Differential Gene Expression in Individual Papilla-Resistant and Powdery Mildew-Infected Barley Epidermal Cells

Resistance and susceptibility in barley to the powdery mildew fungus (Blumeria graminis f. sp. hordei) is determined at the single-cell level. Even in genetically compatible interactions, attacked plant epidermal cells defend themselves against attempted fungal penetration by localized responses leading to papilla deposition and reinforcement of their cell wall. This conveys a race-nonspecific form of resistance. However, this defense is not complete, and a proportion of penetration attempts succeed in infection. The resultant mixture of infected and uninfected leaf cells makes it impossible to relate powdery mildew-induced gene expression in whole leaves or even dissected epidermal tissues to resistance or susceptibility. A method for generating transcript profiles from individual barley epidermal cells was established and proven useful for analyzing resistant and successfully infected cells separately. Contents of single epidermal cells (resistant, infected, and unattacked controls) were collected, and after cDNA synthesis and PCR amplification, the resulting sample was hybridized to dot-blots spotted with genes, including some previously reported to be induced upon pathogen attack. Transcripts of several genes, (e.g., PR1a, encoding a pathogenesis related protein, and GLP4, encoding a germin-like protein) accumulated specifically in resistant cells, while GRP94, encoding a molecular chaperone, accumulated in infected cells. Thus, the single-cell method allows discrimination of transcript profiles from resistant and infected cells. The method will be useful for microarray expression profiling for simultaneous analysis of many genes.

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A Small GTP-Binding Host Protein Is Required for Entry of Powdery Mildew Fungus into Epidermal Cells of Barley

PLANT PHYSIOLOGY ◽

10.1104/pp.010805 ◽

2002 ◽

Vol 128 (4) ◽

pp. 1447-1454 ◽

Cited By ~ 97

Author(s):

Holger Schultheiss ◽

Cornelia Dechert ◽

Karl-Heinz Kogel ◽

Ralph Hückelhoven

Keyword(s):

Powdery Mildew ◽

Epidermal Cells ◽

Host Protein ◽

Powdery Mildew Fungus ◽

Gtp Binding

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Single-cell analysis of early antiviral gene expression reveals a determinant of stochasticIFNB1expression

Integrative Biology ◽

10.1039/c7ib00146k ◽

2017 ◽

Vol 9 (11) ◽

pp. 857-867 ◽

Cited By ~ 7

Author(s):

Sultan Doğanay ◽

Maurice Youzong Lee ◽

Alina Baum ◽

Jessie Peh ◽

Sun-Young Hwang ◽

...

Keyword(s):

Gene Expression ◽

Decision Making ◽

Single Cell ◽

Single Cell Analysis ◽

Decision Making Process ◽

Cell Analysis ◽

Early Expression ◽

Infected Cells ◽

Antiviral Gene

Early expression ofRIG-IandMDA5in a subset of infected cells may contribute to the decision making process for turning on theIFNB1expression.

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Defense reactions of barley cultivars to an inappropriate forma specialis of the powdery mildew fungus of gramineous plants

Canadian Journal of Botany ◽

10.1139/b84-287 ◽

1984 ◽

Vol 62 (10) ◽

pp. 2114-2117 ◽

Cited By ~ 27

Author(s):

Y. Tosa ◽

J. Shishiyama

Keyword(s):

Powdery Mildew ◽

Colony Growth ◽

Epidermal Cells ◽

Colony Development ◽

Powdery Mildew Fungus ◽

Barley Cultivars ◽

Cellular Defense ◽

Defense Reactions ◽

Forma Specialis ◽

Fluorescent Cells

Cellular defense reactions in five barley cultivars against Erysiphe graminis f. sp. tritici were examined in the course of primary and secondary penetrations. In cv. Kairyobozu-mugi, 35% of infection attempts were stopped at fluorescent papillae, and the others (65%) induced fluorescing of epidermal cells, resulting in the failure of the formation of primary haustoria. In the other cultivars ('H.E.S.4', 'Russian No. 12', 'Goseshikoku', and 'Turkey 290') the penetration failures associated with fluorescent papillae reached 50–75%, but the infection attempts that induced the fluorescing of epidermal cells were fewer than 20%. Consequently 10–30% of the germlings that attempted penetration successfully formed normal primary haustoria 48 h after inoculation. In cv. Goseshikoku, cv. Russian No. 12, and cv. H.E.S.4, 50–75% of the epidermal cells that contained the primary haustoria were fluorescent 7 days after inoculation, and colony growth was severely restricted. In cv. Turkey 290 such fluorescent cells scarcely occurred and colonies developed comparatively well. On this cultivar conidia were produced 5–6 days after inoculation, but only in small quantities. This restriction of colony development was mainly attributable to the inhibition of the formation of secondary haustoria by fluorescent papillae. These results indicate that there are differences among barley cultivars in cellular defense reactions against the wheat powdery mildew fungus and suggest that the formation of papillae during the course of primary penetration is not necessarily an essential factor in the resistance of barley to this inappropriate forma specialis.

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Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

Journal of Virology ◽

10.1128/jvi.00166-17 ◽

2017 ◽

Vol 91 (11) ◽

Cited By ~ 13

Author(s):

Tomasz Krzywkowski ◽

Sibel Ciftci ◽

Farzaneh Assadian ◽

Mats Nilsson ◽

Tanel Punga

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Single Cell ◽

Cell Population ◽

Rolling Circle Amplification ◽

Expression Patterns ◽

Human Adenovirus ◽

Viral Dna ◽

Rolling Circle ◽

Infected Cells

ABSTRACT An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.

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HSV-1 single cell analysis reveals anti-viral and developmental programs activation in distinct sub-populations

10.1101/566489 ◽

2019 ◽

Author(s):

Nir Drayman ◽

Parthiv Patel ◽

Luke Vistain ◽

Savaş Tay

Keyword(s):

Gene Expression ◽

Single Cell ◽

Single Cell Analysis ◽

Viral Gene Expression ◽

Population Level ◽

Cellular Reprogramming ◽

Viral Gene ◽

Beta Catenin ◽

Infected Cells ◽

Hsv 1

ABSTRACTViral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that they cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of beta-catenin to the host nucleus and viral replication compartments and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.

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Single-Cell Transcript Profiling of Barley Attacked by the Powdery Mildew Fungus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-3-0235 ◽

2007 ◽

Vol 20 (3) ◽

pp. 235-246 ◽

Cited By ~ 35

Author(s):

Torben Gjetting ◽

Peter H. Hagedorn ◽

Patrick Schweizer ◽

Hans Thordal-Christensen ◽

Timothy L. W. Carver ◽

...

Keyword(s):

Gene Expression ◽

Powdery Mildew ◽

Expression Profiles ◽

Cdna Array ◽

Gene Expression Profiles ◽

Powdery Mildew Fungus ◽

Nonspecific Resistance ◽

Susceptible Host ◽

Leaf Epidermal Cell ◽

Induced Changes

In many plant-pathogen interactions, there are several possible outcomes for simultaneous attacks on the same leaf. For instance, an attack by the powdery mildew fungus on one barley leaf epidermal cell may succeed in infection and formation of a functional haustorium, whereas a neighboring cell attacked at the same time may resist fungal penetration. To date, the mixed cellular responses seen even in susceptible host leaves have made it difficult to relate induced changes in gene expression to resistance or susceptibility in bulk leaf samples. By microextraction of cell-specific mRNA and subsequent cDNA array analysis, we have successfully obtained separate gene expression profiles for specific mildew-resistant and -infected barley cells. Thus, for the first time, it is possible to identify genes that are specifically regulated in infected cells and, presumably, involved in fungal establishment. Further, although much is understood about the genetic basis of effective papilla resistance associated with mutant mlo barley, we provide here the first evidence for gene regulation associated with effective papilla-based nonspecific resistance expressed in nominally “susceptible” wild-type barley.

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Tissue-Specific Superoxide Generation at Interaction Sites in Resistant and Susceptible Near-Isogenic Barley Lines Attacked by the Powdery Mildew Fungus (Erysiphe graminis f. sp. hordei)

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.4.292 ◽

1998 ◽

Vol 11 (4) ◽

pp. 292-300 ◽

Cited By ~ 95

Author(s):

Ralph Hückelhoven ◽

Karl-Heinz Kogel

Keyword(s):

Cell Death ◽

Powdery Mildew ◽

Mesophyll Cell ◽

Epidermal Cells ◽

Erysiphe Graminis ◽

Time Range ◽

Powdery Mildew Fungus ◽

Hypersensitive Cell Death ◽

Interaction Sites ◽

Nbt Reduction

The pathogenesis-related, azide-insensitive generation of superoxide anions (O2 -) was comparatively analyzed in near-isogenic barley (Hordeum vulgare cv. Pallas) lines carrying the powdery mildew (Erysiphe graminis f. sp. hordei) resistance genes Mla12, Mlg, and mlo5, respectively, by the microscopic detection of nitroblue tetra-zolium (NBT) reduction to dark blue formazan dyes. These genes govern fungal arrest at different stages of the interaction: (i) at the penetration stage within cell wall appositions (papillae) leaving the attacked cell alive (mlo); (ii) within papillae of cells that subsequently undergo a hypersensitive cell death (HR) (Mlg); or (iii) after penetration by a subsequent HR (Mla12). The susceptible parent line Pallas showed a transient O2 - generation in penetrated epidermal cells at 18 h after inoculation (hai), whereas epidermal cells of the resistant BCPMla12 produced O2 - over a longer time range (by 18 to 36 hai) preceding cell death. No oxidative burst was detected in association with penetration resistance due to effective papillae (BCPMlg and BCPmlo5) although Mlg specified an HR subsequent to fungal arrest. Hence, O2 - generation in attacked epidermal cells was a result of fungal penetration of the host cell walls and subsequent contact with the host plasma membrane, and not a requirement for HR elicitation. O2 - generation in the mesophyll tissue beneath attacked cells was associated with the response mediated by the genes Mla12 and Mlg. However, only BCPMla12 showed mesophyll cell death. The data indicate that, in barley, O2 - accumulation is not a single key determinant of HR in response to a powdery mildew attack.

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Exogenous Trehalose Induces Defenses in Wheat Before and During a Biotic Stress Caused by Powdery Mildew

Phytopathology ◽

10.1094/phyto-07-13-0191-r ◽

2014 ◽

Vol 104 (3) ◽

pp. 293-305 ◽

Cited By ~ 26

Author(s):

Christine Tayeh ◽

Béatrice Randoux ◽

Dorothée Vincent ◽

Natacha Bourdon ◽

Philippe Reignault

Keyword(s):

Gene Expression ◽

Powdery Mildew ◽

Lipid Transfer Protein ◽

Control Strategies ◽

Lipid Transfer ◽

Wheat Diseases ◽

Expression Of Genes ◽

Pathogenesis Related Protein ◽

Genes Encoding ◽

The Impact

Powdery mildew would be one of the most damaging wheat diseases without the extensive use of conventional fungicides. Some of the alternative control strategies currently emerging are based on the use of resistance inducers. The disacharride trehalose (TR) is classically described as an inducer of defenses in plants to abiotic stress. In this work, the elicitor or priming effect of TR was investigated in wheat both before and during a compatible wheat–powdery mildew interaction through molecular, biochemical, and cytological approaches. In noninoculated conditions, TR elicited the expression of genes encoding chitinase (chi, chi1, and chi4 precursor), pathogenesis-related protein 1, as well as oxalate oxidase (oxo). Moreover, lipid metabolism was shown to be altered by TR spraying via the upregulation of lipoxygenase (lox) and lipid-transfer protein (ltp)-encoding gene expression. On the other hand, the protection conferred by TR to wheat against powdery mildew is associated with the induction of two specific defense markers. Indeed, in infectious conditions following TR spraying, upregulations of chi4 precursor and lox gene expression as well as an induction of the LOX activity were observed. These results are also discussed with regard to the impact of TR on the fungal infectious process, which was shown to be stopped at the appressorial germ tube stage. Our findings strongly suggest that TR is a true inducer of wheat defense and resistance, at least toward powdery mildew.

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Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

Development ◽

10.1242/dev.087551 ◽

2013 ◽

Vol 140 (7) ◽

pp. 1433-1444 ◽

Cited By ~ 62

Author(s):

D. W. M. Tan ◽

K. B. Jensen ◽

M. W. B. Trotter ◽

J. T. Connelly ◽

S. Broad ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Epidermal Cells ◽

Functional Heterogeneity ◽

Human Epidermal Cells ◽

Cell Gene Expression ◽

Cell Gene

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First Report of Powdery Mildew Caused by Podosphaera xanthii on Salvia farinacea in Korea

Plant Disease ◽

10.1094/pdis-07-21-1427-pdn ◽

2021 ◽

Author(s):

In-Young Choi ◽

Ho-Jong Ju ◽

Kui-Jae Lee ◽

Hyeon-Dong Shin

Keyword(s):

Powdery Mildew ◽

Large Subunit ◽

Pcr Amplification ◽

Echinacea Purpurea ◽

The United States ◽

Infected Leaf ◽

Podosphaera Xanthii ◽

Powdery Mildew Fungus ◽

First Report ◽

Perennial Plant

Salvia farinacea Benth. (Lamiaceae) is an herbaceous perennial plant, native to Mexico and southern parts of the United States. This plant is cultivated worldwide for its ornamental value. In November 2019, hundreds of S. farinacea ‘Blue Bedder” grown in a flower garden in Jeju (33°30'57"N 126°32'50"E), Korea have been found to be infected with a powdery mildew fungus. The disease severity was estimated to be 100%. Likewise in October 2020, a similar situation with this plant was also observed in a flower garden in Seoul (37°35'19"N 127°01'07"E), Korea. Leaves, stems and inflorescence of plants were covered by white, thin mycelial felt, bearing an abundance of conidiophores and conidia. Eventually, infected plants lose their ornamental value. Two voucher specimens have been deposited in the Korea University Herbarium (KUS-F31478 and F32164). Fresh materials were examined. Hyphal appressoria were nipple-shaped, but rarely found. Conidiophores (n = 30) were straight, 95 to 160 × 10 to 12 μm and produced 2 to 7 immature conidia in chains with a crenate outline. Foot-cells were cylindric and 36 to 60 μm long. Conidia (n = 30) were ellipsoid-ovoid to barrel-shaped, 32 to 38 × 18 to 24 μm, and contained conspicuous fibrosin bodies. Dark brown chasmothecia were found partly embedded in the mycelial felt on leaves, mostly hypophyllous, spherical, and 82 to 100 µm diameter, with a single ascus in each. Appendages were few, mycelioid, 1- to 4-septate, brown near the base when mature, but paler above. Asci were broadly ellipsoid to subglobose, 56 to 68 × 50 to 62 μm, sessile and 8-spored. Ascospores were colorless, oval to subglobose, and 14 to 18 × 12 to 15 µm. These characteristics were consistent with those of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012). For further confirmation, genomic DNA was extracted from chasmothecia from KUS-F31478 and F32164. PCR amplification was performed using the primer pair ITS1F/PM6 for internal transcribed spacer (ITS) and PM3/TW14 for the large subunit (LSU) of the rDNA (Takamatsu and Kano 2001). Obtained sequences were deposited to the GenBank under the accession numbers MZ359847 and MZ359859 for ITS, MZ359858 and MZ359861 for LSU. For ITS regions 99.80-100% similarity was found with sequences MT131256 (Salvia farinacea), MT131254 (Mazus pumilus) and MT131252 (Erigeron bellioides) of P. xanthii, whereas it was 99.90% with sequences of this fungus on Echinacea purpurea (MT826247 and MT826245) for 28S rDNA gene. Pathogenicity tests were carried out by touching an infected leaf onto healthy leaves of disease-free 30 days old potted ‘Blue Bedder’ using replication of five plants, with five non-inoculated plants used as controls. The typical signs of powdery mildew started to develop on the inoculated leaves in 7 to 10 days, and microscopic examination revealed the morphological identity with the fungus observed from the field. All non-inoculated control plants remained symptomless. Hitherto Golovinomyces powdery mildews on Salvia spp. were reported globally (Farr and Rossman 2021). However, Podosphaera elsholtziae on Salvia sp. and P. xanthii on S. farinacea were reported from China and Taiwan (Zheng and Yu 1987, Yeh et al. 2021). To our knowledge, this is the first report of P. xanthii on S. farinacea in Korea. The occurrence of Podosphaera powdery mildew on S. farinacea could pose a serious threat to the beauty of this plant, causing premature senescence of young leaves and gray to purplish discoloration of the leaves.

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