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

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.

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Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

Scientific Reports ◽

10.1038/s41598-020-80213-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ruiqi Wang ◽

Kun Li ◽

Jifang Yu ◽

Jiaoyu Deng ◽

Yaokai Chen

Keyword(s):

Mycobacterium Tuberculosis ◽

Type Strain ◽

Wild Type Strain ◽

Clinical Isolates ◽

Aminobenzoic Acid ◽

Wild Type ◽

Dihydropteroate Synthase ◽

Expression Levels ◽

Encoding Gene ◽

Increased Susceptibility

AbstractPrevious studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-2-0176 ◽

2010 ◽

Vol 23 (2) ◽

pp. 176-186 ◽

Cited By ~ 28

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.

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Exploring the Pathogenicity of Pseudomonas brassicacearum Q8r1-96 and Other Strains of the Pseudomonas fluorescens Complex on Tomato

Plant Disease ◽

10.1094/pdis-09-19-1989-re ◽

2020 ◽

Vol 104 (4) ◽

pp. 1026-1031 ◽

Cited By ~ 2

Author(s):

Mingming Yang ◽

Dmitri V. Mavrodi ◽

Olga V. Mavrodi ◽

Linda S. Thomashow ◽

David M. Weller

Keyword(s):

Pacific Northwest ◽

Type Strain ◽

Wild Type Strain ◽

Acc Deaminase ◽

The United States ◽

Similar Amount ◽

Wild Type ◽

Tomato Fruits ◽

Plant Microbe Interaction ◽

Pseudomonas Brassicacearum

Pseudomonas brassicacearum and related species of the P. fluorescens complex have long been studied as biocontrol and growth-promoting rhizobacteria involved in suppression of soilborne pathogens. We report here that P. brassicacearum Q8r1-96 and other 2,4-diacetylphloroglucinol (DAPG)-producing fluorescent pseudomonads involved in take-all decline of wheat in the Pacific Northwest of the United States can also be pathogenic to other plant hosts. Strain Q8r1-96 caused necrosis when injected into tomato stems and immature tomato fruits, either attached or removed from the plant, but lesion development was dose dependent, with a minimum of 106 CFU ml−1 required to cause visible tissue damage. We explored the relative contribution of several known plant-microbe interaction traits to the pathogenicity of strain Q8r1-96. Type III secretion system (T3SS) mutants of Q8r1-96, injected at a concentration of 108 CFU ml−1, were significantly less virulent, but not consistently, as compared with the wild-type strain. However, a DAPG-deficient phlD mutant of Q8r1-96 was significantly and consistently less virulent as compared with the wild type. Strain Q8r1-96acc, engineered to over express ACC deaminase, caused a similar amount of necrosis as the wild type. Cell-free culture filtrates of strain Q8r1-96 and pure DAPG also cause necrosis in tomato fruits. Our results suggest that DAPG plays a significant role in the ability of Q8r1-96 to cause necrosis of tomato tissue, but other factors also contribute to the pathogenic properties of this organism.

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DNA Methylation on N6-Adenine Regulates the Hyphal Development during Dimorphism in the Early-Diverging Fungus Mucor lusitanicus

Journal of Fungi ◽

10.3390/jof7090738 ◽

2021 ◽

Vol 7 (9) ◽

pp. 738

Author(s):

Macario Osorio-Concepción ◽

Carlos Lax ◽

Eusebio Navarro ◽

Francisco E. Nicolás ◽

Victoriano Garre

Keyword(s):

Wild Type Strain ◽

Pathogenic Fungi ◽

Infection Process ◽

Deletion Mutants ◽

Polar Growth ◽

Wild Type ◽

C Elegans ◽

Double Deletion ◽

Multiple Processes ◽

Causative Agents

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process.

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DNA Methylation on N6-Adenine Regulates the Hyphal Development During Dimorphism in the Early-Diverging Fungus Mucor lusitanicus

10.20944/preprints202107.0525.v1 ◽

2021 ◽

Author(s):

Macario Osorio-Concepción ◽

Carlos Lax ◽

Eusebio Navarro ◽

Francisco E. Nicolás ◽

Victoriano Garre

Keyword(s):

Wild Type Strain ◽

Pathogenic Fungi ◽

Infection Process ◽

Deletion Mutants ◽

Polar Growth ◽

Wild Type ◽

C Elegans ◽

Double Deletion ◽

Multiple Processes ◽

Causative Agents

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales; causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxyadenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins homologs to C. elegans NMAD-1 and ALKBH1, respectively. The function of the three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase of 6mA levels during the dimorphic transition, suggesting that 6mA regulates dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction of polar growth, suggesting that 6mA also has a role during the spore-macrophage interaction that could be important in the infection process.

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Cysteine desulphurase-encoding gene sufS2 is required for the repressor function of RirA and oxidative resistance in Agrobacterium tumefaciens

Microbiology ◽

10.1099/mic.0.068643-0 ◽

2014 ◽

Vol 160 (1) ◽

pp. 79-90 ◽

Cited By ~ 20

Author(s):

Sakkarin Bhubhanil ◽

Phettree Niamyim ◽

Rojana Sukchawalit ◽

Skorn Mongkolsuk

Keyword(s):

Agrobacterium Tumefaciens ◽

Wild Type Strain ◽

Physiological Function ◽

Response Regulator ◽

Cluster Assembly ◽

Wild Type ◽

Oxidative Resistance ◽

Small Colony ◽

Encoding Gene ◽

Assembly Proteins

The Agrobacterium tumefaciens genome contains a cluster of genes that are predicted to encode Fe–S cluster assembly proteins, and this cluster is known as the sufS2BCDS1XA operon. sufS2 is the first gene in the operon, and it was inactivated to determine its physiological function. The sufS2 mutant exhibited a small colony phenotype, grew slower than the wild-type strain and was more sensitive to various oxidants including peroxide, organic hydroperoxide and superoxide. The sufS2 gene was negatively regulated by iron response regulator (Irr) and rhizobial iron regulator (RirA) under low and high iron conditions, respectively, and was inducible in response to oxidative stress. The oxidant-induced expression of sufS2 was controlled by Irr, RirA and an additional but not yet identified mechanism. sufS2 was required for RirA activity in the repression of a sufS2 promoter-lacZ fusion. RirA may use Fe–S as its cofactor. sufS2 disruption may cause a defect in the Fe–S supply and could thereby affect the RirA activity. The three conserved cysteine residues (C91, C99 and C105) in RirA were predicted to coordinate with the Fe–S cluster and were shown to be essential for RirA repression of the sufS2-lacZ fusion. These results suggested that sufS2 is important for the survival of A. tumefaciens.

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The Role of G Protein Alpha Subunits in the Infection Process of the Gray Mold Fungus Botrytis cinerea

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.11.1293 ◽

2001 ◽

Vol 14 (11) ◽

pp. 1293-1302 ◽

Cited By ~ 150

Author(s):

Christian Schulze Gronover ◽

Daniela Kasulke ◽

Paul Tudzynski ◽

Bettina Tudzynski

Keyword(s):

Botrytis Cinerea ◽

Magnaporthe Grisea ◽

Gray Mold ◽

Infection Process ◽

Colony Morphology ◽

Gene Copy ◽

Wild Type ◽

Mold Fungus ◽

Protease Secretion

To identify signal transduction pathways of the gray mold fungus Botrytis cinerea involved in host infection, we used heterologous hybridization and a polymerase chain reaction (PCR)-based approach to isolate two genes (bcg1 and bcg2) encoding α subunits of heterotrimeric GTP-binding proteins. Both genes have homologues in other fungi: bcg1 is a member of the Gαi class, whereas bcg2 has similarities to the magC gene of Magnaporthe grisea and the gna-2 gene of Neurospora crassa. Reverse-transcription (RT)-PCR experiments showed clearly that both genes are expressed at very early stages in infected bean leaves. Gene replacement experiments were performed for both genes. bcg1 null mutants differ in colony morphology from the wild-type strain, do not secrete extracellular proteases, and show clearly reduced pathogenicity on bean and tomato. Conidia germination and penetration of plant tissue is not disturbed in bcg1 mutants, but the infection process stops after formation of primary lesions. In contrast, bcg2 mutants show wild-type colony morphology in axenic culture and are only slightly reduced in pathogenicity. Complementation of bcg1 mutants with the wild-type gene copy led to the full recovery of colony morphology, protease secretion, and pathogenicity on both host plants. Application of exogenous cyclic AMP restored the wild-type growth pattern of bcg1 mutants, but not the protease secretion, implicating an essential role of BCG1 in different signaling pathways.

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F240 of β2-Tubulin Explains why Fusarium graminearum is Less Sensitive to Carbendazim than Botrytis cinerea

Phytopathology ◽

10.1094/phyto-09-17-0295-r ◽

2018 ◽

Vol 108 (3) ◽

pp. 352-361 ◽

Cited By ~ 8

Author(s):

Yuanye Zhu ◽

Xiaoyu Liang ◽

Yanjun Li ◽

Yabing Duan ◽

Zhitian Zheng ◽

...

Keyword(s):

Botrytis Cinerea ◽

Binding Affinity ◽

Fusarium Graminearum ◽

Type Strain ◽

Wild Type Strain ◽

Fluorescent Protein ◽

Amino Acid Sequences ◽

Wild Type ◽

Benzimidazole Fungicides ◽

Β Tubulin

β-Tubulin is the target of benzimidazole fungicides, the most widely used of which is carbendazim (methyl benzimidazol-2-ylcarbamate [MBC]). MBC sensitivity is determined by the differential affinity of MBC for β-tubulins. However, the mechanism of less sensitivity of Fusarium graminearum to MBC compared with other fungi, including Botrytis cinerea, Colletotrichum gloeosporioides, and Sclerotinia sclerotiorum, remains exclusive. Alignment of β-tubulin amino acid sequences showed that position 240 of β-tubulins is leucine (L) in most pathogenic fungi but is phenylalanine (F) in the Fgβ2-tubulin of the F. graminearum wild type. The effective concentration resulting in 50% inhibition (EC50) value of MBC against the Fgβ2F240L mutant of F. graminearum is 0.047 μg/ml, which was 10-fold lower than that of wild-type strain 2021. Moreover, The EC50 value of MBC against the BcβL“240”F (actually position 232) mutant of Botrytis cinerea was 0.44 μg/ml, which was ninefold higher than that of B. cinerea wild-type strain Bt4-1. In response to MBC treatment (0.15 μg/ml), microtubules were clearly visible in Fgβ2-enhanced green fluorescent protein (EGFP) but not in Fgβ2F240L-EGFP. Moreover, a molecular docking assay indicated that F240L mutation created a pi-pi interaction between Fgβ2-tubulin and MBC and increased the binding affinity of Fgβ2-tubulin to MBC. Our results suggest that F240 is responsible for the naturally less MBC sensitivity in F. graminearum compared with B. cinerea, C. gloeosporioides, and S. sclerotiorum by decreasing the binding affinity between Fgβ2-tubulin and MBC.

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XagR, a LuxR Homolog, Contributes to the Virulence of Xanthomonas axonopodis pv. glycines to Soybean

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-12-0008-r ◽

2012 ◽

Vol 25 (8) ◽

pp. 1104-1117 ◽

Cited By ~ 34

Author(s):

Tiyakhon Chatnaparat ◽

Sutruedee Prathuangwong ◽

Michael Ionescu ◽

Steven E. Lindow

Keyword(s):

Type Strain ◽

Transcriptional Control ◽

Wild Type Strain ◽

Infection Process ◽

Wild Type ◽

In Planta ◽

Xanthomonas Axonopodis ◽

Luxr Homolog ◽

Dry Conditions ◽

Soybean Leaves

A novel luxR homolog, termed XagR, in Xanthomonas axonopodis pv. glycines, the cause of soybean pustule, controls expression of pip, yapH, and at least 77 other genes. Although XagR and Pip are required for full virulence of X. axonopodis pv. glycines to soybean, constitutive overproduction of XagR suppresses infection. The xagR-dependent induction of pip occurs in planta only 2 days or more after inoculation. Although the transcription of xagR appears constitutive, XagR accumulates only in cells that have colonized soybean plants for more than 2 days suggesting that some components produced during the infection process mediate post-transcriptional control, likely by protecting XagR from proteolytic degradation. XagR modulates the adhesiveness of the pathogen during the infection process by suppressing the adhesin YapH. Although yapH mutants incite more infections of soybean leaves than the wild-type strain when topically applied under dry conditions, the mutant causes fewer infections when leaves are subject to simulated rain events after inoculation. Likewise, yapH mutants and cells in which XagR was overexpressed exhibited much more egress from infected leaves than the wild-type strain. Thus, XagR differentially modulates expression of a variety of genes during the infection process in response to feedback from plant molecules elaborated during infection to coordinate processes such as invasion, infection, and cell egress needed to complete the disease cycle.

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Biological Control of Grape Crown Gall by Strain F2/5 Is Not Associated with Agrocin Production or Competition for Attachment Sites on Grape Cells

Phytopathology ◽

10.1094/phyto.1997.87.7.706 ◽

1997 ◽

Vol 87 (7) ◽

pp. 706-711 ◽

Cited By ~ 27

Author(s):

T. J. Burr ◽

C. L. Reid ◽

E. Tagliati ◽

C. Bazzi ◽

S. Süle

Keyword(s):

Biological Control ◽

Crown Gall ◽

Virulence Genes ◽

Wild Type Strain ◽

Wild Type ◽

Gall Formation ◽

Agrobacterium Vitis ◽

Gus Reporter ◽

Attachment Sites ◽

Control Transfer

Agrocin-minus mutants of nontumorigenic Agrobacterium vitis strain F2/5 controlled grape crown gall as well as the wild-type strain, indicating that agrocin is not a major factor in the mechanism of biological control. Relative levels of attachment to grape cells by tumorigenic and biocontrol strains were also measured. Attachment of tumorigenic strains (CG49 and K306) and biological control strains (F2/5 and agrocin-minus mutant 1077) was often reduced when mixtures of the strains were applied. However, high populations (103 to 105 CFU/ml) of all strains attached following mixed inoculations, suggesting that competition for attachment sites is also not a factor in the mechanism of biological control. Transfer of T-DNA to grape by CG49 was prevented or greatly inhibited in the presence of F2/5 or 1077 as measured by expression of the GUS reporter gene. The Ti plasmid virulence genes, however, were induced by exudates from grape shoots that had been inoculated with F2/5. Sonicated and autoclaved preparations of F2/5 and 1077 did not control crown gall or inhibit T-DNA transfer. Control by F2/5 is specific to grape, since gall formation on tomato, sunflower, and Kalanchoe daigremontiana were not inhibited.

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