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

Abstract

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.

INTRODUCTION

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). 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; Foster et al., 2003; Fudal et al., 2007; Xue et al., 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

Isolates of the fungal pathogen and growth conditions

M. grisea strain Guy-11 was cultured on complete medium (Talbot et al., 1993) at 25 °C in a cycle comprising 14 h of light alternating with 10 h of darkness. Conidia were harvested and resuspended (1×10⁵ 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).

Cloning and sequencing of the NAR promoter

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).

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, version 2.2), TSSP (http://www.softberry.com/berry.phtml, prediction of plant promoters), and TFSCAN (http://bioweb.pasteur.fr/seqanal/interfaces/tfscan.html).

Construction of green fluorescent protein (GFP) expression vector and fungal transformation

The promoter DNA fragment of the NAR gene was cut out from the T-vector containing the NAR promoter using restriction endonuclease BamHI and NcoI and inserted into BamHI-NcoI sites of pEGFP (Clontech, USA) to produce an interim vector, namely pEGFP-NAR. Then a hygromycin-resistant gene (HPH) was cut out using SalI from pCB1003 vector (Carroll et al., 1994) and inserted at the SalI 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).

Analyses of GFP expression

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) was also examined.

RESULTS AND DISCUSSION

Cloning of NAR promoter

After searching the phytopathogenic fungi and oomycete EST database in COGEME (http://cogeme.ex.ac.uk) (Soanes and Talbot, 2005), an expressed sequence tag (EST) (unigene ID: DNMag0422) was found, primarily expressed in the hyphae as revealed by EST transcript analysis (Table 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) (Dean et al., 2005) by BLASTN (Altschul et al., 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).

Table 1.

Analysis of transcript abundance of an EST (unigene ID: DNMag0422) from four cDNA libraries (data from http://cogeme.ex.ac.uk)

Library1	Mag02	Mag06	Mag07	Mag08
No. of ESTs2	0	178	4	1
%ESTs3	0	5.649	0.118	0.023

¹Composition 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).

²Number of ESTs representing this unigene sequenced from each cDNA library.

³%ESTs representing this unigene shown as percentage of the total number of ESTs sequenced from each library

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

Neural Network Promoter Prediction (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) and that the transcription start lies at location 847 (Fig.1). TSSP (http://www.softberry.com/berry.phtml) indicated that the promoter position lies at 849 (Fig.1) and “TATA” box at position 813 (Fig.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) predicted that the NAR promoter fragment contains 7 known binding sites for regulatory proteins of Saccharomyces cerevisiae (Table 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.

Fig. 1.

The sequence of NAR promoter. The NAR gene initiation codon “ATG” is marked by a double underline. The first four basic groups “CTCA”, appearing in the ESTs found in GenBank using BLASTN, are enclosed within a frame. The basic promoter sequence predicted by Neural Network Promoter Prediction is marked by a single underline (807~857). The “TATA” box or “CAAT” box is marked with a wavy underline. The transcription start “T” (847, Neural Network Promoter Prediction) or “T” (849, TSSP) is shaded light grey

Table 2.

Putative regulatory protein binding sites of Saccharomyces cerevisiae in NAR promoter DNA fragment predicted by TFSCAN program

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) 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). 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

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), 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). 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).

Fig. 2.

Differential expression pattern of NAR promoter. GFP under the control of the NAR promoter was expressed strongly in hyphae (a), weakly in ungerminated (b) and germinating conidia (c) (2 hpi), and faintly in incipient appressoria (d) (4 hpi) and fully formed appressoria (e) (24 hpi) of M. grisea. The mycelia, conidia, and appressoria were observed by fluorescence microscopy (top) and by light microscopy (bottom) in each panel

CONCLUSION

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. The successful expression of Mgatg1-GFP fusion protein and Mnh6-GFP fusion protein under NAR promoter in M. grisea (Liu et al., 2007) also showed that NAR promoter isolated in this study could be used to express native or extrinsic genes in the mycelia of the rice blast fungus.