Cloning, sequencing and expression analysis of the NAR promoter activated during hyphal stage of Magnaporthe grisea

Cloning, sequencing and expression analysis of the NAR promoter activated during hyphal stage of Magnaporthe grisea 1 first-author †E-mail:jplu@zju.edu.cnfuchenglin@zju.edu.cn †E-mail:jplu@zju.edu.cnfuchenglin@zju.edu.cn jplu@zju.edu.cn 1 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jplu@zju.edu.cnfuchenglin@zju.edu.cn †E-mail:jplu@zju.edu.cnfuchenglin@zju.edu.cn jplu@zju.edu.cn Department of Biology, School of Life Sciences, Zhejiang University, Hangzhou 310058, China Department of Biology, School of Life Sciences, Zhejiang University, Hangzhou 310058, China Biotechnology Institute, Zhejiang University, Hangzhou 310029, China Biotechnology Institute, Zhejiang University, Hangzhou 310029, China The promoter of NAR gene in Magnaporthe grisea was isolated and sequenced. The promoter sequences contained the “TATA” box, the “CAAT” box, and binding sites for fungal regulatory proteins. Programs that predict promoter sequences indicated that promoter sequence lies between locations 430 and 857 of the NAR promoter fragment. GFP expression under the NAR promoter and NAR transcript analysis revealed that this promoter is activated primarily at the mycelial stage in the rice blast fungus and could be used to express native or extrinsic genes in the mycelia of the rice blast fungus. Magnaporthe grisea Promoter NAR Clone Hypha INTRODUCTION 1 The fungus Magnaporthe grisea is the cause of the rice blast disease found the world over wherever rice is grown and is increasingly used as a model system to study host-pathogen interaction (Dean et al., 2005 2005 ). Introducing such extrinsic genes as GFP , or the gene for fusion protein, into the blast fungus under the control of a native promoter is an effective method to study this fungus. The powerful active promoter of M . grisea can also be used for gene expression in other pathogenic fungi or fungi of industrial significance to study the function of a gene or to produce the protein. Although many promoters of this fungus have been obtained during studies on the function of pathogenic genes (Bruno et al., 2004 2004 ; Foster et al., 2003 2003 ; Fudal et al., 2007 2007 ; Xue et al., 2002 2002 ), these promoters were generally expressed weakly in the fungus. Also, a powerful promoter actively expressed during the hyphal stage of this fungus has never been reported. In this paper, we report a typical hypha promoter, which is actively expressed in the hyphae of M . grisea , and its cloning, sequencing, and expression analyses. MATERIALS AND METHODS 1 Isolates of the fungal pathogen and growth conditions 2 M . grisea strain Guy-11 was cultured on complete medium (Talbot et al., 1993 1993 ) at 25 °C in a cycle comprising 14 h of light alternating with 10 h of darkness. Conidia were harvested and resuspended (1×10 5 spores/ml), and a 20-μl drop was placed on each plastic coverslip for studying conidial germination and appressorium formation, following the procedure described before (Lu et al., 2005 2005 ). Cloning and sequencing of the NAR promoter 2 The DNA fragment containing promoter sequences of the NAR gene (putative protein MG10274.5 in the M . grisea genome database) was amplified from Guy-11 genomic DNA by polymerase chain reaction (PCR) using primers 5′-CAGGATCCGGGAAGCGATTGCGTT-3′ and 5′-GGTGCCATGGTGTCGGTTGTGGTG-3′ and cloned into a T-vector, namely pUCm-T (Sangon, Shanghai, China). The recombinant cDNA clones were directly sequenced on an ABI 377 DNA sequencer (Applied Biosystems, USA) with primers M13R and M13F. DNA isolation, PCR, ligation reaction, restriction digest, and gel electrophoresis were carried out following standard procedures (Sambrook et al., 1989 1989 ). The structure of the NAR promoter was predicted using online promoter analysis programs, such as Neural Network Promoter Prediction ( http://www.fruitfly.org/seq_tools/promoter.html http://www.fruitfly.org/seq_tools/promoter.html , version 2.2), TSSP ( http://www.softberry.com/berry.phtml http://www.softberry.com/berry.phtml , prediction of plant promoters), and TFSCAN ( http://bioweb.pasteur.fr/seqanal/interfaces/tfscan.html http://bioweb.pasteur.fr/seqanal/interfaces/tfscan.html ). Construction of green fluorescent protein (GFP) expression vector and fungal transformation 2 The promoter DNA fragment of the NAR gene was cut out from the T-vector containing the NAR promoter using restriction endonuclease Bam HI and Nco I and inserted into Bam HI- Nco I sites of pEGFP (Clontech, USA) to produce an interim vector, namely pEGFP-NAR. Then a hygromycin-resistant gene ( HPH ) was cut out using Sal I from pCB1003 vector (Carroll et al., 1994 1994 ) and inserted at the Sal I sites of pEGFP-NAR to generate pNAR. In the pNAR, the GFP expression construct was under the control of the native NAR promoter. The resulting vector pNAR was transformed into protoplasts of Guy-11 and transformants were selected on the complete medium with 20% sucrose and 200 μg/ml hygromycin B (Roche Diagnostics GmbH, Germany). The hygromycin-resistant transformants were further confirmed by passing them through one more round of selection and then individually confirmed by PCR and DNA gel blot analysis. The transformation procedure including protoplast generation was the same as that described before (Liu et al., 2007 2007 ). Analyses of GFP expression 2 The expression of GFP in cells during the development stage of M . grisea was examined and photographed using an Olympus-BX51 epifluorescence microscope with a cooled CCD camera DP50 (Olympus, Japan). And the expression of GFP in the hyphae grown in CM liquid medium (complete medium) and CM-N liquid medium (complete medium sans the nitrogen source) (Talbot et al., 1993 1993 ) was also examined. RESULTS AND DISCUSSION 1 Cloning of NAR promoter 2 After searching the phytopathogenic fungi and oomycete EST database in COGEME ( http://cogeme.ex.ac.uk http://cogeme.ex.ac.uk ) (Soanes and Talbot, 2005 2005 ), an expressed sequence tag (EST) (unigene ID: DNMag0422) was found, primarily expressed in the hyphae as revealed by EST transcript analysis (Table 1 1 ). The locus (putative protein MG10274.5) of this EST in the genomic DNA of M . grisea strain 70-15 was identified through a homology search of the M . grisea database ( http://www.broad.mit.edu/annotation/fungi/magnaporthe http://www.broad.mit.edu/annotation/fungi/magnaporthe ) (Dean et al., 2005 2005 ) by BLASTN (Altschul et al., 1997 1997 ). The putative protein MG10274.5, a norsolorinic acid reductase (Nar) in M . grisea , is a homolog of the Nar in Aspergillus flavus , which is involved in the aflatoxin biosynthetic pathway (Hua et al., 1999 1999 ). Composition of the cDNA libraries was as follows (numbers in parentheses are the total number of ESTs sequenced from each library): Mag02, 70-15 appressorium (2553); Mag06, Guy-11 mycelium grown in complete medium (3193); Mag07, Guy-11 conidia (3405); Mag08, Guy-11 mycelium grown in nitrogen starvation medium (4310). Number of ESTs representing this unigene sequenced from each cDNA library. %ESTs representing this unigene shown as percentage of the total number of ESTs sequenced from each library Library 1 1 Mag02 Mag06 Mag07 Mag08 No. of ESTs 2 2 0 178 4 1 %ESTs 3 3 0 5.649 0.118 0.023 Primers for cloning the promoter fragment of NAR gene were designed based on the DNA sequence of NAR gene in M . grisea strain 70-15. A 1157-bp PCR product amplified from the genomic DNA of M . grisea strain Guy-11 was cloned to the T-vector and sequenced. The sequence data were analyzed using VecScreen program (NCBI) for vector masking. The promoter sequence of NAR gene was submitted to GenBank (GenBank_Accn: EF486491). Analyses of the NAR promoter 2 Neural Network Promoter Prediction ( http://www.fruitfly.org/seq_tools/promoter.html http://www.fruitfly.org/seq_tools/promoter.html ) indicated that the basic promoter sequence starts at position 807 and ends at position 857 of the NAR promoter DNA sequences (score=0.95, underlined in Fig. 1 1 ) and that the transcription start lies at location 847 (Fig. 1 1 ). TSSP ( http://www.softberry.com/berry.phtml http://www.softberry.com/berry.phtml ) indicated that the promoter position lies at 849 (Fig. 1 1 ) and “TATA” box at position 813 (Fig. 1 1 ) and found 19 binding sites of the transcription factor of NAR promoter sequences (“+” strand), all located between locations 592 and 802. TFSCAN for fungi ( http://bioweb.pasteur.fr/seqanal/interfaces/tfscan.html http://bioweb.pasteur.fr/seqanal/interfaces/tfscan.html ) predicted that the NAR promoter fragment contains 7 known binding sites for regulatory proteins of Saccharomyces cerevisiae (Table 2 2 ) and that 20% of the sites are distributed between positions 59 and 283, 60% between positions 430 and 840, and 20% between positions 915 and 1145. Binding factors DBF-A MCBF GCN4 GCN4 GAL4 GAL4 GAL4 GAL80 RAF ADR1 Sequences TTATC ACGCGT AAGTCA CAGTCA GGGG GATAA GAGGA GAGGA CCGA TCTCC Positions (Start-end) 551-555 701-706 1087-1092 996-1001 68-73 59-64 837-840 836-839 624-627 464-467 463-466 453-456 728-732 456-460 279-283 279-283 1142-1145 915-918 726-729 576-579 136-139 926-930 689-693 554-558 430-434 On searching the EST database in GenBank by BLASTN (Altschul et al., 1997 1997 ) using putative NAR coding sequence (CDS), many ESTs were found, with sequences starting at position 874 of the NAR promoter DNA fragment (e.g. GenBank_Accn: BM862247). This implied that the transcription start lies at a location before 874 (Fig. 1 1 ). Therefore, the promoter sequence of NAR promoter fragment probably lies between locations 430 and 857 and the transcription starts at location 847 or 849. Analyses of GFP expression 2 The pNAR, in which the GFP gene is under the control of the NAR promoter, was introduced into protoplasts of M . grisea strain Guy-11. Twelve hygromycin-resistant transformants were selected and confirmed by PCR and DNA gel blot analysis. The expression of GFP in cells, including hyphal cells (on CM plate), ungerminated spores, germinating spores, and appressoria, was examined under an epifluorescence microscope. The green fluorescence emitted by GFP protein was bright in the hyphae, weaker in ungerminated and germinating spores (2 h post-incubation, hpi), and the weakest in incipient appressoria (4 hpi) and fully formed appressoria (24 hpi) (Fig. 2 2 ), thereby implying that the NAR promoter is activated mainly at the mycelial stage in the rice blast fungus. These results were similar to those of NAR transcript analysis (Table 1 1 ). No transcripts of NAR gene, which was faintly expressed in appressoria, were found in the appressorial cDNA library, perhaps because the transcript was lost during the building of the library. Furthermore, when inoculated in CM-N medium (CM medium sans the nitrogen source), the green fluorescence emitted by GFP protein in hyphae was brighter than those inoculated in complete medium. This cue implied that the NAR promoter was induced by starvation, and was consistent with the predicted results that this promoter was regulated by Gcn4 protein, which is a transcription factor in response to starvation (Tournu et al., 2005 2005 ). CONCLUSION 1 The NAR promoter containing the “TATA” box, the “CAAT” box, and fungal regulatory protein binding sites was isolated from M . grisea . Analyses of GFP expression under the NAR promoter showed that the promoter was primarily activated in hyphae. 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