Isolation of Gene Mutation from a Pathogenicity-Enhanced Mutant of Magnaporthe oryzae

Rice Science, 2010, 17(2): 129í134 Copyright © 2010, China National Rice Research Institute. Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(08)60115-8

Isolation of Gene Mutation from a Pathogenicity-Enhanced Mutant of Magnaporthe oryzae WU Xiao-yan1, 2, WANG Jiao-yu1, ZHANG Zhen1, JING Jin-xue2, DU Xin-fa1, CHAI Rong-yao1, MAO Xue-qin1, QIU Hai-ping1, JIANG Hua1, WANG Yan-li1, SUN Guo-chang1 (1Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; 2College of Plant Protection, Northwest Agriculture and Forest University, Yangling 712100, China)

Abstract: To find new genes involved in fungal pathogenicity, a mutant (B11) exhibiting enhanced pathogenicity was isolated from an Agrobacterium-mediated transformed Magnaporthe oryzae mutant library. Southern blotting analysis showed that T-DNA insertion in the B11 genome was a single copy. TAIL-PCR and sequence alignment analyses revealed that a putative gene locus MG01679 was interrupted by the T-DNA fragment. By using the PCR-based method, the DNA and cDNA of the mutant gene MG01679 was cloned and sequenced. The open reading frame of MG01679 includes one intron and two exons, and the coding sequence is 696 bp in length and encodes a 231 amino acid peptide. Protein similarity analysis indicated that the gene belongs to the ThiJ/Pfp I protein family, and the gene was thus designated MgThiJ1. MgThiJ1 showed 57% similarity to FOXG_09029 from Fusarium oxysporum and 54% similarity to FGSG_08979 from F. graminearum in protein sequence. MgThiJ1 gene might act as a negative regulator in vegetative growth and pathogenesis in filamentous fungi, and its specific mechanism needs to be studied further. Key words: Magnaporthe oryzae; mutant; T-DNA; pathogencity; MgThiJ1 gene; gene function

The ascomycete fungus Magnaporthe oryzae (anamorph Pyricularia grisea) is a model organism for research on pathogenesis of plant fungal pathogen, which causes rice blast, the world-wide serious rice disease. The infection process of M. oryzae is very complicated, which includes conidiation, germination, appressorium formation, penetration, infectious hyphae differentiation and extension. Damages to any step of the infection cycle of M. oryzae will lead to the weakening or completely loss of the pathogenecity [1-3]. So far, the research on functional genomics of plant pathogenic fungi has made considerable progress. Many functional genes have been cloned and analyzed. Strategies used in gene function analysis can be grouped into two types: from phenotype to gene which is also regarded as forward genetics and from gene to phenotype which is often recognized as reverse genetics. As for M. oryzae, the reverse genetics strategy is widely used whereas the forward genetics strategy is seldom reported comparatively. According to the Received: 8 December 2009; Accepted: 3 March 2010 Corresponding author: WANG Jiao-yu ([email protected]); SUN Guo-chang ([email protected]) This is an English version of the paper published in Chinese in Chinese Journal of Rice Science, Vol. 23, No. 6, 2009, Pages 611–615.

reverse genetics, the phenotypic variations of mutants are firstly generated and selected by various means, the mutation-related genes are then identified, and the gene functions are studied successively. The insertion mutagenesis method is widely used in functional genomics of M. oryzae, which mainly involves two means: restriction enzyme mediated integration (REMI) and Agrobacterium tumefaciens mediated transformation (AtMT). The REMI technology has been used in the cloning of more than 10 pathogenecity-related genes in M. oryzae [2, 4-5]. Comparing with REMI, AtMT has the following main advantages: firstly, the operation is simple, A. tumefaciens can directly transform fungal spores, hyphae or even tissues without protoplast preparation; secondly, the integration of T-DNA into the chromosome is random and generally single-copy, which is easier to isolate and identify the insertion locus; thirdly, AtMT is competent for the transformation of high molecular weight exogenous DNA [6]. Therefore, the AtMT is a suitable and efficient means for insertion mutagenesis, genetic mapping and related research in filamentous fungi. In previous studies, we generated a mutant library of rice blast fungus by the AtMT method. From the library, a mutant (B11) which showed enhanced pathogenecity

Rice Science, Vol. 17, No. 2, 2010

130

was selected. The characterization of the mutant, the identification of the T-DNA insertion locus and the cloning of related gene are reported in this paper.

MATERIALS AND METHODS Strains and culture conditions The wild-type strain Guy11 of M. oryzae is commonly used in molecular biology research. The culture, sporulation and appressorium induction of Guy11 and its mutants were referred to Talbot et al [7]. Escherichia coli strain DH5Į was cultured in an LB medium in darkness at 37°C. An A. tumefaciens strain AGL1 was cultured in a YEB medium in darkness at 28°C. Reagents and equipments Taq DNA polymerase, restriction enzymes and T4 DNA ligase were bought from the Sangon Co. Ltd (Shanghai, China) or TaKaRa Co. Ltd (Japan). pGEM-T easy vector (Promega, USA) was used for T/A cloning. The plasmid preparation and DNA gel extraction kits were from Axygen Co. Ltd (USA). Probe labeling and hybridization were performed using the DNA labeling and detection starter kit I (Roche). The instruments used included a DNA engine (model PTC-200, BioRad, USA), a microscope (model DFC 420C, Leica, Germany), and a programmable plant growth chamber (model KBWF 720, Binder, Germany). Identification of T-DNA insertion copies Southern hybridization with the hygromycin resistance gene HPH was carried out to analyze the copy number of T-DNA insertion. The procedures of hybridization were referred to Sambrook et al [8]. TAIL-PCR and primers Thermal asymmetric interlaced PCR (TAIL-PCR) technique [9] was used to identify the flank regions of T-DNA integration. The primers used in the TAILPCR were RB1 (5ƍ-TAATGCATGACGTTATTTATG AGATG-3ƍ), RB2 (5ƍ-GATGGGTTTTTATGATTAGA GTCCCG-3ƍ), RB3 (5ƍ-ATCGCGCGCGGTGTCATC TATGTTAC-3ƍ), and a degenerate primer AD1 (5ƍ-W GTGNAGWANCANAGA-3ƍ). The PCR products were cloned into the pGEM-T easy vector (Promega, USA)

and sequenced. Primer synthesis and DNA sequencing were carried out by the Sangon Co. Ltd (Shanghai, China). Sequence alignment The BlastT program was used to compare the flank sequence with M. oryzae genome to find related genes. The protein sequence of the targeted gene was used to search the homologous genes from the GenBank by the BlastP program. The sequence of the top 7 hits (E < e–20) was selected for sequence alignment and phylogenetic analysis using the softwares DNAstar and GeneDoc. Phenotypic analysis The phenotypes (colony morphology, conidial production, germination and appressorium formation and so on) of the mutant strain B11 and the wild type strain Guy11 of M. oryzae were analyzed [10]. For the pathogenicity test, conidia from 10-day culture plates were collected. Droplets of 20 μL conidium suspension (1×104 CFU/mL or 5×104 CFU/mL) were inoculated on barley leaves, and the droplets of the same volume of ddH2O were used as control. After cultured at 28°C and 85% humidity for 5 days, the symptoms of M. oryzae on the inoculated barley leaves were observed [12]. The ability of sexual reproduction was checked on oatmeal agars (OMA). B11 (or Guy11) and another mating type strain 2539 were cultured on the same OMA plate. After cultured at 25°C for 5 days when two colonies were adjacent to each other, the plates were transferred to 18–20°C conditions with continuous illumination. After 3–4 weeks, perithecia at the junctions between colonies, ascus and ascospores formation of B11 and Guy11 were observed under the microscope. The experiment was repeated three times.

RESULTS Phenotypes of the mutant B11 The mutant B11 grew faster than the wild-type strain Guy11. At the CM cultural plates for 5 days and 10 days, the colony size of B11 was significantly larger than that of Guy11 (P < 0.05) (Table 1 and Fig. 1). No significant differences were found between B11 and Guy11 in spore germination rate and appressorium

WU Xiao-yan, et al. Isolation of Gene Mutation from a Pathogenicity-Enhanced Mutant of Magnaporthe oryzae

131

Fig. 1. Colony (A, 10 d), germination (B, 4 h) and appressorium (C, 12 h) of the strains B11 and Guy11.

formation rate on the induced surface. In addition, B11 could produce normal sexual generation when mating with the strain 2539. The perithecium, ascus and ascospore of B11 were similar with Guy11 (data not shown). However, the pathogenicity of B11 was improved compared with that of Guy11 (Fig. 2). When the conidium suspensions of either B11 or Guy11 were inoculated on barley leaves, the typical spindle-type lesions were generated, distributing evenly on the leaf surface. But under the same conditions, the mutant B11 drove more serious symptom than Guy11, wherever the concentration of conidium suspension for inoculation was 1×104 CFU/mL or 5×104 CFU/mL (Fig. 2). These results indicate that the mutant strain B11 keeps the ability of penetrating the host cuticle and epidermal cell wall, spreading in the cells and destroying the leaf tissues, and the ability is stronger than that of the wild type under the same conditions as well. B11 possesses a single-copy T-DNA integration Southern blot analysis was carried out to investigate

Fig. 2. Pathogenicities of B11 and Guy11 on barley leaves. A, 1×104 CFU/mL; B, 5×104 CFU/mL.

the copy number of T-DNA insertion in the mutant B11. The genomic DNA of B11 was digested with five different restriction enzymes (Sal I, Xba I, BamH I, Hind III and Kpn I), respectively. Then they were dispersed by electrophoresis, transferred to a nylon membrane, and hybridized with an inner hygromycin resistance gene HPT as probe. The probe fragment contained no site of the five used restriction enzymes.

Table 1. Colony size, germination rate and appressorium formation rate of B11 and Guy11. Colony semidiameter (cm) Spore germination rate (4 h) (%) 2d 5d 10 d Guy11 0.63±0.06 a 1.62±0.03 a 3.45±0.05 a 97.92±3.20 a B11 0.68±0.03 a 1.77±0.06 b 3.82±0.03 b 92.20±3.42 a Within a column, data followed by common letters indicate no significant difference at the 0.05 level.

Strain

Appressorium formation rate (12 h) (%) 94.12±2.32 a 93.17±3.24 a

132

Rice Science, Vol. 17, No. 2, 2010

Only one hybridization band in each B11 lane was observed, and no band was found in the control lane, which was loaded with the Sal I-digested genomic DNA of Guy11 (Fig. 3). These results indicate that the mutant B11 possessed a single copy T-DNA insertion. T-DNA flanking sequence analysis The TAIL-PCR method was used to analyze the insertion site of B11. A 500 bp fragment flanking to the 3ƍ terminal of T-DNA was obtained and sequenced. The T-DNA was inserted at the 178 bp 5ƍ upstream of a coding region of the hypothetical gene MG06179. The MG06179 gene was assigned as MgThiJ1 in this study, which encodes a hypothetic protein of ThiJ/Pfp I family. MgThiJ1 sequence analysis A 696 bp fragment of the MgThiJ1 coding region was amplified from a 24 h mature appressorium cDNA library using primers according to the genome data published by Broad Institute of MIT and Harvard, USA (http://www.broadinstitute.org/annotation/genome/ magnaporthe_grisea/MultiHome.html). The MgThiJ1 coding region determined by cDNA corresponded to the MG06179 coding sequence predicted in silico. Comparison between coding sequence and genome sequence revealed

Fig. 3. Analysis of the copy number of T-DNA integration in the M. oryzae strain B11 by Southern blotting. The CK lane was loaded with the Sal I-digested genomic DNA of Guy11.

that the open reading frame of MgThiJ1 included one intron and two exons and encoded a 231 amino acid peptide. Numerous homologous genes of MgThiJ1 were found in different fungi in GenBank by the BlastP program using the protein sequence of MgThiJ1. These fungi involved many important plant pathogens and economic species, such as Fusarium oxysporum (FOXG_09029), F. graminearum (FGSG_08979) and the fungus Neurospora crassa (NCU06603). The first six homologous proteins with the highest scores (E < e–20) in BlastP were aligned to MgThiJ1 (Fig. 4) and the phylogenetic analysis was carried out (Fig. 5). The results suggest that the mechanisms similar to MgThiJ1 probably exist in different fungi.

Fig. 4. Protein sequence alignment of MgThiJ1p and its homologous proteins. AN6796 (from Aspergillus nidulans FGSC A4), ATEG_09753 (from A. terreus NIH2624), FGSG_08979 (Alias: fgd367-300, fg08979; from Fusarium graminearum PH-1), FOXG_09029 (from F. oxysporum f. sp. lycopersici 4286), FVEG_06630 (from F. verticillioi 7600), and NCU06630 (from Neurospora crassa OR74A).

WU Xiao-yan, et al. Isolation of Gene Mutation from a Pathogenicity-Enhanced Mutant of Magnaporthe oryzae

133

Amino acid substitution (h100) Fig. 5. Phylogenetic analysis of MgThiJ1 and other homologous protein sequences AN6796, ATEG_09753, FGSG_08979, FOXG_09029, FVEG_06630 and NCU06630.

DISCUSSION As a model organism of plant pathogenic fungi, more than 60 functional genes have been cloned and characterized in M. oryzae at different stages of life cycle, including MPG1, MHP1, PTH11, PMK1, MPS1, MAGB, MAC1, PLS1, PDE1, ICL1, NPR1 and OSM1 [11-17]. And the reverse genetic strategy has been widely used in these studies. Reverse genetic strategy, which consists of the generation of phenotypic variations, the isolation of caused genes and the functional analysis of relatedgenes, is more sufficient to find new genes and to study their functions. In this study, B11, a pathogenicityenhanced mutant of rice blast fungus, was obtained through the AtMT method. B11 showed stronger ability to expand in leaf tissues and more harmful to host barley leaves compared to the wild-type strain Guy11 when placed under the same conditions. Consistent with the improved pathogenicity, B11 also showed higher vegetative growth rate on CM media. But no other pathogenicity-related phenotypes had significantly changes. Taking these data together, we speculated that the caused gene MgThiJ1 participated in the hypha growth and acted as a suppressor. The loss function of MgThiJ1 in B11 improved the growth of both vegetative hypha in culture media and infection hypha in host tissues. The improved growth of infection hypha then accelerated the process of lesion expansion which induced the enhanced pathogencity. We will carry out the gene replacement experiment to further confirm the function of MgThiJ1 and reveal its role in hypha growth. Up to now, we have constructed a replacement vector (p1300-MgThiJ1) and fungal transformation by AtMT using this vector is ongoing. MgThiJ1 encodes a ThiJ/PfpI family protein. The

ThiJ/PfpI family belongs to DJ-1/Pfp I super family. DJ-1/Pfp I super family includes catalase A, catalase II ES-1, DJ-1, ThiJ/Pfp I and other sub-families. To date, the most intensive studied gene in DJ-1/Pfp I super family is DJ-1, which is related to human autosomal recessive Parkinson’s disease [18]. DJ-1 and its roles in Parkinson’s disease give a good model for studying the nerve cell disease at molecular and cell levels. Other than DJ-1, the DJ-1 sub-family also includes some different-structured bacterial enzymes involved in human metabolism [19]. The exact functions of these enzymes are still unknown. The related-research on ThiJ/Pfp I sub-family is much less. Totally, five ThiJ/Pfp I family genes, i.e. MG01679 (MgThiJ1), MG10466, MG01085, MG13216 and MG01896, have been found in the M. oryzae genome. So far, there is no report on the involvement of ThiJ/Pfp I gene in fungal pathogenicity yet. It will be very interesting and significant to further study the function of ThiJ/Pfp I genes in fungi, including gene interactions, and the roles in fungal morphology, development and pathogenicity by related mutants obtained through gene-knockout strategy.

ACKNOWLEDGEMENTS This work was supported by the Natural Science Foundation of Zhejiang Province, China (Grant No. Y306638); the Project of Zhejiang Science and Technology, China (Grant No. 2007C12905); the National Natural Science Foundation of China (Grant Nos. 30900933 and 30970082).

REFERENCES 1

Urban M, Bhargava T, Hamer J E. An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease.

Rice Science, Vol. 17, No. 2, 2010

134 EMBO J, 1999, 18(3): 512–521. 2

12 Talbot N J, Kershaw M J, Wakley G E, de Vries O M H,

Sweigard J A, Carroll A M, Farrall L, Chumley F G, Valent B.

Wessels J G H, Hamer J E. MPGI encodes a fungal

Magnaporthe grisea pathogenicity genes obtained through

hydrophobin involved in surface interactions during infection-

insertional mutagenesis. Mol Plant Microbe Interact, 1998,

related development of Magnaporthe grisea. Plant Cell, 1996,

11(5): 404–412. 3

4

Hamer J E, Valent B, Chumley F G. Mutations at the SMO

grisea hydrophobin gene, is required for fungal development

blast fungus. Genetics, 1989, 122(2): 351–361.

and plant colonization. Mol Microbiol, 2005, 57(5): 1224–

7

8

DeZwaan T M, Carroll A M, Valent B, Sweigard J A. Magnaporthe grisea Pth11p is a novel plasma membrane

Balhadere P V, Foster A J, Talbot N J. Identification of

protein that mediates appressorium differentiation in response

pathogenicity mutants of the rice blast fungus Magnaporthe

to inductive substrate cues. Plant Cell, 1999, 11(10): 2013–

grisea by insertional mutagenesis. Mol Plant Microbe Interact,

2030. 15 Clergeot P H, Gourgues M, Cots J, Laurans F, Latorse M P,

Mullins E D, Kang S. Transformation: A tool for studying

Pepin R, Tharreau D, Notteghem J L, Lebrun M H. PLS1, a

fungal pathogens of plants. Cell & Mol Life Sci, 2001, 58(14):

gene encoding a tetraspanin-like protein, is required for

2043–2052.

penetration of rice leaf by the fungal pathogen Magnaporthe

Talbot N J, Ebbole D J, Hamer J E. Identification and

grisea. Proc Natl Acad Sci USA, 2001, 98(12): 6963–6968.

characterization of MPG1, a gene involved in pathogenicity

16 Veneault-Fourrey C, Lambou K, Lebrun M H. Fungal Pls1

from the rice blast fungus Magnaporthe grisea. Plant Cell,

tetraspanins as key factors of penetration into host plants: A

1993, 5(11): 1575–1590.

role in re-establishing polarized growth in the appressorium?

Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A

FEMS Microbiol Lett, 2006, 256(2): 179–184. 17

Balhadere P V, Talbot N J. PDE1 encodes a P-type ATPase

Laboratory Press, 1989: 43–45.

involved in appressorium-mediated plant infection by the rice

Liu Y G, Whittier R F. Thermal asymmetric interlaced PCR:

blast fungus Magnaporthe grisea. Plant Cell, 2001, 9(13):

Automatable amplification and sequencing of insert end

1987–2004.

fragments from P1 and YAC clones for chromosome walking. 10

14

Mol Plant Microbe Interact, 1997, 10(2): 187–194.

Laboratory Manual. 2nd ed. New York: Cold Spring Harbor 9

1237.

Xu J R, Urban M, Sweigard J A, Hamer J E. The CPKA gene

1999, 12(2): 129–142. 6

Kim S, Ahn I P, Rho H S, Lee Y H. MHP1, a Magnaporthe

genetic locus affect the shape of diverse cell types in the rice

of Magnaporthe grisea is essential for appressorial penetration. 5

6(8): 985–999. 13

18

Canet-Aviles R M, Wilson M A, Miller D W, Ahmad R,

Genomics, 1995, 25: 674–681.

McLendon C, Bandyopadhyay S, Baptista M J, Ringe D,

Bootman M D, Lipp P, Berridge M J. The organization and

Petsko G A, Vookson M R. The Parkinson’s disease protein

functions of local Ca2+ signals. J Cell Sci, 2001, 114(12):

DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven

2213–2222.

mitochondrial localization. Proc Natl Accd Sci USA, 2004, 15(101): 9103–9108.

11 Wang H K, Lin F C, Li D B. Research advances of genes involved in phytopathogenicity in Magnaporthe grisea.

19

Bandyopadhyay S, Cookson M R. Evolutionary and functional

Mycosystema, 2002, 21(3): 459–464. (in Chinese with an

relationships within the DJ-1 superfamily. BMC Evol Biol,

English abstract)

2004, 4: 6.