FGB1 showed no significant homology to MGB1 gene of M . grisea , which also encoded β subunit involved in appressorium formation of M . grisea (Nishimura et al., 2003 2003 ). We believed that the homologue was a novel member of G protein β subunit of M . grisea and might be related to cell signaling in appressorium maturation. Mitosis is regulated by signals inside cells. There is a single mitosis before appressoium formation and mitosis starts again only in infectious hyphae of M . grisea (Dean, 1997 1997 ). Sgt1 protein is involved in kinetochore assembly of mitosis and regulation of adenylate cyclase activity in cAMP signal transduction pathways in eukaryotic cell (Dubacq et al., 2002 2002 ; Steensgaard et al., 2004 2004 ). In M . grisea , we identified a homologue of SGT1 gene, the transcript abundance of which is highest in mature appressoria. Because there is no mitosis in appressorium maturation, up-regulation of SGT1 might reflect activation of the cAMP pathway responsible for appressorium maturation. Comparison of differentially expressed genes in appressoria with results of previous studies 2 Mature appressoria is dispensable for penetration into host plant cell. We found 45 annotated genes among 77 genes differentially expressed in mature appressoria of M . grisea . We compared our annotated genes with results from previous analyses of subtracted cDNA libraries, SAGE, promotor- egfp (a red-shifted green fluorescent gene) fusion system, microarray in appressorium initiation, formation and maturation of M . grisea (Banno et al., 2003 2003 ; Irie et al., 2003 2003 ; Kamakura et al., 1999 1999 ; Lu et al., 2005 2005 ; Takano et al., 2003 2003 ). Thirty-nine annotated genes were isolated in mature appressoria for the first time in this study, besides 8 homologues of hydrophobin, metallothionein, dTDP-D-glucose 4,6-dehydratase, UVI-1, PPIase, GAPDH, integral membrane protein Pth11 elongation factor 1-alpha and hypothetical protein MG09618.4 identified in previous studies on appressorium development of M . grisea . Among the 8 genes, some genes such as PPIase might be novel homologues different from previous studies. We found four genes encoding different ribosomal proteins up-regulated in mature appressoria, but a gene encoding elongation factor 1-alpha was up-regulated in mycelia and down-regulated in mature appressoria. A similar phenomenon was also found in previous study. It was possible that the translational machinery was not identical between appressoria and mycelia (Takano et al., 2003 2003 ). 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Variability of pathogenicity of Pyricularia oryzae 10.1631/jzus.2007.B0088 Project (No. 2002BA711A15) supported by the National Hi-Tech Research and Development Program (863) of China 2006-04-10 2006-07-11 2022-01-05 Copyright © 2007, Journal of Zhejiang University Science 2007 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 08