Timothy L. W. Carver

The relationship between insoluble silicon and success or failure of attempted primary penetration by powdery mildew (Erysiphe graminis) germlings on barley

Abstract Insoluble silicon (Si) accumulation was quantified in the fungal structures of genetically compatible Erysiphe graminis hordei germlings attempting primary penetration of attached and detached barley leaves and in the leaf cells around the contact sites. This was achieved using previously published procedures of sequential light microscopy, scanning electron microscopy and energy dispersive X-ray microanalysis. Fungal germlings and host cells were sampled and observed at 12, 16, 20 and 24 h after inoculation and the germlings were classified into three categories, viz. successful penetrations, failed penetrations, or indeterminates. With time, relative levels of Si increased in parasite contact regions of host cells, and subsequently in parasite cells, indicating localized accumulation of Si in host cells and Si uptake from host cells into germlings. At 20 h when definite penetration successes and failures by germlings could first be discriminated, far higher Si levels were found in host cell contact sites associated with failed penetration attempts. By 24 h the difference in host cell Si levels between sites of failed and successful penetrations was obscured by continued Si accumulation into haustorial neck and collar regions subtending host cell papilla responses. Thus, the quantity and timing of Si accumulation in host cell regions under attack, is critical to the relationship between Si deposition and failure of fungal germlings to penetrate.

Inhibition of phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase increases quantitative susceptibility of barley to powdery mildew (Erysiphe graminis D.C.)

Seedling leaves of two barley lines, Algerian/4 * (f14) Man (S) and RISO 5678-S, were excised and supplied either with water (controls), the phenylalanine ammonia lyase inhibitors AOA ( α -aminooxy acetic acid) or AOPP ( α -aminooxy-β-phenylpropionic acid), or the cinnamyl alcohol dehydrogenase inhibitor OH-PAS ([[(2-hydroxyphenyl)amino]sulphinyl]acetic acid, 1,1-di-methylethyl ester) by immersing their cut ends in solution. After 24 h leaves were inoculated with compatible Erysiphe graminis f.sp. hordei conidia and returned to the test solution for 36 h incubation. Treatment with AOA had no effect on the percentage of conidial germlings which penetrated the barleys successfully (formed haustoria), nor on the occurrence of localized autofluorescent host cell responses associated with fungal primary germ-tubes or appressoria. However, both AOPP and OH-PAS treatment led to increased quantitative susceptibility of both barley lines as the percentage of appressoria penetrating host cells to form haustoria was approximately doubled. This was associated with reduction in both the frequency and intensity of autoflu orescent responses associated with fungal germ-tubes; in this respect, the effects of AOPP were slightly more pronounced than those of OH-PAS. The results support the view that in barley, as in oats, autofluorogenic compounds accumulating at sites of germ-tube contact with host cells are phenolic compound(s) synthesized de novo following pathogen attack, that these compound(s) are products synthesized as part of the lignin biosynthetic pathway, and that the compound(s) are involved with resistance to attempted penetration from appressoria.

Nitric oxide contributes both to papilla-based resistance and the hypersensitive response in barley attacked by Blumeria graminis f. sp.hordei

SUMMARY Nonspecific penetration resistance due to papilla formation and race-specific hypersensitive response (HR) can both contribute to Blumeria graminis resistance in barley. Some effective papillae form even in the susceptible cv. Pallas and the isoline P01 carries the additional Mla1 allele conditioning HR. The NO-specific stain DAF-2DA (4,5-diaminofluorescein-2-diacetate) revealed a transient NO generation burst commencing 10 h after inoculation (h.a.i.) in close association with sites of papilla formation in both barley lines. In P01 a burst of NO production throughout some attacked cells was initiated around 10-12 h.a.i. and this preceded whole-cell autofluorescence indicative of HR. The specificity of DAF-2DA staining was demonstrated by the suppression of staining following application of the NO scavenger C-PTIO (1H-imidazol-1-yloxy-2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-3-oxide). In addition, C-PTIO application increased penetration frequencies in both barley lines, indicating a role for NO in papilla-based resistance. Furthermore, C-PTIO application slightly delayed HR in P01 whereas, conversely, application of an NO donor, sodium nitroprusside, slightly accelerated HR in P01 and increased cell death frequency in Pallas. Thus, NO generation is one of the earliest responses of barley epidermal cell defence against B. graminis attack and may be important in both the initiation and the development of effective papillae and cell death due to HR.

Effects of two PAL inhibitors on the susceptibility and localized autofluorescent host cell responses of oat leaves attacked by Erysiphe graminis DC

Abstract Seedling leaves of oat (Avena sativa L.) cv. Maldwyn, with adult plant resistance to Erysiphe graminis DC f.sp. avenae ex Merat., were detached and treated with competitive inhibitors of phenylalanine ammonia-lyase (PAL), the enzyme catalysing the first committed step of phenylpropanoid metabolism. The two inhibitors used were α-aminooxy-β-phenylpropionic acid (AOPP) and α-aminooxy acetic acid (AOA). Both chemicals inhibited PAL extracted from seedling leaves, but AOPP was more effective than AOA. To examine effects on host response and pathogen development, cut ends of leaves were immersed in various concentrations of inhibitor for 24 h before inoculation with fungal conidia, and then incubated for a further 36 h. AOPP had no deleterious effects on host tissues or pathogen development, but high (10−3 m ) concentrations of AOA caused loss of leaf turgor, and prevented fungal haustoria from maturing. At high concentrations (⩾ 10−4 m ) both inhibitors increased leaf susceptibility to fungal penetration. Both inhibitors reduced the intensity and frequency of localized autofluorescent host cell responses associated with contact by primary germ tubes and appressorial germ tubes of the fungus. AOPP significantly reduced diameters of autofluorescent host cell responses. The results support the view that the autofluorescent materials are phenolic and that they play a role in the resistance of epidermal cells to penetration. Attempts to reverse the inhibitory effects of AOPP by simultaneous application of cinnamate, the product of PAL activity, were largely unsuccessful.

Of genes and genomes, needles and haystacks: Blumeria graminis and functionality

SUMMARY Here, we consider the barley powdery mildew fungus, Blumeria graminis (DC Speer) f.sp. hordei (Marchal), and review recent research which has added to our understanding of the biology and molecular biology which underpins the asexual life cycle of this potentially devastating pathogen. We focus on the early stages of the host-pathogen interaction and report current understanding in the areas of leaf perception, fungal signal transduction and host-imposed oxidative stress management. Through this, it is becoming increasingly clear how closely and subtly both sides of the relationship are regulated. Collectively, however, this review highlights the high degree of complexity in working with an obligate parasite. Our experiences suggest that we would make more efficient progress towards understanding the basis of susceptibility and resistance to this true obligate biotroph if its genome sequence was available.

Responses of Erysiphe graminis germlings to contact with artificial and host surfaces

Germling development by Erysiphe graminis f. sp. hordei was compared between conidia held in a simulated air-borne state on microthreads constructed from safety-line threads produced by orb--weaving spiders (Araneus diadematus), and conidia inoculated onto glass, agar, or living or dead barley coleoptile epidermes. Suspended conidia germinated but generally produced only multiple short germ tubes. Conidia on living or dead coleoptiles, bathed from beneath with 0.01 m Ca(NO3)2 solution, generally produced one short germ tube and a second germ tube which elongated and formed a normal appressorium. On glass and agar, multiple short germ tubes were sometimes formed but long germ tubes were formed less frequently than on host epidermis. When conidia with short germ tubes were transferred from microthreads to coleoptiles, they produced a long germ tube which differentiated an appressorium. Conidia with a single short germ tube were also transferred from microthreads so that only the tip of the short germ tube was in contact with a leaf epidermal strip layed on agar, whilst the conidium rested on the agar. Long germ tubes were formed more frequently by such conidia than by controls which had no contact with the leaf epidermis. This suggested that a stimulus causing elongation of the second tube was perceived through the short germ tube in contact with the epidermal strip. Where long germ tubes made contact with the epidermal strip, normal appressoria were formed more frequently than where the long tube made contact with the agar surface alone. The results indicate that germlings develop through distinct stages in response to particular stimuli.

Release and visualization of the extracellular matrix of conidia of Blumeria graminis

The time course of release of an extracellular matrix (ECM) from conidia of the barley powdery mildew pathogen Blumeria graminis was investigated by scanning electron and light microscopy. Conidia released the ECM material preferentially onto a hydrophobic surface and release was detected within 20 s of contact of a conidium with the substratum. No such rapid release of an ECM could be detected when conidia were deposited onto the hydrophilic surface of clean glass. A limited amount of ECM was, however, released from conidia onto the hydrophilic surface of a hydrated cellulose membrane. A time study on hydrophobic plastic revealed that although the ECM was clearly present within the first hour after contact of the conidium with the substratum, it was much reduced in quantity after 12 h incubation. The ECM was demonstrated by micromanipulation and light microscopy to be present as a liquid at the contact interface of the conidium and the substratum. The ECM could not be detected by SEM beneath conidia on leaves, but light microscopy and micromanipulation demonstrated that the ECM could be detected beneath some conidia on the waxy surface of an epidermal strip taken from the abaxial surface of a barley leaf. The ECM material stained positively for protein and could not be removed from the conidium interface by vacuum desiccation in the scanning electron microscope indicating that the ECM is not simply water.

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.

Effects of the PAL inhibitor, AOPP, on oat, barley and wheat cell responses to appropriate and inappropriate formae specials of Erysiphe graminis DC☆

Abstract Seedling leaves of oat cv. Selma, barley cv. Golden Promise and wheat cv. Cerco, were excised and infused either with water or with 10−3 m α-aminooxy-β-phenylpropionic acid (AOPP), a specific inhibitor of phenylalanine ammonia lyase (PAL). Seedling leaves of the three cereal species from both treatments were inoculated with an isolate of Erysiphe graminis from each of the formae speciales avenae, hordei and tritici. In all appropriate host-parasite combinations, AOPP treatment decreased the percentage of primary germ tubes and appressoria associated with localized autofluorescent host cell responses, and increased the percentage of appressoria forming haustoria i.e. increased the susceptibility of epidermal cells to haustorium formation. Thus, autofluorogens appear to be phenolic compounds associated with resistance to infection of these cereal species by their appropriate forma specialis of E. graminis. AOPP treatment also decreased the percentage of host cells which showed whole-cell autofluorescence, indicating cell death, as a result of attack by an appropriate fungal isolate. In appropriate combinations, whole-cell autofluorescence was most common in the barley f. sp. hordei combination, where decreased epidermal cell death due to AOPP treatment may have contributed to increased rates of haustorium formation. For appropriate combinations this is the first report showing AOPP-associated increase in quantitative susceptibility (reduction in background resistance) for barley and wheat. In inappropriate combinations, AOPP treatment decreased the percentage of primary germ tube contact sites associated with localized autofluorescence, but did not decrease the percentage of appressoria associated with localized autofluorescence. In only one of six inappropriate combinations (oat f. sp. tritici) did AOPP treatment increase rates of haustorium formation above the very low levels seen in all inappropriate host-parasite combinations in water-treated leaves. The failure of AOPP to suppress localized autofluorescent responses to fungal appressoria and to increase susceptibility, in most inappropriate host-parasite combinations suggests that resistance factors operate in inappropriate combinations which are not present in appropriate combinations. Although AOPP treatment decreased the relatively high percentages of dead, autofluorescent cells resulting from inappropriate interactions, decreased cell death was not accompanied by increased haustorium formation i.e. cell susceptibility was not increased.

Suppression of host cinnamyl alcohol dehydrogenase and phenylalanine ammonia lyase increases oat epidermal cell susceptibility to powdery mildew penetration

Seedling leaves of oat cvs Maldwyn and Selma have no known major resistance genes to powdery mildew caused by Erysiphe graminis f.sp. avenae , but their susceptibility to infection is quantitative. Thus, only a portion of fungal germlings successfully overcome cell defences to penetrate host epidermis to form haustoria. OH-PAS ([[(2-hydroxyphenyl)amino] sulphinyl]acetic acid, 1,1-dimethylethyl ester) is a potent, specific suicide inhibitor of CAD (cinnamyl alcohol dehydrogenase), an enzyme specifically involved with synthesis oflignin precursors. OH-PAS was shown to inhibit CAD from oat in vitro . For in vivo assays of effects on epidermal cell defences, the cut ends of excised seedling leaves were immersed in OH-PAS solution for 24 h to allow uptake before inoculation with E. graminis conidia. Inoculated leaves were allowed OH-PAS uptake during a further 36 h incubation period. Initial experiments established that OH-PAS at 10 −3 m decreased the frequency and intensity of localized autofluorescent host epidermal cell responses associated with primary germ-tubes (PGTs) and appressoria. Concurrently, OH-PAS treatment doubled the proportion of appressoria forming haustoria, i.e., it increased quantitative susceptibility by suppressing host cell defences. Similar results were obtained with 10 −3 m AOPP ( α -aminooxy- β -phenyl propionic acid), a competitive inhibitor of PAL (phenylalanine ammonia lyase) which catalyzes the first committed step in phenylpropanoid metabolism. Both inhibitors doubled the proportion of appressoria penetrating epidermal cells and forming haustoria. Both inhibitors reduced the frequency and intensity of localized autofluorescent epidermal host cell responses to PGTs and appressoria, although the effect of AOPP was somewhat greater than that of OH-PAS. Neither OH-PAS nor AOPP had any deleterious effects on fungal development. Results support the idea that host autofluorogens accumulating at sites of fungal germ-tube contact with epidermal cells are phenolic compounds. In addition, the study provides experimental evidence pointing to involvement of products synthesized as part of the lignin biosynthetic pathway in oat epidermal cell defence against attempted penetration by appressoria of E. graminis f.sp. avenae .