Expression of the Histidine Kinase Gene Sshk Correlates with Dimethachlone Resistance in Sclerotinia sclerotiorum

Histidine kinases (HK) are implicated in virulence, vegetative mycelial growth, and osmotic and oxidative responses in pathogenic fungi. Our previous work showed that transcriptional levels of the group III HK gene Sshk are higher in field dimethachlone-resistant isolates of Sclerotinia sclerotiorum compared with sensitive isolates. However, it is not clear whether the overexpression of Sshk is the major mechanism for resistance to dimethachlone. In this study, we constructed Sshk silencing and overexpression vectors and assessed dimethachlone resistance levels, virulence, mycelial growth, and sensitivity to osmotic stress for the Sshk-silenced and -overexpression transformants. Overexpression of Sshk resulted in resistance to dimethachlone and increased sensitivity to various stresses and to the cell-wall-perturbing agents sodium dodecyl sulfate (SDS) and Congo red (CR). Compared with the parent isolate, Sshk-silenced transformants had reduced resistance to dimethachlone, significantly higher (P < 0.05) mycelial growth and virulence, and lower sclerotium production, and were less sensitive to various exogenous stresses such as sodium chloride. Compared with the parent sensitive isolate HLJMG1, dimethachlone resistance ratios of the three overexpression transformants ∆C101, ∆C21, and ∆C10 increased 168.1-, 189.5-, and 221.2-fold, respectively. The three overexpression transformants were more sensitive to CR and SDS than their parent isolate. These findings suggest that overexpression of Sshk is a major mechanism for dimethachlone resistance in some isolates of S. sclerotiorum, and that Sshk plays an important role in maintaining the integrity of the cell wall. Our findings reveal a novel molecular mechanism for dimethachlone resistance in plant-pathogenic fungi.

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Stimulatory Effects of Sublethal Doses of Dimethachlon on Sclerotinia sclerotiorum

Plant Disease ◽

10.1094/pdis-10-13-1059-re ◽

2014 ◽

Vol 98 (10) ◽

pp. 1364-1370 ◽

Cited By ~ 21

Author(s):

Feng Zhou ◽

Hong-Jie Liang ◽

Ya-Li Di ◽

Hong You ◽

Fu-Xing Zhu

Keyword(s):

Oilseed Rape ◽

Sclerotinia Sclerotiorum ◽

Mycelial Growth ◽

Plant Disease ◽

Pathogenic Fungi ◽

Potato Dextrose Agar ◽

Detached Leaves ◽

Sublethal Doses ◽

Fungal Phytopathogens ◽

Plant Disease Management

Growth and virulence stimulations of sublethal doses of fungicides on plant-pathogenic fungi and oomycetes have been reported and the stimulatory effects are potentially relevant to plant disease management. Sclerotinia sclerotiorum is one of the most devastating and economically important necrotrophic fungal phytopathogens, capable of infecting more than 400 species of plants worldwide. In order to study stimulatory effects of sublethal doses of fungicides on S. sclerotiorum, 55 dimethachlon-sensitive isolates and 3 dimethachlon-resistant isolates of S. sclerotiorum were assayed to determine effects of sublethal doses of dimethachlon on mycelial growth rate on potato dextrose agar (PDA) media and virulence on oilseed rape plants. Results showed that all 3 dimethachlon-resistant isolates and 13 of the 55 sensitive isolates exhibited stimulatory responses to sublethal doses of dimethachlon. Dimethachlon-resistant isolates grew significantly (P < 0.05) faster on PDA media amended with dimethachlon at 0.5 to 4 μg/ml than on fungicide-free PDA media. As for virulence on detached leaves of oilseed rape plants, lesion diameters of dimethachlon-resistant isolates after growth on PDA media amended with dimethachlon at 0.5 to 2 μg/ml were significantly larger (P < 0.05) than the control. The maximum stimulatory effects were 42.40 to 59.80%. In pot experiments, for both dimethachlon-sensitive and -resistant isolates, significant (P < 0.05) virulence stimulations were observed after spraying with dimethachlon at a concentration of 2 μg/ml. After growing on dimethachlon-amended PDA media, H2O2 sensitivity of S. sclerotiorum decreased significantly (P < 0.05) compared with the nonamended PDA control.

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Deletion of YLR358C Contributes to Reduce Cell Wall Integrity in Saccharomyces Cerevisiae

10.21203/rs.3.rs-1235133/v1 ◽

2022 ◽

Author(s):

Yu Zhang ◽

Mengyan Li ◽

Hanying Wang ◽

Juqing Deng ◽

Jianxing Liu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Sodium Dodecyl ◽

Wall Stress ◽

Pathogenic Fungi ◽

Cell Wall Integrity ◽

Calcofluor White ◽

Fungal Cell ◽

Reading Frame ◽

Stress Signals

Abstract The mechanism of fungal cell wall synthesis and assembly is still unclear. Saccharomyces cerevisiae (S. cerevisiae) and pathogenic fungi are conserved in cell wall construction and response to stress signals, and often respond to cell wall stress through activated cell wall integrity (CWI) pathways. Whether the YLR358C open reading frame regulates CWI remains unclear. This study found that the growth of S. cerevisiae with YLR358C knockout was significantly inhibited on the medium containing different concentrations of cell wall interfering agents Calcofluor White (CFW), Congo Red (CR) and sodium dodecyl sulfate (SDS). CFW staining showed that the cell wall chitin was down-regulated, and transmission electron microscopy also observed a decrease in cell wall thickness. Transcriptome sequencing and analysis showed that YLR358C gene may be involved in the regulation of CWI signaling pathway. It was found by qRT-PCR that WSC3, SWI4 and HSP12 were differentially expressed after YLR358C was knocked out. The above results suggest that YLR358C may regulate the integrity of the yeast cell walls and has some potential for application in fermentation.

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Baseline Sensitivity and Toxic Actions of Prochloraz to Sclerotinia sclerotiorum

Plant Disease ◽

10.1094/pdis-01-18-0148-re ◽

2018 ◽

Vol 102 (11) ◽

pp. 2149-2157 ◽

Cited By ~ 11

Author(s):

Ran Zhang ◽

Qianru Xu ◽

Yuchao Zhang ◽

Fuxing Zhu

Keyword(s):

Cell Wall ◽

Sclerotinia Sclerotiorum ◽

Mycelial Growth ◽

Dose Range ◽

Cell Membrane Permeability ◽

Ergosterol Biosynthesis ◽

Baseline Sensitivity ◽

Necrotrophic Pathogen ◽

Frequency Distributions ◽

Significant Difference

The ergosterol biosynthesis inhibitor prochloraz is a broad-spectrum fungicide and has been registered in China since 2007 for control of the economically important necrotrophic pathogen Sclerotinia sclerotiorum. In this study, relative baseline sensitivity and toxic actions of prochloraz on S. sclerotiorum were investigated. The mean EC50 values (effective concentrations causing 50% mycelial growth inhibition) for isolates collected in 2008 (n = 73) and 2014 (n = 76) were 0.0463 and 0.0434 µg/ml, respectively. There was no significant difference (P = 0.348) in EC50 values between the two years. Both frequency distributions of EC50 values for 2008 and 2014 were unimodal. The curative efficacy of prochloraz was significantly higher (P < 0.05) than that of the reference fungicide carbendazim. Prochloraz in potato dextrose agar (PDA) at concentrations from 0.01 to 0.36 µg/ml had no significant (P = 0.574) effects on the weight of sclerotia, but the number of sclerotia per plate increased for treatments with prochloraz at 0.15 and 0.36 µg/ml. Light microscopic observations showed that prochloraz in PDA at 0.03 µg/ml increased the number of hyphal offshoots. Observations with a transmission electron microscope showed that the cell wall of the prochloraz-treated hyphae became thicker and darker than the nontreated control. Prochloraz at 0.01 and 0.04 µg/ml significantly (P < 0.001) reduced rather than increased cell membrane permeability. Prochloraz significantly (P = 0.041) increased the mannan content in the cell wall of S. sclerotiorum. The observed mycelial growth inhibitions for the mixtures of prochloraz at 0.03 µg/ml and Congo red at a dose range from 0.05 to 0.4% (w/v) were lower than the expected inhibitions, indicating prochloraz might reduce the content of chitin in S. sclerotiorum. These results demonstrate that prochloraz has significant effects on the morphology and components of the cell wall of S. sclerotiorum and thus will advance our understanding of the toxic actions of prochloraz on phytopathogenic fungi.

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Calcineurin Is Required for Sclerotial Development and Pathogenicity of Sclerotinia sclerotiorum in an Oxalic Acid-Independent Manner

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-0682 ◽

2006 ◽

Vol 19 (6) ◽

pp. 682-693 ◽

Cited By ~ 81

Author(s):

A. Harel ◽

S. Bercovich ◽

O. Yarden

Keyword(s):

Cell Wall ◽

Oxalic Acid ◽

Sclerotinia Sclerotiorum ◽

Cell Wall Degrading Enzymes ◽

Independent Manner ◽

Worldwide Distribution ◽

Cation Homeostasis ◽

Increased Sensitivity ◽

Synthase Inhibitor ◽

Sclerotial Development

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Calcineurin, a Ser/Thr phosphatase linked to several signal-transduction pathways, plays a key role in the regulation of cation homeostasis, morphogenesis, cell-wall integrity, and pathogenesis in fungi. We demonstrate that calcineurin expression in S. sclerotiorum is altered in a phase-specific manner during sclerotial development. Inhibition of calcineurin by FK506, cysclosporin A, or inducible antisense calcineurin expression impaired sclerotial development at the prematuration phase and increased germination of preformed sclerotia. Induction of antisense calcineurin expression in S. sclerotiorum resulted in reduced pathogenesis on tomato and Arabidopsis. However, secretion of oxalic acid, a key virulence factor of S. scle-rotiorum, was not altered. Inhibition of calcineurin conferred a reduction in cell wall β-1,3-glucan content and increased sensitivity to cell-wall-degrading enzymes and to the glucan synthase inhibitor caspofungin. Thus, calcineurin plays a major role in both sclerotial development and pathogenesis of S. sclerotiorum and, most likely, other phyto-pathogens.

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Hormetic Effects of Flusilazole Preconditioning on Mycelial Growth and Virulence of Sclerotinia sclerotiorum

Plant Disease ◽

10.1094/pdis-10-17-1638-re ◽

2018 ◽

Vol 102 (6) ◽

pp. 1165-1170 ◽

Cited By ~ 5

Author(s):

Xiaoming Lu ◽

Shun He ◽

Hongju Ma ◽

Jianhong Li ◽

Fuxing Zhu

Keyword(s):

Sclerotinia Sclerotiorum ◽

Mycelial Growth ◽

Dose Range ◽

Pathogenic Fungi ◽

Phytopathogenic Fungi ◽

Dependent Manner ◽

Inhibitory Effects ◽

Mycelia Growth ◽

Dmi Fungicides ◽

Dose Dependent

Hormetic effects of fungicides are highly relevant to fungicide applications and management of plant-pathogenic fungi. Preconditioning (i.e., early exposure to relatively low doses of a toxicant) is a special form of hormesis, and fungicide preconditioning of phytopathogenic fungi is inevitable in the field. The present study showed that spraying the demethylation inhibitor (DMI) fungicide flusilazole at 0.1 µg/ml had stimulatory effects on the virulence of Sclerotinia sclerotiorum inoculated at 1 and 24 h after spraying. Flusilazole sprayed at 10 µg/ml showed inhibitory effects on the virulence of S. sclerotiorum inoculated during the first 3 days after spraying. Inoculations on the 5th, 7th, and 10th day after spraying did not show any significant inhibitory or stimulatory effects on the virulence. After growing for 2 days on potato dextrose agar (PDA) amended with flusilazole at a dose range from 0.0005 to 0.25 µg/ml as preconditioning treatments, mycelia were transferred onto PDA without fungicide and subsequent mycelial growth was slower than the nonpreconditioned control. However, after the preconditioned colonies were transferred onto PDA supplemented with flusilazole at 0.2 µg/ml, percent stimulations of mycelia growth compared with the control had a parabolic shape across the preconditioning flusilazole concentration range. Similarly, the mycelial growth of the preconditioned mycelial plugs on PDA amended with other DMI fungicides (prochloraz or tebuconazole) also showed a typical hormetic response, whereas mycelial growth on PDA amended with carbendazim or dimethachlone was inhibited in a dose-dependent manner. Preconditioning S. sclerotiorum with flusilazole on rapeseed plants elicited virulence stimulations in a dose-dependent manner similar to those on mycelial growth on PDA. After disease lesions developed on rapeseed leaves sprayed with flusilazole as the preconditioning treatment were inoculated onto rapeseed plants, virulence was inhibited on leaves without fungicide or sprayed with carbendazim or dimethachlone compared with the nonpreconditioned control, whereas virulence was stimulated on leaves sprayed with flusilazole, prochloraz, or tebuconazole, and the maximum percent stimulation was 10.2%. These results will advance our understanding of hormetic effects of fungicides and of preconditioning hormesis in particular.

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The Regulatory Function of the Molecular Chaperone Hsp90 in the Cell Wall Integrity of Pathogenic Fungi

Current Proteomics ◽

10.2174/1570164615666180820155807 ◽

2018 ◽

Vol 16 (1) ◽

pp. 44-53

Author(s):

Marina Campos Rocha ◽

Camilla Alves Santos ◽

Iran Malavazi

Keyword(s):

Cell Wall ◽

Antifungal Therapy ◽

Molecular Chaperone ◽

Regulatory Protein ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Cell Wall Integrity ◽

Regulatory Function ◽

Loss Of Function ◽

Hsp90 Inhibition

Different signaling cascades including the Cell Wall Integrity (CWI), the High Osmolarity Glycerol (HOG) and the Ca2+/calcineurin pathways control the cell wall biosynthesis and remodeling in fungi. Pathogenic fungi, such as Aspergillus fumigatus and Candida albicans, greatly rely on these signaling circuits to cope with different sources of stress, including the cell wall stress evoked by antifungal drugs and the host’s response during infection. Hsp90 has been proposed as an important regulatory protein and an attractive target for antifungal therapy since it stabilizes major effector proteins that act in the CWI, HOG and Ca2+/calcineurin pathways. Data from the human pathogen C. albicans have provided solid evidence that loss-of-function of Hsp90 impairs the evolution of resistance to azoles and echinocandin drugs. In A. fumigatus, Hsp90 is also required for cell wall integrity maintenance, reinforcing a coordinated function of the CWI pathway and this essential molecular chaperone. In this review, we focus on the current information about how Hsp90 impacts the aforementioned signaling pathways and consequently the homeostasis and maintenance of the cell wall, highlighting this cellular event as a key mechanism underlying antifungal therapy based on Hsp90 inhibition.

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An R2R3-type myeloblastosis transcription factor MYB103 is involved in phosphorus remobilization

Food Production, Processing and Nutrition ◽

10.1186/s43014-020-00038-6 ◽

2020 ◽

Vol 2 (1) ◽

Author(s):

Fangwei Yu ◽

Shenyun Wang ◽

Wei Zhang ◽

Hong Wang ◽

Li Yu ◽

...

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Abiotic Stresses ◽

Myb Transcription Factor ◽

Ethylene Signaling ◽

Biotic And Abiotic Stresses ◽

P Deficiency ◽

P Concentration ◽

Increased Sensitivity

Abstract The members of myeloblastosis transcription factor (MYB TF) family are involved in the regulation of biotic and abiotic stresses in plants. However, the role of MYB TF in phosphorus remobilization remains largely unexplored. In the present study, we show that an R2R3 type MYB transcription factor, MYB103, is involved in phosphorus (P) remobilization. MYB103 was remarkably induced by P deficiency in cabbage (Brassica oleracea var. capitata L.). As cabbage lacks the proper mutant for elucidating the mechanism of MYB103 in P deficiency, another member of the crucifer family, Arabidopsis thaliana was chosen for further study. The transcript of its homologue AtMYB103 was also elevated in response to P deficiency in A. thaliana, while disruption of AtMYB103 (myb103) exhibited increased sensitivity to P deficiency, accompanied with decreased tissue biomass and soluble P concentration. Furthermore, AtMYB103 was involved in the P reutilization from cell wall, as less P was released from the cell wall in myb103 than in wildtype, coinciding with the reduction of ethylene production. Taken together, our results uncover an important role of MYB103 in the P remobilization, presumably through ethylene signaling.

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Co-operative action by endo- and exo-β-(1→3)-glucanases from parasitic fungi in the degradation of cell-wall glucans of Sclerotinia sclerotiorum (Lib.) de Bary

Biochemical Journal ◽

10.1042/bj1400047 ◽

1974 ◽

Vol 140 (1) ◽

pp. 47-55 ◽

Cited By ~ 57

Author(s):

David Jones ◽

Alex. H. Gordon ◽

John S. D. Bacon

Keyword(s):

Cell Wall ◽

Sclerotinia Sclerotiorum ◽

Culture Filtrate ◽

Cell Walls ◽

Trichoderma Viride ◽

Major Constituent ◽

Coniothyrium Minitans ◽

Parasitic Fungi ◽

Complete Breakdown

1. Two fungi, Coniothyrium minitans Campbell and Trichoderma viride Pers. ex Fr., were grown on autoclaved crushed sclerotia of the species Sclerotinia sclerotiorum, which they parasitize. 2. in vitro the crude culture filtrates would lyse walls isolated from hyphal cells or the inner pseudoparenchymatous cells of the sclerotia, in which a branched β-(1→3)-β-(1→6)-glucan, sclerotan, is a major constituent. 3. Chromatographic fractionation of the enzymes in each culture filtrate revealed the presence of several laminarinases, the most active being an exo-β-(1→3)-glucanase, known from previous studies to attack sclerotan. Acting alone this brought about a limited degradation of the glucan, but the addition of fractions containing an endo-β-(1→3)-glucanase led to almost complete breakdown. A similar synergism between the two enzymes was found in their lytic action on cell walls. 4. When acting alone the endo-β-(1→3)-glucanase had a restricted action, the products including a trisaccharide, tentatively identified as 62-β-glucosyl-laminaribiose. 5. These results are discussed in relation to the structure of the cell walls and of their glucan constituents.

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Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

Eukaryotic Cell ◽

10.1128/ec.00040-09 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1475-1485 ◽

Cited By ~ 33

Author(s):

Thanyanuch Kriangkripipat ◽

Michelle Momany

Keyword(s):

Cell Wall ◽

Aspergillus Nidulans ◽

Double Mutant ◽

Elevated Temperatures ◽

Cell Wall Integrity ◽

Wild Type ◽

Spatial Cues ◽

Developmental Patterning ◽

In The Wild ◽

Increased Sensitivity

ABSTRACT Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.

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