scholarly journals Molecular Evidence that the Extracellular Cutinase Pbc1 Is Required for Pathogenicity of Pyrenopeziza brassicae on Oilseed Rape

2003 ◽  
Vol 16 (6) ◽  
pp. 545-552 ◽  
Author(s):  
Donghui Li ◽  
Alison M. Ashby ◽  
Keith Johnstone
Keyword(s):  
Oilseed Rape ◽  
Single Copy ◽  
Leaf Spot Disease ◽  
Molecular Evidence ◽  
Wild Type ◽  
In Planta ◽  
Coding Region ◽  
Causal Organism ◽  
Type Gene ◽  
Culture Supernatants

Recent evidence has suggested that cutinase is required for cuticular penetration and, therefore, is essential for pathogenicity of Pyrenopeziza brassicae, the causal organism of light leaf spot disease of oilseed rape and other brassicas. In order to acquire molecular evidence for the role of cutinase in pathogenesis, the single-copy P. brassicae cutinase gene Pbc1 was disrupted by a transformation-mediated approach. Southern hybridization analysis revealed that in one mutant, NH10-1224, the disruption was due to a tandem insertion of two copies of the disruption vector into the 5′ coding region of Pbc1. In contrast to the wild type, no expression of Pbc1 was detected during in planta growth or in cutin-induced mycelium of NH10-1224 and no cutinase activity was detected in culture supernatants from NH10-1224 using pnitrophenyl butyrate as substrate. Scanning electron microscopy of Brassica napus cotyledons infected with wild-type P. brassicae confirmed that entry into the host is by direct penetration of the cuticle. In contrast, the cutinase-deficient mutant NH10-1224 failed to penetrate the cuticular layer and was unable to develop disease symptoms. This evidence is consistent with the hypothesis that Pbc1 is required for P. brassicae to penetrate the plant cuticle. Demonstration that complementation of NH10-1224 with the Pbc1 wild-type gene restores both cutinase activity and pathogenicity will be required to definitively establish that cutinase is required for successful pathogenesis of brassicas by P. brassicae.

scholarly journals Molecular Evidence for Hybrid Origin and Phenotypic Variation of Rosa Section Chinenses

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 996
Author(s):  
Chenyang Yang ◽  
Yujie Ma ◽  
Bixuan Cheng ◽  
Lijun Zhou ◽  
Chao Yu ◽  
...  
Keyword(s):  
Genetic Markers ◽  
Genetic Background ◽  
Morphological Traits ◽  
Taxonomic Status ◽  
Single Copy ◽  
Hybrid Origin ◽  
Biological Research ◽  
Molecular Evidence ◽  
Germplasm Resources ◽  
Wild Type

Rosa sect. Chinenses (Rosaceae) is an important parent of modern rose that is widely distributed throughout China and plays an important role in breeding and molecular biological research. R. sect. Chinenses has variable morphological traits and mixed germplasm. However, the taxonomic status and genetic background of sect. Chinenses varieties remain unclear. In this study, we collected germplasm resources from sect. Chinenses varieties with different morphological traits. Simple sequence repeat (SSR) markers, chloroplast markers, and single copy nuclear markers were used to explore the genetic background of these germplasm resources. We described the origin of hybridization of rose germplasm resources by combining different molecular markers. The results showed that the flower and hip traits of different species in R. sect. Chinenses were significantly different. The SSR analysis showed that the two wild type varieties have different genetic backgrounds. The double petal varieties of R. sect. Chinenses could be hybrids of two wild type varieties. A phylogenetic analysis showed that the maternal inheritance of sect. Chinenses varieties had two different origins. To some extent, variation in the morphological traits of double petal species of R. sect. Chinenses reflects the influence of cultivation process. This study emphasizes that different genetic markers vary in their characteristics. Therefore, analyzing different genetic markers in could provide an insight into highly heterozygous species.

scholarly journals Functional mapping of the translation-dependent instability element of yeast MATalpha1 mRNA.

1996 ◽  
Vol 16 (7) ◽  
pp. 3833-3843 ◽  
Author(s):  
A N Hennigan ◽  
A Jacobson
Keyword(s):  
Stability Boundary ◽  
Mrna Translation ◽  
Rare Codon ◽  
Molecular Evidence ◽  
Wild Type ◽  
Coding Region ◽  
Sequence Element ◽  
Protein Coding ◽  
Cis Acting ◽  
Mrna Stabilization

The determinants of mRNA stability include specific cis-acting destabilizing sequences located within mRNA coding and noncoding regions. We have developed an approach for mapping coding-region instability sequences in unstable yeast mRNAs that exploits the link between mRNA translation and turnover and the dependence of nonsense-mediated mRNA decay on the activity of the UPF1 gene product. This approach, which involves the systematic insertion of in-frame translational termination codons into the coding sequence of a gene of interest in a upf1delta strain, differs significantly from conventional methods for mapping cis-acting elements in that it causes minimal perturbations to overall mRNA structure. Using the previously characterized MATalpha1 mRNA as a model, we have accurately localized its 65-nucleotide instability element (IE) within the protein coding region. Termination of translation 5' to this element stabilized the MATalpha1 mRNA two- to threefold relative to wild-type transcripts. Translation through the element was sufficient to restore an unstable decay phenotype, while internal termination resulted in different extents of mRNA stabilization dependent on the precise location of ribosome stalling. Detailed mutagenesis of the element's rare-codon/AU-rich sequence boundary revealed that the destabilizing activity of the MATalpha1 IE is observed when the terminal codon of the element's rare-codon interval is translated. This region of stability transition corresponds precisely to a MATalpha1 IE sequence previously shown to be complementary to 18S rRNA. Deletion of three nucleotides 3' to this sequence shifted the stability boundary one codon 5' to its wild-type location. Conversely, constructs containing an additional three nucleotides at this same location shifted the transition downstream by an equivalent sequence distance. Our results suggest a model in which the triggering of MATalpha1 mRNA destabilization results from establishment of an interaction between translating ribosomes and a downstream sequence element. Furthermore, our data provide direct molecular evidence for a relationship between mRNA turnover and mRNA translation.

scholarly journals Amino Acid Transport and Metabolism in Mycobacteria: Cloning, Interruption, and Characterization of anl-Arginine/γ-Aminobutyric Acid Permease inMycobacterium bovis BCG

2000 ◽  
Vol 182 (4) ◽  
pp. 919-927 ◽  
Author(s):  
Anjali Seth ◽  
Nancy D. Connell
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Nitrogen Sources ◽  
Single Copy ◽  
Aminobutyric Acid ◽  
Cosmid Library ◽  
Wild Type ◽  
Carbon And Nitrogen ◽  
Genes Encoding ◽  
Type Gene

ABSTRACT Genes encoding l-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter fromMycobacterium bovis BCG and the effects of its deletion onl-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to therocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using 14C-labeled substrates. Greatly reduced uptake of l-arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow),l-[14C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenousl-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. l-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.

scholarly journals The Endo-β-1,4-Xylanase Xyn11A Is Required for Virulence in Botrytis cinerea

2006 ◽  
Vol 19 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Nélida Brito ◽  
José Juan Espino ◽  
Celedonio González
Keyword(s):  
Extracellular Enzymes ◽  
Lesion Size ◽  
Phytopathogenic Fungi ◽  
Glycosyl Hydrolases ◽  
Plant Cell Walls ◽  
Wild Type ◽  
In Planta ◽  
Polymer Backbone ◽  
Beechwood Xylan ◽  
Type Gene

Phytopathogenic fungi can degrade xylan, an abundant hemicellulose in plant cell walls, by the coordinate action of a group of extracellular enzymes. Among these, endo-β-1,4-xylanases carry out the initial breakdown by cleaving internal bonds in the polymer backbone. We have isolated and characterized a gene, xyn11A coding for an endo-β-1,4-xylanase belonging to family 11 of glycosyl hydrolases. xyn11A was shown to be induced by xylan and repressed by glucose and to be expressed in planta. The disruption of xyn11A caused only a moderate decrease, about 30%, in the level of extracellular endo-β-1-4-xy-lanase activity and in the growth rate, with beechwood xylan as the only carbon source. However, deletion of the gene had a more pronounced effect on virulence, delaying the appearance of secondary lesions and reducing the average lesion size by more than 70%. Reintroducing the wild-type gene into the mutant strains reversed this phenotype back to wild type.

scholarly journals Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

1998 ◽  
Vol 180 (11) ◽  
pp. 2931-2935 ◽  
Author(s):  
Toru Nakayashiki ◽  
Hachiro Inokuchi
Keyword(s):  
Escherichia Coli ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Suppressor Activity ◽  
Temperature Sensitive Mutants ◽  
Wobble Position ◽  
Group I ◽  
Group Ii ◽  
Type Gene

ABSTRACT Escherichia coli has only a single copy of a gene for tRNA6 Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O 2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4 Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O 2-methyluridine) as its anticodon, tRNA6 Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6 Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6 Leu. These tRNA6 Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaAgene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4 Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4 Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA6 Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6 Leu remains to be investigated.

scholarly journals Arginine vasopressin secretion with mutants of wild-type and Brattleboro rats AVP gene.

1997 ◽  
Vol 8 (12) ◽  
pp. 1863-1869
Author(s):  
J K Kim ◽  
S N Summer ◽  
W M Wood ◽  
J L Brown ◽  
R W Schrier
Keyword(s):  
Arginine Vasopressin ◽  
Stop Codon ◽  
Bb Rat ◽  
Wild Type ◽  
Coding Region ◽  
Cos Cells ◽  
Coding Regions ◽  
C Terminus ◽  
Type Gene ◽  
Avp Gene

Defects in peptide processing are associated with several disorders, including central diabetes insipidus (CDI). In the Brattleboro (BB) rat with CDI, the mRNA and protein of arginine vasopressin (AVP) are present in the hypothalamus, but no circulating AVP is detectable, thus suggesting a processing defect. The present study examined AVP secretion in cultured COS cells transfected with various constructs from wild-type and mutated Brattleboro AVP gene precursors. The precursor contains three exons encoding for vasopressin (VP), neurophysin (NP), and glycopeptide (GP). The Brattleboro rat has a deletion of a single base, guanine (G), in the NP coding region that leads to a frameshift, resulting in the loss of normal stop codon. The wild-type pcVP (22.0 +/- 5.2 pg/10[-2] U beta-galactosidase [beta-gal]), but not the mutated BB AVP gene pcBB (1.2 +/- 0.4 pg/10[-2] U beta-gal), was associated with AVP secretion from the COS cells as measured by RIA. The wild-type AVP gene without the GP coding region was associated with AVP release greater (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05, versus pcVP) than the pcVP with intact VP, NP, and GP coding regions. However, the wild-type AVP gene with VP coding region alone was not processed and secreted. Normalizing the pcBB total length with the insertion of a stop codon at the site of the normal stop codon was not associated with AVP secretion (3.0 +/- 1.4 pg/10[-2] U beta-gal). However, insertion of a stop codon so that the pcBB length equaled the length of VP and NP coding regions of the wild type was associated with AVP secretion (13.5 +/- 4.0 pg/10[-2] U beta-gal). When a stop codon was inserted into the wild-type NP coding region at the same site as the G deletion in the pcBB, the AVP secretion was significantly lower (15.1 +/- 5.0 pg/10[-2] U beta-gal) than pcVP with VP + NP but no GP coding regions (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05). In summary, (1) both VP and intact NP, but not GP, coding regions are necessary for AVP processing and secretion; (2) decreasing the length of the NP coding region diminishes but does not abolish AVP processing and secretion; and (3) shortening of the pcBB length with a stop codon at a site comparable to wild-type VP + NP allows AVP secretion, albeit less than with wild-type gene precursor. Thus, the CDI in BB rats is caused by the G deletion in NP coding region. This defect leads to abnormalities that contribute to the abnormal AVP processing. Specifically, the frameshift and absence of a stop codon cause a mutated extended C terminus, which, along with the mutated NP, contribute to the abnormal steps of AVP processing, transport, and secretion in the BB rat. These defects no doubt impair the folding and configuration necessary for normal processing of the AVP gene precursor.

The DNA Binding Protein Rfg1 Is a Repressor of Filamentation inCandida albicans

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1503-1512 ◽  
Author(s):  
Roy A Khalaf ◽  
Richard S Zitomer
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Hyphal Growth ◽  
Dna Binding Protein ◽  
Homozygous Deletion ◽  
Wild Type ◽  
Pathogenic Yeast ◽  
Repression Complex ◽  
Type Gene ◽  
Repression Domain

AbstractWe have identified a repressor of hyphal growth in the pathogenic yeast Candida albicans. The gene was originally cloned in an attempt to characterize the homologue of the Saccharomyces cerevisiae Rox1, a repressor of hypoxic genes. Rox1 is an HMG-domain, DNA binding protein with a repression domain that recruits the Tup1/Ssn6 general repression complex to achieve repression. The C. albicans clone also encoded an HMG protein that was capable of repression of a hypoxic gene in a S. cerevisiae rox1 deletion strain. Gel retardation experiments using the purified HMG domain of this protein demonstrated that it was capable of binding specifically to a S. cerevisiae hypoxic operator DNA sequence. These data seemed to indicate that this gene encoded a hypoxic repressor. However, surprisingly, when a homozygous deletion was generated in C. albicans, the cells became constitutive for hyphal growth. This phenotype was rescued by the reintroduction of the wild-type gene on a plasmid, proving that the hyphal growth phenotype was due to the deletion and not a secondary mutation. Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene. All these data indicate that this gene, now designated RFG1 for Repressor of Filamentous Growth, is a repressor of genes required for hyphal growth and not a hypoxic repressor.

scholarly journals Occurrence of Repeat Induced Point Mutation in Long Segmental Duplications of Neurospora

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 125-136 ◽  
Author(s):  
David D Perkins ◽  
Brian S Margolin ◽  
Eric U Selker ◽  
S D Haedo
Keyword(s):  
Point Mutation ◽  
Sexual Cycle ◽  
Segmental Duplications ◽  
Wild Type ◽  
Single Chain ◽  
Base Pairs ◽  
Gene Size ◽  
Type Gene ◽  
Repeat Induced Point Mutation

Abstract Previous studies of repeat induced point mutation (RIP) have typically involved gene-size duplications resulting from insertion of transforming DNA at ectopic chromosomal positions. To ascertain whether genes in larger duplications are subject to RIP, progeny were examined from crosses heterozygous for long segmental duplications obtained using insertional or quasiterminal translocations. Of 17 distinct mutations from crossing 11 different duplications, 13 mapped within the segment that was duplicated in the parent, one was closely linked, and three were unlinked. Half of the mutations in duplicated segments were at previously unknown loci. The mutations were recessive and were expressed both in haploid and in duplication progeny from Duplication × Normal, suggesting that both copies of the wild-type gene had undergone RIP. Seven transition mutations characteristic of RIP were found in 395 base pairs (bp) examined in one ro-11 allele from these crosses and three were found in ~750 bp of another. A single chain-terminating C to T mutation was found in 800 bp of arg-6. RIP is thus responsible. These results are consistent with the idea that the impaired fertility that is characteristic of segmental duplications is due to inactivation by RIP of genes needed for progression through the sexual cycle.

scholarly journals Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

2003 ◽  
Vol 185 (23) ◽  
pp. 6801-6808 ◽  
Author(s):  
Shannon A. Carroll ◽  
Torsten Hain ◽  
Ulrike Technow ◽  
Ayub Darji ◽  
Philippos Pashalidis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Separation ◽  
Bacterial Species ◽  
Surface Protein ◽  
Wild Type ◽  
Terminal Domain ◽  
Cell Wall Hydrolase ◽  
Characteristic Features ◽  
Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

scholarly journals Essential Function of the Pseudorabies Virus UL36 Gene Product Is Independent of Its Interaction with the UL37 Protein

2004 ◽  
Vol 78 (21) ◽  
pp. 11879-11889 ◽  
Author(s):  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Pseudorabies Virus ◽  
Rabbit Kidney ◽  
Tegument Protein ◽  
Wild Type ◽  
Coding Region ◽  
Ordered Structures ◽  
Ca 50 ◽  
Essential Function ◽  
Full Length Clone

ABSTRACT The large tegument protein encoded by the UL36 gene of pseudorabies virus (PrV) physically interacts with the product of the adjacent UL37 gene (B. G. Klupp, W. Fuchs, H. Granzow, R. Nixdorf, and T. C. Mettenleiter, J. Virol. 76:3065-3071, 2002). To analyze UL36 function, two PrV recombinants were generated by mutagenesis of an infectious PrV full-length clone in Escherichia coli: PrV-ΔUL36F exhibited a deletion of virtually the complete UL36 coding region, whereas PrV-UL36BSF contained two in-frame deletions of 238 codons spanning the predicted UL37 binding domain. Coimmunoprecipitation experiments confirmed that the mutated gene product of PrV-UL36BSF did not interact with the UL37 protein. Like the previously described PrV-ΔUL37 (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) but in contrast to PrV-ΔUL36F, PrV-UL36BSF was able to replicate in rabbit kidney (RK13) cells, although maximum virus titers were reduced ca. 50-fold and plaque diameters were reduced by ca. 45% compared to wild-type PrV. PrV-ΔUL36F was able to productively replicate after repair of the deleted gene or in a trans-complementing cell line. Electron microscopy of infected RK13 cells revealed that PrV-UL36BSF and phenotypically complemented PrV-ΔUL36F were capable of nucleocapsid formation and egress from the nucleus by primary envelopment and deenvelopment at the nuclear membrane. However, reenvelopment of nucleocapsids in the cytoplasm was blocked. Only virus-like particles without capsids were released efficiently from cells. Interestingly, cytoplasmic nucleocapsids of PrV-UL36BSF but not of PrV-ΔUL36F were found in large ordered structures similar to those which had previously been observed with PrV-ΔUL37. In summary, our results demonstrate that the interaction between the UL36 and UL37 proteins is important but not strictly essential for the formation of secondary enveloped, infectious PrV particles. Furthermore, UL36 possesses an essential function during virus replication which is independent of its ability to bind the UL37 protein.

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