scholarly journals Overexpression of a Redox-Regulated Cutinase Gene, MfCUT1, Increases Virulence of the Brown Rot Pathogen Monilinia fructicola on Prunus spp.

2010 ◽  
Vol 23 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Miin-Huey Lee ◽  
Chiu-Min Chiu ◽  
Tatiana Roubtsova ◽  
Chien-Ming Chou ◽  
Richard M. Bostock
Keyword(s):  
Gene Expression ◽  
Redox Regulation ◽  
Brown Rot ◽  
Gus Expression ◽  
Apple Fruit ◽  
Gus Activity ◽  
Monilinia Fructicola ◽  
Gus Reporter ◽  
Flanking Regions ◽  
Prunus Spp

A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUT1, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUT1 carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUT1 expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUT1 gene expression in cutin-induced cultures, and in the present study, where H2O2 enhanced MfCUT1 gene expression. A β-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUT1 under the control of the MfCUT1 promoter, and this construct was then used to generate an MfCUT1-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUT1-GUS fusion protein was expressed correctly under the control of the MfCUT1 promoter. MfCUT1-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUT1 expression within 5 h of inoculation in both almond and peach petals. MfCUT1-GUS transformants expressed MfCUT1 transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUT1 contributing to the virulence of M. fructicola.

scholarly journals Cutinase A of Botrytis cinerea is Expressed, but not Essential, During Penetration of Gerbera and Tomato

1997 ◽  
Vol 10 (1) ◽  
pp. 30-38 ◽  
Author(s):  
J. A. L. van Kan ◽  
J. W. van 't Klooster ◽  
C. A. M. Wagemakers ◽  
D. C. T. Dees ◽  
C. J. B. van der Vlugt-Bergmans
Keyword(s):  
Botrytis Cinerea ◽  
Wild Type Strain ◽  
Gus Expression ◽  
Infection Process ◽  
Gus Activity ◽  
Wild Type ◽  
Tomato Fruits ◽  
Fusion Construct ◽  
Gus Reporter ◽  
Encoding Gene

The plant pathogen Botrytis cinerea can infect undamaged plant tissue directly by penetration of the cuticle. This penetration has been suggested to be enzyme-mediated, and an important role for cutinase in the infection process has been proposed. In this study the expression of the cutinase encoding gene cutA of B. cinerea was analyzed using a cutA promoter-GUS reporter gene fusion. Transformants containing the fusion construct were examined for GUS expression on gerbera flowers and tomato fruits. High GUS activity was detected from the onset of conidial germination and during penetration into epidermal cells, indicating that cutA is expressed during the early stages of infection. To determine the biological relevance of cutinase A for successful penetration, cutinase A-deficient mutants were constructed by gene disruption. Pathogenicity of two transformants lacking a functional cutA gene was studied on gerbera flowers and tomato fruits. Their ability to penetrate and cause symptoms was unaltered compared to the wild-type strain. These results exclude an important role for cutinase A during direct penetration of host tissue by B. cinerea.

scholarly journals Mating System in the Brown Rot Pathogens Monilinia fructicola, M. laxa, and M. fructigena

2018 ◽  
Vol 108 (11) ◽  
pp. 1315-1325 ◽  
Author(s):  
Domenico Abate ◽  
Rita M. De Miccolis Angelini ◽  
Caterina Rotolo ◽  
Stefania Pollastro ◽  
Francesco Faretra
Keyword(s):  
Mating System ◽  
Population Biology ◽  
Constitutive Expression ◽  
Brown Rot ◽  
Antisense Transcription ◽  
Inter Simple Sequence Repeat ◽  
Monilinia Fructicola ◽  
Specific Pcr ◽  
Rot Disease ◽  
Flanking Regions

Monilinia fructicola, M. laxa, and M. fructigena are the most important pathogens responsible for brown rot disease of stone and pome fruits. Information on their mating system and sexual behavior is scant. A mating-type-specific PCR-based assay was developed and applied to 155 Monilinia isolates from 10 countries and 10 different host plants. We showed that single isolates carry only one of two opposite idiomorphs at the MAT1 locus consistent with a heterothallic mating system for all three species. MAT1-1 and MAT1-2 mating types were detected in similar proportions in samples of isolates of each species and hence there do not appear to be genetic obstacles to the occurrence of sexual reproduction in their populations. Inter simple sequence repeat markers suggested that asexual reproduction is prevalent, but that sexual recombination occurs in M. fructicola populations in Italy. The genetic architectures of the MAT1 loci of the three pathogens were analyzed. MAT1-1 and MAT1-2 idiomorphs are flanked upstream and downstream by the APN2 and SLA2 genes and resemble those of Botrytis cinerea and other heterothallic fungi in the family Sclerotiniaceae. Each idiomorph contains a specific couple of genes, MAT1-1-1 (with alpha-box domain) and MAT1-1-5 in MAT1-1, and MAT1-2-1 (with HMG-box domain) and MAT1-2-10 in MAT1-2. Small gene fragments (dMAT1-1-1 and dMAT1-2-1) from the opposite idiomorph were detected close to their flanking regions. Constitutive expression of the four MAT1 genes during vegetative growth was ascertained by transcriptomic analysis (RNA-Seq). Antisense transcription of the MAT1-1-1 and MAT1-2-1 genes and intergenic transcribed regions of the MAT1 locus were detected. These results represent new insights into the mating systems of these three economically-important pathogens which could contribute to improve the knowledge on their population biology.

scholarly journals Detection and Identification of Six Monilinia spp. Causing Brown Rot Using TaqMan Real-Time PCR from Pure Cultures and Infected Apple Fruit

Plant Disease ◽  
2018 ◽  
Vol 102 (8) ◽  
pp. 1527-1533 ◽  
Author(s):  
Jing-ru Wang ◽  
Li-yun Guo ◽  
Chang-lin Xiao ◽  
Xiao-qiong Zhu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Related Species ◽  
Severe Disease ◽  
Brown Rot ◽  
Apple Fruit ◽  
Monilinia Fructicola ◽  
The Real ◽  
Pcr Assays ◽  
Monilia Polystroma

Brown rot is a severe disease affecting stone and pome fruit. This disease was recently confirmed to be caused by the following six closely related species: Monilinia fructicola, M. laxa, M. fructigena, Monilia polystroma, M. mumecola, and M. yunnanensis. Because of differences in geographic distributions, some of these species are important quarantine pathogens in certain countries. In this study, we developed TaqMan real-time polymerase chain reaction (PCR) assays to detect and identify the six species. Primer pairs and probes were designed for Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma based on sequence differences in the laccase-2 genes. Additionally, based on sequence differences in the elongation factor genes, primer pairs and probes were designed for Monilia mumecola and M. yunnanensis. The real-time PCR assays were able to specifically identify the target pathogens, with detection limits of 10 to 100 fg of DNA, which is equivalent to one to seven conidia. The assays were also able to detect the target pathogens in a mixed DNA sample comprising all six Monilinia spp. and related species. The real-time PCR assays accurately detected target fungi from infected apple fruit. Furthermore, the identification results were consistent with those of traditional morphological methods.

scholarly journals The Point Mutation G461S in the MfCYP51 Gene is Associated with Tebuconazole Resistance in Monilinia fructicola Populations in Brazil

2017 ◽  
Vol 107 (12) ◽  
pp. 1507-1514 ◽  
Author(s):  
Paulo S. F. Lichtemberg ◽  
Yong Luo ◽  
Rafael G. Morales ◽  
Juliana M. Muehlmann-Fischer ◽  
Themis J. Michailides ◽  
...  
Keyword(s):  
Gene Expression ◽  
Point Mutation ◽  
Molecular Mechanisms ◽  
Brown Rot ◽  
Polymerase Chain Reaction Method ◽  
Stone Fruit ◽  
Monilinia Fructicola ◽  
Nucleotide Position ◽  
Gene Encoding ◽  
Elevated Expression

The ascomycete Monilinia fructicola is the causal agent of brown rot of stone fruit in Brazil, causing major pre- and postharvest losses. For many years, the demethylation inhibitor (DMI) fungicide tebuconazole has been used as the most effective active ingredient for controlling brown rot and, as a result, strains of M. fructicola resistant to this ingredient have emerged in many Brazilian states producing stone fruit. The aim of this study was to investigate the mechanisms associated with the resistance of M. fructicola to DMI tebuconazole. By sequencing the M. fructicola CYP51 (MfCYP51) gene, encoding the azole target sterol 14α-demethylase, a mutation was identified at the nucleotide position 1,492, causing the amino acid substitution from glycine to serine at the codon position 461, associated with reduced tebuconazole sensitivity. In addition, it was observed that MfCYP51 gene expression could play a secondary role in DMI fungicide resistance of M. fructicola strains in Brazil. However, for the specific isolate found to exhibit elevated expression levels of MfCYP51, no insertions that would trigger gene expression were found. Based on the point mutation associated with tebuconazole resistance, an allele-specific polymerase chain reaction method was developed to quickly identify resistant genotypes within the Brazilian population. This is the first report determining molecular mechanisms for DMI resistance identification for M. fructicola isolates from Brazil. This information provides an important advancement for risk assessment of DMI fungicides used to manage brown rot of stone fruit.

Factors determining the length of the incubation period of Monilinia fructicola (wint.) Honey in fruits of Prunus spp.

1963 ◽  
Vol 14 (1) ◽  
pp. 51 ◽  
Author(s):  
JB Corbin
Keyword(s):  
Relative Humidity ◽  
Incubation Period ◽  
Brown Rot ◽  
Monilinia Fructicola ◽  
Inoculum Potential ◽  
Prunus Species ◽  
Brown Rot Fungus ◽  
Time Required ◽  
Dry Out ◽  
Prunus Spp

The length of the median incubation period of Monilinia fruticola (Wint.) Honey was determined on fruits of several Prunus species at various maturity, injury, and inoculum levels; the median incubation period being defined as the interval between application of conidia to fruits and the first appearance of sporodochia on 50% of fruits. Results were expressed as the time required for 20% and 50% of fruits to produce sporodochia. The following points were demonstrated: (1) Differences in the median incubation period between varieties for similar inoculum dosage were greatest when fruits were least matured and non-injured. (2) These differences became less apparent as inoculum dosage increased, as fruits matured, and as skin injuries increased. (3) Green fruits could be infected by high inoculum dosages when non-injured and by low inoculum dosages when injured. (4) Ripe fruits could be infected by low inoculum dosages whether non-injured or injured. (5) Free ("precipitated") moisture was necessary for infection. High relative humidity (R.H.) maintained this moisture after application of spore suspensions, thus permitting infection, whilst low R.H. allowed this moisture film to dry out, preventing infection. (6) After infection occurred, the R.H. level did not appear to affect the sporulation intensity.The above results are discussed in relation to the concept of inoculum potential, as defined by Garrett (1956), and to the development of epiphytotics and other aspects of the epidemiology of the brown rot fungus.

scholarly journals First Report of Brown Rot on Apple Fruit Caused by Monilinia fructicola in Pennsylvania

Plant Disease ◽  
2015 ◽  
Vol 99 (8) ◽  
pp. 1179-1179 ◽  
Author(s):  
K. A. Peter ◽  
V. L. Gaskins ◽  
B. Lehman ◽  
W. M. Jurick
Keyword(s):  
Brown Rot ◽  
Apple Fruit ◽  
Monilinia Fructicola ◽  
First Report

scholarly journals Induction, Regulation, and Role in Pathogenesis of Appressoria in Monilinia fructicola

2006 ◽  
Vol 96 (10) ◽  
pp. 1072-1080 ◽  
Author(s):  
Miin-Huey Lee ◽  
Richard M. Bostock
Keyword(s):  
Brown Rot ◽  
Large Degree ◽  
Immature Stage ◽  
Epidermal Cells ◽  
Monilinia Fructicola ◽  
Appressorium Formation ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Prunus Spp

Monilinia fructicola, which causes brown rot in stone fruit, forms appressoria on plant and artificial surfaces. On nectarine, the frequency of appressoria produced by conidial germlings depends to a large degree on the stage of fruit development, with numerous appressoria formed on immature (stage II) nectarine fruit, and no appressoria observed on fully mature fruit (late stage III). On polystyrene surfaces, appressorium formation was increased from <10% of germinated conidia to >95% of germinated conidia when the conidia were washed to remove residual nutrients and self-inhibitors. M. fructicola appressorium formation also appears to be regulated by the topography of the plant surface. On fruit, appressoria formed on stomatal guard cell lips, on the grooves of lateral cells adjacent to stomata or between two epidermal cells, and on the convex surfaces of epidermal cells. Pharmacological effectors indicate that cyclic AMP-, MAP kinase-, and calcium/calmodulin-dependent signaling pathways are involved in the induction and development of appressoria. KN-93, an inhibitor of calmodulin-dependent protein kinase II, did not inhibit conidial germination but did inhibit appressorium formation and brown rot development on flower petals, suggesting that appressoria are required for full symptom development on Prunus spp. petals.

Improved electroporation buffer enhances transient gene expression in Arachis hypogaea protoplasts

Genome ◽  
1995 ◽  
Vol 38 (5) ◽  
pp. 858-863 ◽  
Author(s):  
Zhijian Li ◽  
Ming Cheng ◽  
James W. Demski ◽  
Robert L. Jarret
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Arachis Hypogaea ◽  
Transient Gene Expression ◽  
Gus Expression ◽  
Histochemical Staining ◽  
Media Analysis ◽  
Gus Activity ◽  
Arachis Hypogaea L ◽  
Protoplast Viability

An electroporation medium containing 50 mM glycine or 10 mM glycylglycine (glygly), 70 mM potassium glutamate, and 0.4 M mannitol was evaluated for its ability to improve transient β-glucuronidase (GUS) expression in immature cotyledonary protoplasts of Arachis hypogaea L. GUS activity in electroporated protoplasts was 8- to 430-fold greater than that obtained using any of other four commonly employed poration media. Analysis of viability and histochemical staining of protoplasts indicated that electroporation using the glycine- or glygly-based poration medium resulted in increased protoplast viability and GUS expression when compared with other poration media. Replacement of glygly with MES or HEPES buffers significantly reduced the level of GUS expression in electroporated protoplasts.Key words: transient expression, electroporation, Arachis, protoplasts, GUS.

scholarly journals Characterization of two cis-acting elements, P1BS and W-box, in the regulation of OsPT6 responsive to phosphors deficiency

2021 ◽  
Author(s):  
Qingchun Zhao ◽  
Zhenzhen Luo ◽  
Jiadong Chen ◽  
Hongfang Jia ◽  
Penghui Ai ◽  
...  
Keyword(s):  
Gus Expression ◽  
Element Analysis ◽  
P Deficiency ◽  
Targeted Deletion ◽  
Cis Acting ◽  
Gus Reporter ◽  
Pi Starvation ◽  
Expression Activity ◽  
Underlying Mechanisms ◽  
Diverse Plant Species

AbstractPhosphorus (P) deficiency is one of the major nutrient stresses restricting plant growth. The uptake of P by plants from soil is mainly mediated by the phosphate (Pi) transporters belonging to the PHT1 family. Multiple PHT1 genes from diverse plant species have been shown to be strongly up-regulated upon Pi starvation, however, the underlying mechanisms for the Pi-starvation-induced (PSI) up-regulation have not been well deciphered for most Pi transporter genes. Here, we reported a detailed dissection of the promoter activity of a PSI rice Pi transporter gene OsPT6, using the β-glucuronidase (GUS) reporter gene. OsPT6 promoter could drive GUS expression strongly in both roots and blades of rice plants grown under low P, but not high P. Cis-acting element analysis identified one copy of the P1BS motif and two copies of the W-box motif in OsPT6 promoter. Targeted deletion of the P1BS motif caused almost complete abolition of GUS induction in response to Pi starvation, irrespective of the presence or absence of the W-box motif, Four repeats of the P1BS motif fused to the CaMV35S minimal promoter was sufficient to induce GUS expression responsive to Pi starvation. Targeted deletion of the upstream W-box motif (W1) did not affect the GUS expression activity compared with the full-length OsPT6 promoter, while targeted deletion of the downstream W-box motif (W2) or both of the W-box motifs remarkably reduced the GUS induction rate upon Pi starvation. Our results proposed that the PSI response of OsPT6 was positively regulated by at least two elements, the sole P1BS and the downstream W-box, in its promoter, and the W-box-mediated up-regulation of OsPT6 might be highly dependent on the P1BS motif.

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