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PubMed2005

Sequence analysis and expression pattern of MGTA1 gene in rice blast pathogen Magnaporthe grisea.

MGTA1, a putative fungal Zn(II)(2)Cys(6) transcriptional activator-encoding gene, was isolated from rice blast pathogen Magnaporthe grisea, which is homologous to CLTA1 from Colletotrichum lindemuthianum with 51% identity at protein level. MGTA1 cassette contains a 2370 bp open reading frame, consisting of 6 exons, coding a 790 amino acid peptide. MGTA1 gene exists as a single copy in genomes of 7 strains of M. grisea, and is expressed in tip hyphae, conidia, and mature appressoria of strain Guy11.

Amino Acid Sequence↗
PubMed2006

Promoter trapping in Magnaporthe grisea.

Application of promoter trapping based on transformation in Magnaporthe grisea is reported in this paper. Two promoter-trapping vectors, designated as pCBGFP and pEGFPHPH, were constructed and transformed into protoplasts of M. grisea. A library of 1,077 transformants resistant to hygromycin B was generated. Of which, 448 transformants were found to express eGFP gene in different structures of M. grisea. Three transformants grew slowly, 5 transformants decreased in conidiation and 7 transformants reduced in pathogenicity greatly among these 448 transformants. Eleven transformants were checked by genomic southern blot randomly, and 9 of which were single-copy insertions. The promoter trapping technique has been applied successfully in M. grisea and can be used as a tool for functional genomic analysis.

Fungal Proteins↗
PubMed2000

Neobulgarones A approximately F from cultures of Neobulgaria pura, new inhibitors of appressorium formation of Magnaporthe grisea.

Six new dimeric anthraquinone derivatives, neobulgarones A (3a), B (3b), C (4a), D (4b), E (5a) and F (5b), were isolated from the mycelia of the ascomycete Neobulgaria pura together with the monomeric carviolin (1) and 1-O-methylemodin (2). All new compounds inhibited the formation of appressoria in germinating conidia of Magnaporthe grisea on inductive (hydrophobic) surface. The compounds exhibited moderate cytotoxic, but no antifungal, antibacterial, or phytotoxic activities.

Anthracenes↗
PubMed1999

Physical map and organization of chromosome 7 in the rice blast fungus, Magnaporthe grisea.

The rice blast fungus Magnaporthe grisea is a highly destructive plant pathogen and one of the most important for studying various aspects of host-plant interactions. It has been widely adopted as a model organism because it is ideally suited for genetic and biological studies. To facilitate map-based cloning, chromosome walking, and genome organization studies of M. grisea, a complete physical map of chromosome 7 was constructed using a large-insert (130 kb) bacterial artificial chromosome (BAC) library. Using 147 chromosome 7-specific single-copy BAC clones and 20 RFLP markers on chromosome 7, 625 BAC clones were identified by hybridization. BAC clones were digested with HindIII, and fragments were size separated on analytical agarose gels to create DNA fingerprints. Hybridization contigs were constructed using a random cost algorithm, whereas fingerprinting contigs were constructed using the software package FPC. Results from both methods were generally in agreement, but numerous anomalies were observed. The combined data produced five robust anchored contigs after gap closure by chromosomal walking. The genetic and physical maps agreed closely. The final physical map was estimated to cover >95% of the 4.2 Mb of chromosome 7. Based on the contig maps, a minimum BAC tile containing 42 BAC clones was created, and organization of repetitive elements and expressed genes of the chromosome was investigated.

Algorithms↗
PubMed2001

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea.

An effective way to study the infection mechanisms of fungal pathogens is to disrupt their genes via transformation in both targeted and random manners. This isolates the mutants that exhibit altered virulence. In this paper, we report the successful transformation of Magnaporthe grisea, the causal agent for rice blast, that is mediated by Agrobacterium tumefaciens. Employing the binary vector pBHt2, which carries the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 500 to > 1,000 hygromycin B-resistant transformants per 1 x 10(6) conidia of M. grisea. The transformation efficiency is correlated with the number of A. tumefaciens cells used, pre-treating bacterial cells with acetosyringone prior to co-cultivation with fungal spores, and the duration of co-cultivation. All of the transformants tested remained mitotically stable, maintaining their hygromycin B resistance after several generations of growth in the absence of hygromycin B. A genomic Southern blot analysis showed that over 60% of the transformants contained a single T-DNA insert on their genome. Considering the efficiency and flexibility of A. tumefaciens-mediated transformation (ATMT), this technique offers highly efficient means for characterizing the genes that are important for the pathogenicity of M. grisea.

Acetophenones↗
PubMed2000

[Studies on monoclonal antibodies of Magnaporthe grisea and its interfering effect on appressorium formation].

Selecting by indirect ELISA eleven hybridoma cell lines, secreting monoclonal antibodies (McAbs) against Magnaporthe grisea were produced by fusing mouse myeloma cells(SP2/0) with spleen cells from BALB/c mice immunized with the mixture of conidia, germ tubes and appressoria of M. grisea using 50% PEG. The result of IFTC test showed that four of McAbs, named as 2B4, 4A1, 1D1, and 2H4 specifically bound to the cell wall surface of the fungus; Western blotting revealed that 2B4, 4A1, 1D1 were recognized different protein antigens from the surface of conidia and germ tubes; These four McAbs could effectively interfere with the appressorium formation both on cellophane membrane and the surface of onion epidermis and inhibit the disease leaf lesions development in vitro test.

Animals↗
PubMed2002

[Genetic analysis and molecular marker of Avr-Pi1, Avr-Pi2 and Avr-Pi4a of Magnaporthe grisea].

The isolate 81278ZB15 is a fertile strain of predominant virulence of Magnaporthe grisea in Fujian province, China. Its rep-PCR (repetitive element-based polymerase chain reaction) DNA fingerprint and virulence spectrum are different from those of GUY11, a hermophrodite. Ascospores of the cross 81278ZB15 and GUY11 were randomly isolated and their virulence was tested on CO39 NILs (near isogenic lines, C101LAC Pi-1, C101A51 Pi-2, C104PKT Pi-3, C101PKT Pi-4a, C105TTP-4L-23 Pi-4b and CO39). The results showed that 81278ZB15 has Avr-Pil, Avr-Pi2, Avr-Pi4a and Avr-Pi4b genes. The segregation of avirulence and virulence on Pi-1, Pi-2, Pi-4a in the progeny fits 1:1, but there were 8 recombinant ascospore progeny in these three avirulence loci. The results suggest that Avr-Pil, Avr-Pi2 and Avr-Pi4a in 81278ZB15 are three tightly linked but independent loci. A 1.2 kb DNA fragment based on rep-PCR fingerprint (RPF1.2) was found to link to these avirulence loci. The genetic distance between RPF1.2 and Avr-Pil, Avr-Pi2, and Avr-Pi4a is estimated to be 5.9 cM, 2.2 cM and 2.2 cM respectively. Furthermore, some progenies are avirulent on Pi-3, CO39 and 8 prognies are virulent on Pi-4b although both parents are virulent on Pi-3, CO39 and avirulent on Pi-4b. The possible causes of irregular recombination were also discussed.

Chromosome Mapping↗
PubMed2002

[Analysis of gene expression profiles during host-Magnaporthe grisea interactions in a pair of near isogenic lines of rice].

A pair of near isogenic lines G205 and G71 were selected from recombinant inbred lines (RIL) of Zhong156 x Gumei2. On the resistance locus Pi-25(t), G205 had the resistant allele that was from Gumei 2 while G71 had the susceptible allele that was from Zhong156. For the genetic background, different alleles were detected on only 24 loci out of the 672 RFLP or SSLP loci surveyed. The expression profiles of G205 and G71 in response to Magnaporthe grisea were investigated using cDNA microarray containing 2200 Expression Sequence Tags (ESTs). The leaves were inoculated with the pathogen for 12 hours at 4-leaf stage and 998 genes were identified in total. Three genes were up-regulated significantly by the fungus in G205 only. The functions of two genes were known but that of the third gene were unknown. The two genes encoded casein kinase II alpha subunit and retrotransponson TOS17 insertion element respectively. Other thirty-five genes had similar expression patterns between NILs. Among them, 17 genes were up-regulated while 18 genes were down-regulated by the inoculation. The functions of 33 out of the 35 genes were known. BLAST analysis showed that all thirty-five. BLAST analysis showed that all thirty-five genes with known functions were relative to defense reactions, signal transduction, stress response, photosynthesis and sugar metabolism. Northern blot confirmed that four of five differentially displayed genes randomly selected had the same expression patterns as those detected in cDNA microarray. Two of them were up-regulated genes encoding casein kinase II alpha subunit and glycine-rich protein (Grp), and the other two down-regulated genes encoding nitrilase-associated protein and 18S small subnit ribosomal RNA gene respectively. Northern blot also revealed that the expression of Grp was consistently up-regulated from 0 to 36 h after the inoculation of the fungus. These results showed that cDNA microarray was a useful tool to study the molecular mechanisms of disease resistance in plants.

Blotting, Northern↗
PubMed2003

[A study on introduction of chitinase gene and beta-1,3-glucanase gene into restorer line of dian-type hybrid rice (Oryza sativa L.) and enhanced resistance to blast (Magnaporthe grisea)].

Plasmid pBLGC containing chitinase gene from Phaseolus limensis and beta-1,3-glucanase gene from Nicotiana tabacum was bombarded into the restorer line "Nan29" of Dian-type hybrid rice (Oryza sativa L. ssp. japonica) from Yunnan province of South-west China. 93 regenerants were obtained from the calli that were resistant to G418 (100 to 150 mg/L) on NB medium. Using beta-1,3 glucanase gene as the probe, 17 of the regenerants were identified to be transgenic lines by dot blotting and the foreign genes construction were integrated into the genomes of T1 lines by Southern blotting hybridization. Two foreign genes were inherited stably to T4 generation according to PCR results of the lines. The resistance to rice blast of six transgenic lines were evaluated by inoculating four violent biological races of Magnaporthe grisea from Yunnan province and inducing the disease in the field. The results indicated that the resistance to rice blast of transgenic lines were enhanced to varying degrees compared with the receptor line and the transgenic lines could be used in rice blast resistant breeding.

Blotting, Southern↗
PubMed2004

Extracellular matrix protein gene, EMP1, is required for appressorium formation and pathogenicity of the rice blast fungus, Magnaporthe grisea.

Magnaporthe grisea, the causal fungus of rice blast, forms a specialized infection structure called an appressorium that is crucial for host plant penetration. A cDNA clone of M. grisea, showing strong sequence homology to FEM1 of Fusarium oxysporum and encoding an extracellular matrix protein, was isolated during an expressed sequence tag (EST) analysis of an appressorium cDNA library and named extracellular matrix protein 1 (EMP1). Sequence analysis of the corresponding genomic clone revealed that EMP1 contains an open reading frame of 685 nucleotides encoding 207 amino acids. The estimated molecular weight of the protein product was 20.5 kDa with a pI of 7.84. It contains an 18 amino acid N-terminal secretion signal sequence, as well as four potential N-glycosylation sites. At its C-terminus, the protein contains a 16 amino acid sequence with the characteristics of a glycosylphosphatidylinositol (GPI) anchor addition signal. Northern blot analysis showed that EMP1 transcripts accumulate during appressorium formation but not during vegetative growth. An EMP1 null mutant, emp1, generated by targeted gene disruption, exhibited reduced levels of appressorium formation and pathogenicity but no effect on mycelial growth rate or conidiation ability. These data suggest that EMP1 plays important roles in appressorium formation and the pathogenicity of M. grisea.

Amino Acid Sequence↗
PubMed2004

[Membrane lipid peroxidation and hypersensitive reaction induced by a glycoprotein elicitor from Magnaporthe grisea in rice leaves].

With a series of four near-isogenic lines (NIL) of rice, studies were carried out in the interaction between rice and a specific glycoprotein elicitor from Magnaporthe grisea. After treatment with the elicitor, O(-.)(2) generation rate increased more remarkably on the leaves of incompatible interaction of rice, than compatible interaction of rice. Change in SOD activity and CAT activity was similar in leaves of rice with different resistance after inoculation with the elicitor. On the contrary, POD activity was positive relative to rice resistance. And changes in LOX activity were increased more remarkably and rapidly on the leaves of incompatibly interacting rice. These changes led to lipid peroxidation in the leaves of incompatibly interacting rice. Relative conductance rate and MDA content were also increased more remarkably and rapidly in leaves of incompatibly interacting rice. Our results also showed that elicitor could induce hypersensitive reaction (HR reaction) in leaves of completely or highly incompatible interacting rice, but not in the two NIL rice varieties (one with moderately incompatible interactions and another with compatible ones). The results suggest that lipid peroxidation and HR reaction in rice leaves induced by the elicitor may play an important role in the resistance of rice seedlings.

Catalase↗
PubMed2004

[Cellular responses of rice to Magnaporthe grisea at the early stages of disease development].

This paper presents a detailed investigation of the cytological and cytochemical events in rice cells infected by Magnaporthe grisea during the early stages of disease development. It was demonstrated that the spatial and temporal development of cytoplasmic aggregation, host cell autofluorescence, callose deposition and phospholipase D (PLD) accumulation were some of the earliest responses of host cells to M. grisea attacking and showed different patterns between the compatible and incompatible interaction. In the cv. IR64 and strain BN111 interaction (resistant), the earliest cellular response observed in the inner epidermis of rice leaf sheath was aggregation of the cytoplasm. Then, the attacked host cells turned brown and the cell cytoplasm collapsed (HR). In the moderately resistant reactions of cv. IR64 to strain PO66, the granule formation was delayed, and no apposition was observed in host cells. In the compatible reactions of cv. IR64 to strain Ca89, no visible cellular response was detected until 40 h after inoculation. As to auto-fluorescence of host cells, some penetration sites showed faint fluorescence under blue light as early as 12 h after inoculation in the cv. IR64-strain BN111 interaction. As the disease developed, the percentage of attacked epidermal cells showing autofluorescence increased quickly from 20 to 24 h after inoculation and the penetrated host cells showed strong autofluorescence. In the moderately resistant interaction, autofluorescence had been detected until 24 h after inoculation. In the compatible interaction, little autofluorescent cell was detected during the disease development. Patterns of callose deposition and phospholipase Dgamma (PLDgamma) showed the similar dynamic characteristics as did the phenolic compounds.

Cytoplasm↗
PubMed2004

Fertility status and distribution of mating type alleles of the rice blast fungus, Magnaporthe grisea in northern Iran.

This study was carried out using 155 monoconidial isolates collected from different areas of two major rice growing provinces in northern Iran, including 94 isolates from Guilan and 59 isolates from Mazandaran. Among 94 isolates from Guilan, 92 and two isolates recovered from rice and crabgrass (Digitaria sp.), respectively. All 61 rested isolates from Mazandaran were recovered from rice. All isolates were evaluated for in vitro sexual fertility and mating type status by pairing with Mat 1-1 and Mat 1-2 fertile standard hermaphrodite isolates including Br48 and Th12 (Mat 1-1) and KA9 and TH16 (Mat 1-2). Of 155 isolates, 98 (63.2%) were fertile and 57 (36.8%) were infertile and produced no perithecium when mated with standard isolates. Among 98 fertile isolates, 96 isolates were identified as Mat 1-1 and two isolates as Mat 1-2. All Mat 1-1 isolates were obtained from rice and two Mat 1-2 isolates obtained from crab grass. No Mat 1-2 isolate was identified from rice in this study. Both mating types were found in Guilan but all isolates recovered from Mazandaran were identified as Mat 1-1. Male fertility predominated in fertile Mat 1-1 and Mat 1-2 isolates from all sampling sites in northern Iran, and no female fertility was detected. This is the first report of existence of Mat 1-2 allele in Magnaporthe grisea population in Iran.

Fertility↗
PubMed2003

[Optimization of T-dNA insertional mutagenesis and analysis of mutants of Magnaporthe grisea].

The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice around the world. Significant progresses have been made recently in genomics studies of the fungus, opening new era of the functional genomics which requires to generate a large scale of gene knockout mutants. It has been demonstrated that T-DNA insertional mutagenesis is a powerful tool of functional genomics not only for plants but also for fungi. In this paper, we optimized the conditions for T-DNA insertional mutagenesis of M. grisea using Agrobacterium tumefaciens-mediated transformation (ATMT) approach. We employed the binary vector pBHtl constructed by Dr. S. Kang's laboratory at the Pennsylvania State University, which carries the bacterial hygromycin B phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker to transform the conidia of M. grisea. We optimized the conditions for T-DNA insertional mutagenesis including the medium, dosage of hygromycin B, cefotaxime and carbenicillin to select the transformants and inhibit the growth of A. tumefaciens after co-culturing. The dosage to inhibit non-transformants could vary from 200-600microg/mL among different M. grisea isolates so that the optimal dosage of the antibiotics should be decided according to isolates. Rice polished agar medium was found the best selection medium which would facilitate the mutant sporulation and minimize the contamination chance. In average, about 500 transformants could be obtained when transforming 1 x 10(6) spores at the optimum condition, among which 85% had T-DNA insertion detected by polymerase chain reaction (PCR) and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). Fifteen out of 1520 transformants showed mutation in colony morphology. Within 58 randomly selected mutants, it was found that there were 4 sporulation-decreased mutants, 8 less germination mutants and 9 appressorium defective mutants. Several virulent mutants to C101LAC(Pi-1)and 75-1-127(Pi-9)were also obtained which would facilitate cloning the corresponding avirulence genes.

Agrobacterium tumefaciens↗
PubMed2005

Study on genetic diversity of Magnaporthe grisea using PCR and determination of the mating type alleles distribution in Mazandaran province, Iran.

The population structure of Magnaporthe grisea, the causal agent of the rice blast, was analyzed in Mazandaran province, using DNA fingerprinting based on RAPD-PCR by means of three primers including "I", "D" and "H". Total DNA of 47 isolates was extracted and amplified according to a specific PCR program. As a result, variable length fragments were generated. Each isolate was subjected to DNA fingerprinting and clonal lineages were determined. Phenetic analysis differentiated three distinct fingerprint lineages. In order to study on fertility status and distribution of the mating type idiomorphs (alleles), 72 monoconidial isolates from Mazandaran province were paired with four standard fertile hermaphrodite isolates. The mating type of 36 isolates was determined as Mat 1-1. The others (36 isolates) did not form any perithecia in pairing with standard isolates

DNA Fingerprinting↗
PubMed1997

A large-insert (130 kbp) bacterial artificial chromosome library of the rice blast fungus Magnaporthe grisea: genome analysis, contig assembly, and gene cloning.

Magnaporthe grisea (Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant-pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitate M. grisea genome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (> 25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that approximately 26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis of M. grisea. Copies of colony hybridization membranes are available upon request.

Ascomycota↗
PubMed1997

Mutants of the phytopathogenic fungus magnaporthe grisea deficient in alternative, cyanide-resistant, respiration

The phytopathogenic fungus Magnaporthe grisea has a cyanide-resistant respiratory pathway. The fungicide SSF-126 ((E)-2-methoxyimino-N-methyl-2-(2-phenoxyphenyl) acetamide) blocks the cytochrome electron transport of M. grisea and induces the alternative respiratory pathway. Twelve mutants of M. grisea more susceptible to SSF-126 than wild type were identified after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Five mutants retained a reduced alternative respiration activity, and seven mutants lacked alternative pathway activity. A monoclonal antibody against the maize alternative oxidase cross-reacted against a 40-kDa mitochondrial protein of M. grisea, indicating that the 40-kDa protein is an alternative oxidase. Immunoblot analysis indicated that the seven completely deficient mutants grouped into two classes: four mutants produced the 40-kDa proteins while the other three mutants failed to produce the functional protein. Copyright 1997 Academic Press. Copyright 1997 Academic Press

Journal Article↗
PubMed2001

Organization and distribution pattern of MGLR-3, a novel retrotransposon in the rice blast fungus Magnaporthe grisea.

A specific telomere was deleted in spontaneous, gain-of-virulence mutants derived from a rice pathogen of Magnaporthe grisea. Three different types of transposons, including Pot2, Mg-SINE, and a novel, 6-kb-long LTR (long terminal repeat)-type retrotransposon designated MGLR-3, were identified on this chromosomal end. The 114-bp-long telomeric repeat is immediately followed by the 3' LTR of MGLR-3. A truncated copy of Pot2 immediately flanks the 5' LTR, suggesting that this telomere was generated by a transposition event of MGLR-3 into this Pot2 element, causing the breakage of a chromosome. The subsequent addition of a telomeric repeat to the 3' LTR of MGLR-3 most probably repaired the broken end of the chromosome. Mg-SINE is located 25 bp away from the truncated Pot2 element. MGLR-3 exhibited strong homology to various gypsy-class retrotransposons, including grh and MAGGY in M. grisea. MGLR-3 is ubiquitous regardless of the host of origin.

Amino Acid Sequence↗