Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogen Ascochyta rabiei

AbstractPolarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during the hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified an F-BAR domain protein (ArF-BAR) in the necrotrophic fungus Ascochyta rabiei and demonstrate its involvement in endosome-dependent fungal virulence on the host plant, Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is essential for virulence. The loss-of-function of ArF-BAR results in delayed formation of first septa from the hyphal tip, crucial for host penetration and proliferation. ArF-BAR was induced in response to oxidative stress and infection and localized to endocytic vesicles within the fungal hyphae. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formation in vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1) as key regulator for transcriptional reprogramming of ArF-BAR. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate fungal pathogenesis through a mechanism that involves membrane remodeling and actin cytoskeleton regulation.Author summaryBAR-domain superfamily is known to mold amorphous lipid bilayer into defined tubular shapes and critical for endosome formation and trafficking. Although these processes are studied earlier in the context of their structural and biochemical properties, there is limited evidence on the direct role of F-BAR domain proteins in the pathophysiological development of other economically important fungi. Our study assumes functional significance for plant infection as we identified an F-BAR domain-containing protein that is regulated by a distinct transcriptional network. We characterized F-BAR in a necrotrophic fungal pathogen, Ascochyta rabiei that causes the Ascochyta blight (AB) disease in chickpea plants. Additionally, we have also identified a calcium-regulated CRZ1 transcription factor that regulates the transcription of ArF-BAR. Our study will help to understand the complex interplay underlying the endosome formation required for fungal virulence.

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Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogen Ascochyta rabiei

PLoS Genetics ◽

10.1371/journal.pgen.1009137 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1009137

Author(s):

Manisha Sinha ◽

Ankita Shree ◽

Kunal Singh ◽

Kamal Kumar ◽

Shreenivas Kumar Singh ◽

...

Keyword(s):

Gene Knockout ◽

Hyphal Growth ◽

Actin Dynamics ◽

Ascochyta Rabiei ◽

Endoplasmic Reticulum Membrane ◽

Fungal Virulence ◽

Bar Domain ◽

Early Endosomes ◽

Host Penetration ◽

Hyphal Tip

Polarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified a F-BAR domain protein (ArF-BAR) in the necrotrophic fungus Ascochyta rabiei and demonstrate its involvement in endosome-dependent fungal virulence on the host plant Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is necessary for virulence. The loss-of-function of ArF-BAR gene results in delayed formation of apical septum in fungal cells near growing hyphal tip that is crucial for host penetration, and impaired secretion of a candidate effector having secretory signal peptide for translocation across the endoplasmic reticulum membrane. The mRNA transcripts of ArF-BAR were induced in response to oxidative stress and infection. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formation in vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1), as key transcriptional regulator of ArF-BAR expression. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate A. rabiei virulence through a mechanism that involves endocytosis, effector secretion, and actin cytoskeleton regulation.

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Role of Transcription Factor CaNdt80p in Cell Separation, Hyphal Growth, and Virulence in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00325-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 634-644 ◽

Cited By ~ 47

Author(s):

Adnane Sellam ◽

Christopher Askew ◽

Elias Epp ◽

Faiza Tebbji ◽

Alaka Mullick ◽

...

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Candida Albicans ◽

Cell Separation ◽

Hyphal Growth ◽

Functional Domain ◽

Transcription Factor Family ◽

Content Type ◽

Genes Encoding

ABSTRACT The NDT80/PhoG transcription factor family includes ScNdt80p, a key modulator of the progression of meiotic division in Saccharomyces cerevisiae. In Candida albicans, a member of this family, CaNdt80p, modulates azole sensitivity by controlling the expression of ergosterol biosynthesis genes. We previously demonstrated that CaNdt80p promoter targets, in addition to ERG genes, were significantly enriched in genes related to hyphal growth. Here, we report that CaNdt80p is indeed required for hyphal growth in response to different filament-inducing cues and for the proper expression of genes characterizing the filamentous transcriptional program. These include noteworthy genes encoding cell wall components, such as HWP1, ECE1, RBT4, and ALS3. We also show that CaNdt80p is essential for the completion of cell separation through the direct transcriptional regulation of genes encoding the chitinase Cht3p and the cell wall glucosidase Sun41p. Consistent with their hyphal defect, ndt80 mutants are avirulent in a mouse model of systemic candidiasis. Interestingly, based on functional-domain organization, CaNdt80p seems to be a unique regulator characterizing fungi from the CTG clade within the subphylum Saccharomycotina. Therefore, this study revealed a new role of the novel member of the fungal NDT80 transcription factor family as a regulator of cell separation, hyphal growth, and virulence.

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Role of Ammonia Secretion and pH Modulation on Pathogenicity of Colletotrichum coccodes on Tomato Fruit

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-8-1058 ◽

2008 ◽

Vol 21 (8) ◽

pp. 1058-1066 ◽

Cited By ~ 45

Author(s):

N. Alkan ◽

R. Fluhr ◽

A. Sherman ◽

D. Prusky

Keyword(s):

Tomato Fruit ◽

Critical Factor ◽

Colletotrichum Coccodes ◽

Infection Site ◽

Fungal Virulence ◽

A Value ◽

Ammonia Accumulation ◽

Ph Buffer ◽

Hyphal Tip

Colletotrichum coccodes was found to alkalinize the decaying tissue of tomato fruit via accumulation and secretion of ammonia. Alkalinization dynamics caused by ammonia secretion from growing hyphae was examined microscopically using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein. Values of pH of 7.9 observed in the host tissue close to the hyphal tips declined to pH 6.0 at 10 mm away from the hyphal tip, which was a value that was still higher than that detected in the healthy tissue, pH 4.2. Ammonia accumulation at the infection site depended on the initial environmental pH. Treatments with low (4.0) pH buffer at the infection site resulted in high levels of ammonia secretion and increased virulence of C. coccodes compared with similar treatments with buffer at pH 7.0. Significantly, mutants of C. coccodes defective in nitrogen utilization, nit–, and areA– were impaired in ammonia secretion and showed reduced decay development. The reduced infection rate of nit– mutants could be complemented by adding glutamine at the infection site. Thus, ammonia accumulation is a critical factor contributing to C. coccodes pathogenicity on tomato fruit. The results show that the initial acidic pH of the fruit is conducive to ammonia secretion and the subsequent alkalinization of the infection site, and facilitates fungal virulence and the transformation from the quiescent-biotrophic to active-necrotrophic state.

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The WOPR family protein Ryp1 is a key regulator of gene expression, development, and virulence in the thermally dimorphic fungal pathogen Coccidioides posadasii

10.1101/2021.07.26.453804 ◽

2021 ◽

Author(s):

Alejandra Mandel ◽

Sinem Beyhan ◽

Mark Voorhies ◽

Lisa F. Shubitz ◽

John Galgiani ◽

...

Keyword(s):

Transcription Factor ◽

Fungal Pathogens ◽

Hyphal Growth ◽

Outer Wall ◽

Coccidioides Posadasii ◽

Family Protein ◽

Regulated Expression ◽

Isotropic Expansion ◽

Mutant Cells

Coccidioides spp. are mammalian fungal pathogens endemic to the southwestern US and other desert regions of Mexico, central and South America, with the bulk of US infections occurring in California and Arizona. In the soil, Coccidioides grows in a hyphal form that differentiates into 3-5 micron asexual spores (arthroconidia). When arthroconidia are inhaled by mammals they undergo a unique developmental transition from polar hyphal growth to isotropic expansion with multiple rounds of nuclear division, prior to segmentation, forming large spherules filled with endospores. Very little is understood about the molecular basis of spherule formation. Here we characterize the role of the conserved transcription factor Ryp1 in Coccidioides development. We show that Coccidioides Δryp1 mutants have altered colony morphology under hypha-promoting conditions and are unable to form mature spherules under spherule-promoting conditions. We analyze the transcriptional profile of wild-type and Δryp1 mutant cells under hypha- and spherule-promoting conditions, thereby defining a set of hypha- or spherule-enriched transcripts (“morphology-regulated” genes) that are dependent on Ryp1 for their expression. Forty percent of morphology-regulated expression is Ryp1-dependent, indicating that Ryp1 plays a dual role in both hyphal and spherule development. Ryp1-dependent transcripts include key virulence factors such as SOWgp, which encodes the spherule outer wall glycoprotein. Concordant with its role in spherule development, we find that the ?ryp1 mutant is completely avirulent in the mouse model of coccidioidomycosis, indicating that Ryp1-dependent pathways are essential for the ability of Coccidioides to cause disease. Vaccination of C57BL/6 mice with live Δryp1 spores does not provide any protection from lethal C. posadasii intranasal infection, consistent with our findings that the Δryp1 mutant fails to make mature spherules and likely does not express key antigens required for effective vaccination. Taken together, this work identifies the first transcription factor that drives mature spherulation and virulence in Coccidioides.

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The Role of BAR Domain Proteins in the Regulation of Membrane Dynamics

Acta Naturae ◽

10.32607/20758251-2016-8-4-60-69 ◽

2016 ◽

Vol 8 (4) ◽

pp. 60-69 ◽

Cited By ~ 18

Author(s):

T. B. Stanishneva-Konovalova ◽

N. I Derkacheva ◽

S. V. Polevova ◽

O. S. Sokolova

Keyword(s):

Membrane Dynamics ◽

Actin Dynamics ◽

Membrane Remodeling ◽

Bar Domain ◽

Cellular Processes ◽

Functional Differences ◽

Bar Domain Proteins ◽

Domain Protein ◽

Bar Domains

Many cellular processes are associated with membrane remodeling. The BAR domain protein family plays a key role in the formation and detection of local membrane curvatures and in attracting other proteins, including the regulators of actin dynamics. Based on their structural and phylogenetic properties, BAR domains are divided into several groups which affect membrane in various ways and perform different functions in cells. However, recent studies have uncovered evidence of functional differences even within the same group. This review discusses the principles underlying the interactions of different groups of BAR domains, and their individual representatives, with membranes.

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Understanding the Role of Amphipathic Helices in N-BAR Domain Driven Membrane Remodeling

Biophysical Journal ◽

10.1016/j.bpj.2012.12.006 ◽

2013 ◽

Vol 104 (2) ◽

pp. 404-411 ◽

Cited By ~ 67

Author(s):

Haosheng Cui ◽

Carsten Mim ◽

Francisco X. Vázquez ◽

Edward Lyman ◽

Vinzenz M. Unger ◽

...

Keyword(s):

Membrane Remodeling ◽

Bar Domain ◽

Amphipathic Helices

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Ahr1 and Tup1 Contribute to the Transcriptional Control of Virulence-Associated Genes in Candida albicans

mBio ◽

10.1128/mbio.00206-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 4

Author(s):

Sophia Ruben ◽

Enrico Garbe ◽

Selene Mogavero ◽

Daniela Albrecht-Eckardt ◽

Daniela Hellwig ◽

...

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Transcriptional Control ◽

Cell Damage ◽

Iron Acquisition ◽

Hyphal Growth ◽

Fungal Virulence ◽

Content Type ◽

Human Fungal Pathogen ◽

Full Expression

ABSTRACT The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1, which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in C. albicans. However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1. In contrast to NRG1, overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1. In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of ALS3 and ECE1 and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different C. albicans morphologies. IMPORTANCE Candida albicans is a major human fungal pathogen and the leading cause of systemic Candida infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of ALS3 and ECE1 in C. albicans hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in C. albicans.

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