Chasing stress signals – Exposure to extracellular stimuli differentially affects the redox state of cell compartments in the wild type and signaling mutants of Botrytis cinerea

To investigate whether the ech42 gene in Clonostachysrosea can improve the biocontrol efficacy of Bacillus amyloliquefaciens and its molecular mechanism. Compared to the wild type, the B. amyloliquefaciens transformed with the ech42 gene exhibited higher chitinase activity. The B. amyloliquefaciens-ech42 also showed significantly higher biocontrol efficiency compared to Botrytiscinerea when tomato plants were pre-treated with B. amyloliquefaciens-ech42. No significant difference in biocontrol efficiency was observed between the wild type and B.amyloliquefaciens-ech42 when tomato plants were first infected by Botrytiscinerea. In addition, the activity of the defense-related enzyme polyphenol oxidase, but not superoxide dismutase, was significantly higher in B. amyloliquefaciens-ech42 than in the wild type. The ech42 enhances the biocontrol efficiency of B.amyloliquefaciens by increasing the capacity of preventative/curative effects in plants, rather than by killing the pathogens.

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NADPH Oxidase Is Crucial for the Cellular Redox Homeostasis in Fungal Pathogen Botrytis cinerea

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-19-0124-r ◽

2019 ◽

Vol 32 (11) ◽

pp. 1508-1516

Author(s):

Hua Li ◽

Shiping Tian ◽

Guozheng Qin

Keyword(s):

Nadph Oxidase ◽

Botrytis Cinerea ◽

Fungal Pathogen ◽

Molecular Mechanisms ◽

Redox Homeostasis ◽

Signal Transduction Pathway ◽

Regulatory Subunit ◽

Wild Type ◽

Mutant Proteins ◽

In The Wild

During interactions, both plants and pathogens produce reactive oxygen species (ROS). Plants generate ROS for defense induction, while pathogens synthesize ROS for growth, sporulation, and virulence. NADPH oxidase (NOX) complex in the plasma membrane represents a main protein complex for ROS production in pathogens. Although NOX plays a crucial role in pathogenicity of pathogens, the underlying molecular mechanisms of NOX, especially the proteins regulated by NOX, remain largely unknown. Here, we applied an iodoacetyl tandem mass tag-based redox proteomic assay to investigate the protein redox dynamics in deletion mutant of bcnoxR, which encodes a regulatory subunit of NOX in the fungal pathogen Botrytis cinerea. In total, 214 unique peptidyl cysteine (Cys) thiols from 168 proteins were identified and quantified in both the wild type and ∆bcnoxR mutant. The Cys thiols in the ∆bcnoxR mutant were generally more oxidized than those in the wild type, suggesting that BcNoxR is essential for maintaining the equilibrium of the redox state in B. cinerea. Site-specific thiol oxidation analysis indicated that 142 peptides containing the oxidized thiols changed abundance significantly in the ∆bcnoxR mutant. Proteins containing these differential peptides are classified into various functional categories. Functional analysis revealed that one of these proteins, 6-phosphate dehydrogenase, played roles in oxidative stress response and pathogenesis of B. cinerea. These results provide insight into the potential target proteins and the ROS signal transduction pathway regulated by NOX.

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A capsule-associated gene of Cryptococcus neoformans, CAP64, is involved in pH homeostasis

Microbiology ◽

10.1099/mic.0.001029 ◽

2021 ◽

Vol 167 (6) ◽

Author(s):

Yumi Imanishi-Shimizu ◽

Yukina Kamogawa ◽

Yukino Shimada ◽

Kiminori Shimizu

Keyword(s):

Cryptococcus Neoformans ◽

Type Strain ◽

Intracellular Transport ◽

Wild Type Strain ◽

Intracellular Localization ◽

Wild Type ◽

Ph Homeostasis ◽

Cell Compartments ◽

In The Wild ◽

Acidic Organelles

The CAP64 gene is known to be involved in capsule formation in the basidiomycete yeast Cryptococcus neoformans. A null mutant of CAP64, Δcap64, lacks a capsule around the cell wall and its acidic organelles are not stained with quinacrine. In order to clarify whether the Cap64 protein indeed maintains vacuole or vesicle acidification, so that the vesicle containing the capsule polysaccharide or DBB substrate are transported to the cell membrane side, the relationship between CAP64 and intracellular transport genes and between CAP64 and enzyme-secretion activity were analysed. Laccase activity was higher in the Δcap64 strain than in the wild-type strain, and the transcriptional levels of SAV1 and VPH1 were also higher in the Δcap64 strain than in the wild-type strain. The intracellular localization of the Cap64 protein was analysed by overexpressing an mCherry-tagged Cap64 and observing its fluorescence. The Cap64 protein was accumulated within cells in a patch-like manner. The quinacrine-stained cells were observed to analyse the acidified cell compartments; quinacrine was found to be accumulated in a patch-like manner, with the patches overlapping the fluorescence of CAP64-mCherry fusion protein. Quinacrine was thus accumulated in a patch-like fashion in the cells, and the mCherry-tagged Cap64 protein position was consistent with the position of quinacrine accumulation in cells. These results suggest that CAP64 might be involved in intracellular acidification and vesicle secretion via exocytosis.

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A Thioredoxin of Sinorhizobium meliloti CE52G Is Required for Melanin Production and Symbiotic Nitrogen Fixation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-8-0986 ◽

2007 ◽

Vol 20 (8) ◽

pp. 986-993 ◽

Cited By ~ 14

Author(s):

Susana Castro-Sowinski ◽

Ofra Matan ◽

Paula Bonafede ◽

Yaacov Okon

Keyword(s):

Nitrogen Fixation ◽

Sinorhizobium Meliloti ◽

Redox State ◽

Wild Type Strain ◽

Symbiotic Nitrogen Fixation ◽

Mrna Level ◽

Wild Type ◽

Melanin Production ◽

In The Wild ◽

Reducing Activity

A miniTn5-induced mutant of a melanin-producing strain of Sinorhizobium meliloti (CE52G) that does not produce melanin was mapped to a gene identified as a probable thioredoxin gene. It was proved that the thiol-reducing activity of the mutant was affected. Addition to the growth medium of substrates that induce the production of melanin (l-tyrosine, guaiacol, orcinol) increased the thioredoxin-like (trxL) mRNA level in the wild-type strain. The mutant strain was affected in the response to paraquat-induced oxidative stress, symbiotic nitrogen fixation, and both laccase and tyrosinase activities. The importance of thioredoxin in melanin production in bacteria, through the regulation of laccase or tyrosinase activities, or both, by the redox state of structural or catalytic SH groups, is discussed.

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Sensitivity of Populations of Botrytis cinerea from Pear-Related Sources to Benzimidazole and Dicarboximide Fungicides

Plant Disease ◽

10.1094/pdis.2003.87.6.645 ◽

2003 ◽

Vol 87 (6) ◽

pp. 645-649 ◽

Cited By ~ 19

Author(s):

Cheryl L. Lennox ◽

Robert A. Spotts

Keyword(s):

Botrytis Cinerea ◽

Pacific Northwest ◽

Wild Type ◽

Baseline Sensitivity ◽

Benzimidazole Fungicides ◽

Postharvest Application ◽

The Pacific ◽

In The Wild ◽

Type Population ◽

High Level

Botrytis cinerea is responsible for a major portion of postharvest decay in winter pears in the Pacific Northwest. The baseline sensitivity levels (mean EC50 values) of a wild-type B. cinerea population to thiabendazole and iprodione were 6.66 and 0.56 mg/liter, respectively. B. cinerea from commercial orchards not treated with a benzimidazole had significantly lower incidence of resistance (0.59%) to a discriminatory concentration of thiabendazole at 10 mg/liter than did isolates from orchards in which benomyl had been applied for experimental purposes (16.0%), unsprayed control trees in benomyl-sprayed orchards (5.34%), and isolates from packinghouses where thiabendazole was applied as a prestorage drench or packingline spray (3.23%). The mean EC50 value of isolates in the wild-type population was lower than those of resistant isolates from all other sources. High-level thiabendazole resistance (EC50 > 100 mg/liter) was found in 0.20% of isolates from unsprayed commercial orchards, 9.33% of isolates from benomyl-sprayed orchards, and 2.67% of isolates from unsprayed control trees in these benomyl-sprayed orchards. In isolates from packinghouses where a thiabendazole line spray was applied, 1.52% had high-level thiabendazole resistance. All isolates from all pear-related sources tested were sensitive to iprodione at 10 mg/liter. This study provides evidence supporting current recommendations of a single postharvest application of a benzimidazole to control decay caused by B. cinerea, and no application of benzimidazole fungicides in the orchard.

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Microarray analysis of Arabidopsis WRKY33 mutants in response to the necrotrophic fungus Botrytis cinerea

10.31226/osf.io/rw2av ◽

2018 ◽

Author(s):

Synan AbuQamar ◽

Khaled Moustafa

Keyword(s):

Transcription Factor ◽

Botrytis Cinerea ◽

Defense Responses ◽

Plant Responses ◽

Wild Type Plant ◽

The Novel ◽

Wild Type ◽

Genes Encoding ◽

In The Wild ◽

Molecular Components

The WRKY33 transcription factor was reported for resistance to the necrotrophic fungus Botrytis cinerea. Using microarray-based analysis, we compared Arabidopsis WRKY33 overexpressing lines and wrky33 mutant that showed altered susceptibility to B. cinerea with their corresponding wild-type plants. In the wild-type, about 1660 genes (7% of the transcriptome) were induced and 1054 genes (5% of the transcriptome) were repressed at least twofold at early stages of inoculation with B. cinerea, confirming previous data of the contribution of these genes in B. cinerea resistance. In Arabidopsis wild-type plant infected with B. cinerea, the expressions of the differentially expressed genes encoding for proteins and metabolites involved in pathogen defense and non-defense responses, seem to be dependent on a functional WRKY33 gene. The expression profile of 12-oxo-phytodienoic acid- and phytoprostane A1-treated Arabidopsis plants in response to B. cinerea revealed that cyclopentenones can also modulate WRKY33 regulation upon inoculation with B. cinerea. These results support the role of electrophilic oxylipins in mediating plant responses to B. cinerea infection through the TGA transcription factor. Future directions toward the identification of the molecular components in cyclopentenone signaling will elucidate the novel oxylipin signal transduction pathways in plant defense.

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Freeze-substituted fungal cells of Nectria haematococca: A comparison of the macroconidial walls of the adhesion-competent wild type with an adhesion-reduced mutant

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160996 ◽

1990 ◽

Vol 48 (3) ◽

pp. 688-689

Author(s):

Thecan Caesar-Ton That ◽

Lynn Epstein

Keyword(s):

Freeze Substitution ◽

Uranyl Acetate ◽

Wild Type ◽

Nectria Haematococca ◽

Fruit Extract ◽

Dialysis Tubing ◽

Tube Emergence ◽

Contrast Microscopy ◽

In The Wild ◽

Freeze Damage

Nectria haematococca mating population I (anamorph, Fusarium solani) macroconidia attach to its host (squash) and non-host surfaces prior to germ tube emergence. The macroconidia become adhesive after a brief period of protein synthesis. Recently, Hickman et al. (1989) isolated N. haematococca adhesion-reduced mutants. Using freeze substitution, we compared the development of the macroconidial wall in the wild type in comparison to one of the mutants, LEI.Macroconidia were harvested at 1C, washed by centrifugation, resuspended in a dilute zucchini fruit extract and incubated from 0 - 5 h. During the incubation period, wild type macroconidia attached to uncoated dialysis tubing. Mutant macroconidia did not attach and were collected on poly-L-lysine coated dialysis tubing just prior to freezing. Conidia on the tubing were frozen in liquid propane at 191 - 193C, substituted in acetone with 2% OsO4 and 0.05% uranyl acetate, washed with acetone, and flat-embedded in Epon-Araldite. Using phase contrast microscopy at 1000X, cells without freeze damage were selected, remounted, sectioned and post-stained sequentially with 1% Ba(MnO4)2 2% uranyl acetate and Reynold’s lead citrate. At least 30 cells/treatment were examined.

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Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

Molecular and Cellular Biology ◽

10.1128/mcb.00524-07 ◽

2007 ◽

Vol 28 (3) ◽

pp. 897-906 ◽

Cited By ~ 21

Author(s):

Thomas J. Pohl ◽

Jac A. Nickoloff

Keyword(s):

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Single Strand ◽

Homology Search ◽

Wild Type ◽

Dsb Repair ◽

Single Strand Annealing ◽

In The Wild ◽

Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

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Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.2.383 ◽

1996 ◽

Vol 142 (2) ◽

pp. 383-391 ◽

Cited By ~ 2

Author(s):

Yasumasa Tsukamoto ◽

Jun-ichi Kato ◽

Hideo Ikeda

Keyword(s):

Saccharomyces Cerevisiae ◽

Quantitative Analysis ◽

Structural Analysis ◽

Recombination Rate ◽

Type Strain ◽

Negative Selection ◽

Wild Type Strain ◽

Illegitimate Recombination ◽

Wild Type ◽

In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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Transcriptome Analysis Provides Insights into the Mechanism of Astaxanthin Enrichment in a Mutant of the Ridgetail White Prawn Exopalaemon carinicauda

Genes ◽

10.3390/genes12050618 ◽

2021 ◽

Vol 12 (5) ◽

pp. 618

Author(s):

Yue Jin ◽

Shihao Li ◽

Yang Yu ◽

Chengsong Zhang ◽

Xiaojun Zhang ◽

...

Keyword(s):

Transcriptome Analysis ◽

Underlying Mechanism ◽

Biological Processes ◽

Wild Type ◽

Expression Levels ◽

In The Wild ◽

Cuticle Proteins ◽

Apolipoprotein D ◽

High Level ◽

Lower Expression

A mutant of the ridgetail white prawn, which exhibited rare orange-red body color with a higher level of free astaxanthin (ASTX) concentration than that in the wild-type prawn, was obtained in our lab. In order to understand the underlying mechanism for the existence of a high level of free astaxanthin, transcriptome analysis was performed to identify the differentially expressed genes (DEGs) between the mutant and wild-type prawns. A total of 78,224 unigenes were obtained, and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant in comparison with the wild-type prawns. Based on Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis, as well as further investigation of annotated DEGs, we found that the biological processes related to astaxanthin binding, transport, and metabolism presented significant differences between the mutant and the wild-type prawns. Some genes related to these processes, including crustacyanin, apolipoprotein D (ApoD), cathepsin, and cuticle proteins, were identified as DEGs between the two types of prawns. These data may provide important information for us to understand the molecular mechanism of the existence of a high level of free astaxanthin in the prawn.

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