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ABSTRACT: The rice blast fungus Magnaporthe oryzae infects plants with a specialized cell called an appressorium, which uses turgor to drive a rigid penetration peg through the rice leaf cuticle. Here, we show that NADPH oxidases (Nox) are necessary for septin-mediated reorientation of the F-actin cytoskeleton to facilitate cuticle rupture and plant cell invasion. We report that the Nox2-NoxR complex spatially organizes a heteroligomeric septin ring at the appressorium pore, required for assembly of a toroidal F-actin network at the point of penetration peg emergence. Maintenance of the cortical F-actin network during plant infection independently requires Nox1, a second NADPH oxidase, which is necessary for penetration hypha elongation. Organization of F-actin in appressoria is disrupted by application of antioxidants, whereas latrunculin-mediated depolymerization of appressorial F-actin is competitively inhibited by reactive oxygen species, providing evidence that regulated synthesis of reactive oxygen species by fungal NADPH oxidases directly controls septin and F-actin dynamics.
Proceedings of the National Academy of Sciences 02/2013; · 9.68 Impact Factor
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ABSTRACT: An insertional mutagenesis screen in the rice blast fungus, Magnaporthe oryzae, identified a novel mutant, A2-12-3, which is defective in infection-related morphogenesis and pathogenicity. Analysis of the mutation confirmed an insertion into MoLDB1, which putatively encodes an 806-amino-acid protein with a predicted LIM binding domain. Targeted gene deletion mutants of MoLDB1 were unable to produce asexual or sexual spores and were significantly impaired in vegetative growth and fungal virulence. The Δmoldb1 mutants also showed reduced expression of genes coding hydrophobic proteins (e.g. MPG1 and MHP1), resulting in an easily wettable phenotype in vegetative culture. Moreover, the expression of four genes encoding LIM proteins predicted from the M. oryzae genome was significantly downregulated by deletion of MoLDB1. Analysis of an M. oryzae strain expressing a MoLbd1-green fluorescent protein gene fusion was consistent with the protein being nuclear localized. When considered together, MoLdb1 appears to be involved in regulation of cell wall proteins, including hydrophobins and LIM proteins, and is essential for conidiation, sexual development, appressorium formation, and pathogenicity in M. oryzae.
Molecular Plant-Microbe Interactions 10/2010; 23(10):1260-74. · 4.43 Impact Factor
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ABSTRACT: Magnaporthe oryzae 2539 was previously found to be avirulent to most rice cultivars and, therefore, was assumed to carry many avirulence (AVR) genes. However, only one AVR gene, AVR1-CO39, which corresponds to a resistance (R) gene Pi-CO39(t) in rice cv. CO39, has been found from 2539 thus far. In order to identify more AVR genes, we isolated 228 progeny strains from a cross between 2539 and Guy11, an M. oryzae strain with strong virulence on rice, and inoculated these strains onto 23 rice accessions (22 individual cultivars and a mixture of 14 cultivars) that are all resistant to 2539 but susceptible to Guy11. Unexpectedly, the experimental results indicated that the avirulence of 2539 on these rice cultivars appeared to be controlled only by the AVR1-CO39 locus. Consistent with this result, we further found that all except one of the rice cultivars were resistant to two transformed Guy11 strains carrying a 1.05-kb fragment containing the AVR1-CO39 gene from 2539. These results suggest that AVR1-CO39 is a predominant locus controlling the broad avirulence of 2539 on cultivated rice. Based on the results of this study and other previous studies, we infer that AVR1-CO39 is a species-wise rather than a cultivar-wise host-specific AVR locus of M. oryzae for rice.
Molecular Plant-Microbe Interactions 09/2010; 24(1):13-7. · 4.43 Impact Factor
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ABSTRACT: Magnaporthe oryzae starts its infection by the attachment of pyriform conidia on rice tissues, and severity of the disease epidemic is proportional to the quantity of conidia produced in the rice blast lesions. However, the mechanism of conidial production is not well understood. Homeodomain proteins play critical roles in regulating various growth and developmental processes in fungi and other eukaryotes. Through targeted gene replacement, we find that deletion of HTF1, one of seven homeobox genes in the fungal genome, does not affect mycelial growth but causes total defect of conidial production. Further observation revealed that the Deltahtf1 mutant produces significantly more conidiophores, which curve slightly near the tip but could not develop sterigmata-like structures. Although the Deltahtf1 mutant fails to form conidia, it could still develop melanized appressoria from hyphal tips and infect plants. The expression level of HTF1 is significantly reduced in the Deltamgb1 G-beta and DeltacpkA deletion mutant, and the ACR1 but not CON7 gene that encodes transcription factor required for normal conidiogenesis is significantly downregulated in the Deltahtf1 mutant. These data suggest that the HTF1 gene is essential for conidiogenesis, and may be functionally related to the trimeric G-protein signaling and other transcriptional regulators that are known to be important for conidiation in M. oryzae.
Molecular Plant-Microbe Interactions 04/2010; 23(4):366-75. · 4.43 Impact Factor
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Wei Li,
Baohua Wang,
Jun Wu,
Guodong Lu,
Yajun Hu,
Xing Zhang,
Zhengguang Zhang,
Qiang Zhao,
Qi Feng,
Hongyan Zhang,
Zhengyi Wang,
Guoliang Wang,
Bin Han, Zonghua Wang,
Bo Zhou
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ABSTRACT: The Magnaporthe oryzae avirulence gene AvrPiz-t activates immunity in a gene-for-gene fashion to rice mediated by the blast resistance gene Piz-t. To dissect the molecular mechanism underlying their recognition, we initiated the cloning of AvrPiz-t using a map-based cloning strategy. The AvrPiz-t gene was delimited to an approximately 21-kb genomic fragment, in which six genes were predicted. Complementation tests of each of these six candidate genes led to the final identification of AvrPiz-t, which encodes a 108-amino-acid predicted secreted protein with unknown function and no homologues in M. oryzae or in other sequenced fungi. We found that AvrPiz-t is present in the virulent isolate GUY11 but contains a Pot3 insertion at a position 462 bp upstream from the start codon. Complementation tests of AvrPiz-t genes driven by promoters of varying length revealed that a promoter larger than 462 bp is essential to maintain the AvrPiz-t function. These results suggest that a Pot3 insertion in GUY11 might interfere with the proper function of AvrPiz-t. Additionally, we found that AvrPiz-t can suppress the programmed cell death triggered by mouse BAX protein in Nicotiana benthamiana, identifying a mechanism by which AvrPiz-t may contribute virulence of M. oryzae.
Molecular Plant-Microbe Interactions 05/2009; 22(4):411-20. · 4.43 Impact Factor
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ABSTRACT: Laccase is found to be involved in pathogenicity of Cryphonectria parasitica and Cryptococcus neoformans. In this report we demonstrate that laccase is not necessary for pathogenicity in Magnaporthe grisea, which might be due to functional redundancy in some or all of the laccase genes. The major laccase activity in M. grisea is not encoded by either of the MGG_00551.5 and MGG_02876.5 genes, because targeted deletion of each gene shows only a slight decrease in laccase activity compared to wild-type strains.
The MGG_00551.5 and MGG_02876.5 mutants share the same growth rate, conidiation and pathogenicity as wild-type strains. Taken together, our findings provide
evidence that these genes are not essential for the differentiation and development of M. grisea.
12/2008: pages 51-57;
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ABSTRACT: Rac1 is a small GTPase involved in actin cytoskeleton organization and polarized cell growth in many organisms. In this study, we investigate the biological function of MgRac1, a Rac1 homolog in Magnaporthe grisea. The MgRac1 deletion mutants are defective in conidial production. Among the few conidia generated, they are malformed and defective in appressorial formation and consequently lose pathogenicity. Genetic complementation with native MgRac1 fully recovers all these defective phenotypes. Consistently, expression of a dominant negative allele of MgRac1 exhibits the same defect as the deletion mutants, while expression of a constitutively active allele of MgRac1 can induce abnormally large conidia with defects in infection-related growth. Furthermore, we show the interactions between MgRac1 and its effectors, including the PAK kinase Chm1 and NADPH oxidases (Nox1 and Nox2), by the yeast two-hybrid assay. While the Nox proteins are important for pathogenicity, the MgRac1-Chm1 interaction is responsible for conidiogenesis. A constitutively active Chm1 mutant, in which the Rac1-binding PBD domain is removed, fully restores conidiation of the MgRac1 deletion mutants, but these conidia do not develop appressoria normally and are not pathogenic to rice plants. Our data suggest that the MgRac1-Chm1 pathway is responsible for conidiogenesis, but additional pathways, including the Nox pathway, are necessary for appressorial formation and pathogenicity.
PLoS Pathogens 12/2008; 4(11):e1000202. · 9.13 Impact Factor
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Yan Zheng,
Geng Zhang,
Fucheng Lin, Zonghua Wang,
Gulei Jin,
Long Yang,
Ying Wang,
Xi Chen,
Zhaohua Xu,
Xiangqian Zhao,
Hongkai Wang,
Jianping Lu,
Guodong Lu,
Weiren Wu
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ABSTRACT: Magnaporthe grisea is the most destructive fungal pathogen of rice and a model organism for studying plant-pathogen interaction. Molecular markers and genetic maps are useful tools for genetic studies. In this study, based on the released genome sequence data of M. grisea, we investigated 446 simple sequence repeat (SSR) loci and developed 313 SSR markers, which showed polymorphisms among nine isolates from rice (including a laboratory strain 2539). The number of alleles of each marker ranged 2-9 with an average of 3.3. The polymorphic information content (PIC) of each marker ranged 0.20-0.89 with an average of 0.53. Using a population derived from a cross between isolates Guy11 and 2539, we constructed a genetic map of M. grisea consisting of 176 SSR markers. The map covers a total length of 1247 cM, equivalent to a physical length of about 35.0 Mb or 93% of the genome, with an average distance of 7.1cM between adjacent markers. A web-based database of the SSR markers and the genetic map was established (http://ibi.zju.edu.cn/pgl/MGM/index.html).
Fungal Genetics and Biology 08/2008; 45(10):1340-7. · 3.74 Impact Factor
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ABSTRACT: Multiform single chain variable fragments (scFvs) including different length linker scFvs and bispecific scFv were constructed. The linker lengths of 0, 3, 5, 8, 12, and 15 amino acids between V(H) and V(L) of antideoxynivalenol (anti-DON) scFv were used to analyze the affinities of scFvs. The affinity constants of these scFvs increased when the linker was lower than 12 amino acids. The affinity constant would not change when the linker was longer than 12 amino acids. Fusion gene of anti-DON scFv and antizearalenone (anti-ZEN) scFv was also constructed through connection by a short peptide linker DNA to express a bispecific scFv. The affinity constants assay showed that the two scFvs of fusion bispecific scFv remained their own affinity compared to their parental scFvs. Competitive direct enzyme linked immunosorbent assay was used to detect DON and ZEN in contaminated wheat (Triticum aestivum L.) samples, and the results indicated that this bispecific scFv was applicable in DON and ZEN detection. This work confirmed that bispecific scFv could be successfully obtained, and might also have an application in diagnosing fungal infection, and breeding transgenic plants.
Journal of Genetics and Genomics 06/2008; 35(5):313-6. · 1.88 Impact Factor
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ABSTRACT: Oxalic acid (OA) is inhibitory to many fungal plant pathogens. To further characterize the molecular mechanism of OA involved in fungal pathogenesis, OA insensitive mutants were screened from a chemical inducible Arabidopsis mutant library (about 6000 lines) using MS medium (calcium free) containing 1.2 mmol/L OA and 10 micromol/L estradiol. Harvested putative mutants were collected separately. Individual lines of mutants were screened again on modified MS medium containing OA. Mutants D33, D74, D154, D282 and D630 with enhanced OA resistance were obtained. The T-DNA flanking sequences were amplified by TAIL-PCR. The sequences were blasted against TAIR database. The result indicated that the T-DNA of mutant D33 was inserted between At2g39720 (zinc finger) and At2g39730 (Rubisco activase), and the T-DNA junctions of the other four mutants were the same, all inserted in the same site of the first intron of At5g10450 (14-3-3 protein GF14 lambda).
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 03/2008; 24(2):203-8.
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ABSTRACT: The small GTPase Rho3 is conserved in fungi and plays a key role in the control of cell polarity and exocytosis in yeast. In this report, we show that a Rho3 homolog, MgRho3, is dispensable for polarized hyphal growth in the rice blast fungus Magnaporthe grisea. However, MgRho3 is required for plant infection. Appressoria formed by the Mgrho3 deletion mutants are morphologically abnormal and defective in plant penetration. Conidia of the Mgrho3 deletion mutants are narrower than those of the wild-type strain and delayed in germination. Transformants expressing a dominant negative Mgrho3 allele exhibit similar phenotypes as the Mgrho3 deletion mutant, while transformants expressing a constitutively active allele of MgRho3 can produce normal conidia but remain defective in appressorium formation and plant infection. In contrast, overexpression of wild-type MgRho3 increases the infectivity of M. grisea. Our results reveal a new role for the conserved Rho3 as a critical regulator of developmental processes and pathogenicity of M. grisea.
Eukaryotic Cell 01/2008; 6(12):2240-50. · 3.60 Impact Factor
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Wu Zheng,
Zhiying Zhao,
Jisheng Chen,
Wende Liu,
Haiyan Ke,
Jie Zhou,
Guodong Lu,
Alan G. Darvill,
Peter Albersheim,
Shengcheng Wu, Zonghua Wang
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ABSTRACT: Cdc42, a member of the Rho-family small GTP-binding proteins, is a pivotal signaling switch that cycles between active GTP-bound and inactive GDP-bound forms, controlling actin cytoskeleton organization and cell polarity. In this report, we show that MgCdc42, a Cdc42 ortholog in Magnaporthe grisea, is required for its plant penetration. Consequently, the deletion mutants show dramatically decreased virulence to rice due to the arrest of penetration and infectious growth, which may be attributed to the defect of turgor and superoxide generation during the appressorial development in Mgcdc42 deletion mutants. In addition, the mutants also exhibit pleotropic defects including gherkin-shaped conidia, delayed germination as well as decreased sporulation. Furthermore, dominant negative mutation leads to a similar phenotype to that of the deletion mutants, lending further support to the conclusion that MgCdc42 is required for the penetration and virulence of M. grisea.
Fungal Genetics and Biology.
