Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.     

Karyotypic Variation within Clonal Lineages of the Rice Blast Fungus, Magnaporthe grisea

We have analyzed the karyotype of the rice blast fungus, Magnaporthe grisea, by using pulsed-filed gel electrophoresis. We tested whether the electrophoretic karyotype of an isolate was related to its pathotype, as determined by infection assays, or its genetic lineage, as determined by DNA fingerprinting. Highly reproducible electrophoretic karyotypes were obtained for a collection of U.S. and Chinese isolates representing a diverse collection of pathotypes and genetic lineages. Chromosomes ranged in size from 3 to 10 Mb. Although chromosome number was largely invariant, chromosome length polymorphisms were frequent. Minichromosomes were also found, although their presence was not ubiquitous. They ranged in number from 1 to 3 and in size from 470 kb to 2.2 Mb. Karyotypes were sufficiently variable as to obscure the obvious relatedness of isolates on the basis of pathogenicity assays or genetic lineage analysis by DNA fingerprinting. We documented that the electrophoretic karyotype of an isolate can change after prolonged serial transfer in culture and that this change did not alter the isolate's pathotype. The mechanisms bringing about karyotype variability involve deletions, translocations, and more complex rearrangements. We conclude that karyotypic variability in the rice blast fungus is a reflection of the lack of sexuality in wild populations which leads to the maintenance of neutral genomic rearrangements in clones of the fungus.     

Crystal Structure of Manganese Lipoxygenase of the Rice Blast Fungus Magnaporthe oryzae

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese. Little is known about metal selection by LOX and the adjustment of the redox potentials of their protein-bound catalytic metals. Thirteen three-dimensional structures of animal, plant, and prokaryotic FeLOX are available, but none of MnLOX. The MnLOX of the most important plant pathogen, the rice blast fungus Magnaporthe oryzae (Mo), was expressed in Pichia pastoris. Mo-MnLOX was deglycosylated, purified to homogeneity, and subjected to crystal screening and x-ray diffraction. The structure was solved by sulfur and manganese single wavelength anomalous dispersion to a resolution of 2.0 Å. The manganese coordinating sphere is similar to iron ligands of coral 8R-LOX and soybean LOX-1 but is not overlapping. The Asn-473 is positioned on a short loop (Asn-Gln-Gly-Glu-Pro) instead of an α-helix and forms hydrogen bonds with Gln-281. Comparison with FeLOX suggests that Phe-332 and Phe-525 might contribute to the unique suprafacial hydrogen abstraction and oxygenation mechanism of Mo-MnLOX by controlling oxygen access to the pentadiene radical. Modeling suggests that Arg-525 is positioned close to Arg-182 of 8R-LOX, and both residues likely tether the carboxylate group of the substrate. An oxygen channel could not be identified. We conclude that Mo-MnLOX illustrates a partly unique variation of the structural theme of FeLOX.     

Host Species-specific Repetitive DNA Sequence in the Genome of Magnaporthe grisea,the Rice Blast Fungus

We cloned a repetitive sequence to show RFLPs in the genome of Magnaporthe grisea, a fungal pathogen responsible for rice blast. As the sequence was 0.8 kb in length and dispersed in the genome, it was named MGSR1 (for Magnaporthe grisea short repeat 1). MGSR1 was conserved highly in the genome of rice pathogens, but poorly in the genome of pathogens of grasses other than rice. And the RFLPs, displayed with the sequence, could distinguish between clonal lineages in rice-pathogenic isolates. The nucleotide sequence showed the presence of an internal promoter of RNA polymerase III, a 3?-poly(T), and an 8-bp direct repeat in it.     

Polyubiquitin Is Required for Growth, Development and Pathogenicity in the Rice Blast Fungus Magnaporthe oryzae

Protein ubiquitination, which is highly selective, regulates many important biological processes including cellular differentiation and pathogenesis in eukaryotic cells. Here, we integrated pharmacological, molecular and proteomic approaches to explore the role of ubiquitination in Magnaporthe oryzae, the leading fungal disease of rice world-wide. Inhibition of ubiquitin-mediated proteolysis using the 26S proteasome inhibitor, Bortezomib, significantly attenuated conidia germination, appressorium formation and pathogenicity in M. oryzae. Gene expression analysis revealed that many genes associated with protein ubiquitination were developmentally regulated during conidia germination. Only a few, including a polyubiquitin encoding gene, MGG_01282, were more abundantly expressed during appressorium formation and under nitrogen starvation. Targeted gene deletion of MGG_01282, in addition to a significant reduction in protein ubiquitination as determined by immuno blot assays, resulted in pleiotropic effects on M. oryzae including reduced growth and sporulation, abnormal conidia morphology, reduced germination and appressorium formation, and the inability to cause disease. Mutants were also defective in sexual development and were female sterile. Using mass spectrometry, we identified 63 candidate polyubiquitinated proteins under nitrogen starvation, which included overrepresentation of proteins involved in translation, transport and protein modification. Our study suggests that ubiquitination of target proteins plays an important role in nutrient assimilation, development and pathogenicity of M. oryzae.     

A Lipoxygenase Pathway Is Activated in Rice after Infection with the Rice Blast Fungus Magnaporthe grisea

Lipoxygenase (LOX) and lipid hydroperoxide-decomposing activity (LHDA) markedly increased in the fifth leaves of rice (Oryza sativa cv Aichiasahi) after infection with the rice blast fungus, Magnaporthe grisea. The increases in the enzyme activities were significantly higher in response to infection with an incompatible strain (race 131) compared with infection with a compatible strain (race 007) of the fungus. Using ion-exchange chromatography, we isolated three LOX activities (leaf LOX-1, -2, -3) from both uninoculated and infected leaves. The activity of leaf LOX-3, in particular, increased in the incompatible race-infected leaves. The leaf LOX-3 had a pH optimum of 5.0 and produced preferentially 13-l-hydroperoxy-9,11 (Z,E)-octadecadienoic acid (13-HPODD) from linoleic acid. 13-HPODD and 13-l-hydroxy-9,11 (Z,E)-octadecadienoic acid, one of the reaction products from 13-HPODD by LHDA, were highly inhibitory to the germination of conidia of the fungus. The present study provides correlative evidence for important roles of LOX and LHDA in the resistance response of rice against the blast fungus.     

Regulatory Genes Controlling MPG1 Expression and Pathogenicity in the Rice Blast Fungus Magnaporthe grisea

MPG1, a pathogenicity gene of the rice blast fungus Magnaporthe grisea, is expressed during pathogenesis and in axenic culture during nitrogen or glucose limitation. We initiated a search for regulatory mutations that would impair nitrogen metabolism, MPG1 gene expression, and pathogenicity. First, we developed a pair of laboratory strains that were highly fertile and pathogenic toward barley. Using a combinatorial genetic screen, we identified mutants that failed to utilize a wide range of nitrogen sources (e.g., nitrate or amino acids) and then tested the effect of these mutations on pathogenicity. We identified five mutants and designated them Nr- (for nitrogen regulation defective). We show that two of these mutations define two genes, designated NPR1 and NPR2 (for nitrogen pathogenicity regulation), that are essential for pathogenicity and the utilization of many nitrogen sources. These genes are nonallelic to the major nitrogen regulatory gene in M. grisea and are required for expression of the pathogenicity gene MPG1. We propose that NPR1 and NPR2 are major regulators of pathogenicity in M. grisea and may be novel regulators of nitrogen metabolism in fungi.     

Methionine Biosynthesis is Essential for Infection in the Rice Blast Fungus Magnaporthe oryzae

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.     

受稻瘟病原真菌侵染的水稻早期表达基因的cDNA克隆

以亲和性与非亲和性两个稻瘟病原真菌小种（Ｍａｇｎａｐｏｒｔｈｅ ｇｒｉｓｅａ（Ｈｅｂｅｒｔ）Ｂａｒｒ）感染同一水稻品种（Ｏｒｙｚａｓａｔｉｖａ Ｌ．ｃｖ．Ｓｈｅｎｘｉａｎｇｇｅｎｇ Ｎｏ．４）的植株产生明显不同的致病和抗病反应,由此建立了有效的感染系统。应用差异显示技术获得两个在侵染早期具有诱导表达特征的ｃＤＮＡ克隆,其中一个同时在致病和抗病反应中进行早期诱导表达,但在抗病反应中的诱导相对早于其在     

Isolation of the Mating-Type Genes of the Phytopathogenic Fungus Magnaporthe Grisea Using Genomic Subtraction

Using genomic subtraction, we isolated the mating-type genes (Mat1-1 and Mat1-2) of the rice blast fungus, Magnaporthe grisea. Transformation of M. grisea strains of one mating type with a linearized cosmid clone carrying the opposite mating-type gene resulted in many ``dual maters,' strains that contain both mating-type genes and successfully mate with both Mat1-1 and Mat1-2 testers. Dual maters differed in the frequency of production of perithecia in pure culture. Ascospores isolated from these homothallic crosses were either Mat1-1 or Mat1-2, but there were no dual maters. Most conidia from dual maters also had one or the other of the mating-type genes, but not both. Thus, dual maters appear to lose one of the mating-type genes during vegetative growth. The incidence of self-mating in dual maters appears to depend on the co-occurrence of strains with each mating type in vegetative cultures. In rare transformants, the incoming sequences had replaced the resident mating-type gene. Nearly isogenic pairs produced from three M. grisea laboratory strains were mated to investigate their fertility. One transformant with switched mating type appears to have a mutation that impairs the development of asci when its mating partner has a similar genetic background. The M. grisea Mat1-1 and Mat1-2 genes are idiomorphs approximately 2.5 and 3.5 kb in length, respectively.     

Molecular Cloning of Early-expressed cDNAs from Rice in Response to Infection by Rice Blast Fungus Magnaporthe grisea

Compatible and incompatible reactions in rice plants (Oryza sativa L. cv. Shenxianggen No.4) were resulted from inoculation with two different virulent races of rice blast fungus (Magnaporthe grisea (Hebert) Barr), and thus an effective infecting system was established between rice plants and the rice blast pathogen. Two cDNA clones that showed induced and temporal patterns in expression in the very early stage in response to infection of the fungus were obtained from the plants by use of differential display. Of the two cDNA clones, Fastresp-a was induced to express in both compatible and incompatible interactions although it was expressed earlier in the former reaction. The second one, Fastresp-b, was only expressed in incompatible interaction. Southern blot analysis of the rice genomic DNA indicated that both of the two clones were from genome of the plant. No significant homology to the two genes was found from the rice gene database. This suggested that they were novel genes in rice and may play important roles in rice resistant response to infection of rice blast fungus.     

Comparative Analysis of the Genomes of Two Field Isolates of the Rice Blast Fungus Magnaporthe oryzae

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice worldwide. The fungal pathogen is notorious for its ability to overcome host resistance. To better understand its genetic variation in nature, we sequenced the genomes of two field isolates, Y34 and P131. In comparison with the previously sequenced laboratory strain 70-15, both field isolates had a similar genome size but slightly more genes. Sequences from the field isolates were used to improve genome assembly and gene prediction of 70-15. Although the overall genome structure is similar, a number of gene families that are likely involved in plant-fungal interactions are expanded in the field isolates. Genome-wide analysis on asynonymous to synonymous nucleotide substitution rates revealed that many infection-related genes underwent diversifying selection. The field isolates also have hundreds of isolate-specific genes and a number of isolate-specific gene duplication events. Functional characterization of randomly selected isolate-specific genes revealed that they play diverse roles, some of which affect virulence. Furthermore, each genome contains thousands of loci of transposon-like elements, but less than 30% of them are conserved among different isolates, suggesting active transposition events in M. oryzae. A total of approximately 200 genes were disrupted in these three strains by transposable elements. Interestingly, transposon-like elements tend to be associated with isolate-specific or duplicated sequences. Overall, our results indicate that gain or loss of unique genes, DNA duplication, gene family expansion, and frequent translocation of transposon-like elements are important factors in genome variation of the rice blast fungus.     

Spatial Uncoupling of Mitosis and Cytokinesis during Appressorium-Mediated Plant Infection by the Rice Blast Fungus Magnaporthe oryzae

To infect plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we report that appressorium development in the rice blast fungus Magnaporthe oryzae involves an unusual cell division, in which nuclear division is spatially uncoupled from the site of cytokinesis and septum formation. The position of the appressorium septum is defined prior to mitosis by formation of a heteromeric septin ring complex, which was visualized by spatial localization of Septin4:green fluorescent protein (GFP) and Septin5:GFP fusion proteins. Mitosis in the fungal germ tube is followed by long-distance nuclear migration and rapid formation of an actomyosin contractile ring in the neck of the developing appressorium, at a position previously marked by the septin complex. By contrast, mutants impaired in appressorium development, such as Δpmk1 and ΔcpkA regulatory mutants, undergo coupled mitosis and cytokinesis within the germ tube. Perturbation of the spatial control of septation, by conditional mutation of the SEPTATION-ASSOCIATED1 gene of M. oryzae, prevented the fungus from causing rice blast disease. Overexpression of SEP1 did not affect septation during appressorium formation, but instead led to decoupling of nuclear division and cytokinesis in nongerminated conidial cells. When considered together, these results indicate that SEP1 is essential for determining the position and frequency of cell division sites in M. oryzae and demonstrate that differentiation of appressoria requires a cytokinetic event that is distinct from cell divisions within hyphae.