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Abstract.

Over 90% of the germ tubes of

Puccinia graminis tritici

(wheat stem rust) and

Puccinia hordei

(barley brown rust) dierentiate appressoria on encoun-tering stomata.There has been controversy as to the roleof host topographical signals in the highly precise andecient induction of these infection structures overstomata by cereal rusts. In the present study, polystyrenereplicas of microfabricated silicon wafers, bearing pre-cise microtopographies of de®ned dimensions, were usedto investigate the in¯uence of ridge spacing and heighton infection-structure induction by

P. graminis tritici

and

P. hordei

. It was found that arti®cial topographicalsignals alone can induce a reproducibly high percentage(83±86%) of germ tubes to dierentiate infection struc-tures. Multiple, closely spaced (1.5

m) ridges whichwere 2.0

m high provided the most inductive topogra-phy. Dierentiation on ¯at surfaces and over singleridges was < 4%. Appressorium induction commonlyinitiated a cascade of dierentiation events involving theformation of infection pegs, vesicles, infection hyphae,and occasionally haustorial mother cells. It is suggestedthat the close spacing of cell junctions associated withthe dumbbell-shaped guard cells of cereal stomatalcomplexes provide inductive signals for infection-struc-ture formation by cereal rusts in vivo.

Key words:

Appressorium ± Cereal rusts ± Infectionstructure ±

Puccinia

± Topography-sensing

Introduction

The germ tubes of rust fungi possess a highly sophisti-cated capability for sensing host topographical signalsand this plays an important role in achieving successfulinfection of a host plant (Hoch and Staples 1991; Readet al. 1992). During the pre-penetration phase on the leaf surface, rusts respond in speci®c ways to dierenttopographical stimuli. The best characterised of thesecontact-mediated responses has been the induction of appressorium dierentiation over stomata on beanleaves by

Uromyces appendiculatus.

Essential evidenceto demonstrate that any response on a host plant iscontact-mediated requires the response to be mimickedon an arti®cial substratum in the absence of chemicalsignals from the host. Various substrata bearing topo-graphical signals have been used to induce appressoria ina wide variety of rust species. However, the best arti®cialsubstrata for these studies have been polystyrene replicasof microfabricated silicon wafers because they are highlyprecise and reproducible in their three-dimensionalmicrotopographies (Read et al. 1992; Hoch et al. 1996).Wafer replicas were ®rst used to analyse rust topog-raphy sensing by Hoch et al. (1987). These workersdemonstrated that

U. appendiculatus

is optimally in-duced to dierentiate appressoria over steps with aheight of 0.5

m. This topographical stimulus wasclosely correlated with the size of the guard cell lip or``ledge'' of the host plant

Phaseolus vulgaris

(Hoch et al.1987; Allen et al. 1991b; Terhune et al. 1991, 1993). In asurvey of 27 dierent rust species, Allen et al. (1991a)found that most species responded to single ridges byforming appressoria, but the patterns of response to thedierent ridge heights varied signi®cantly between spe-cies. In addition, a notable group of rusts were observedwhich did not dierentiate over single ridges. Theyincluded a number of economically important cerealrusts (

Puccinia graminis tritici

P. graminis avenae

and

P. coronata

). Appressorium formation by

P. graministritici

(the most studied cereal rust) on scratched,arti®cial substrata, or plastic leaf replicas, has been veryvariable ranging from 0% to 69% dierentiation (Wynnand Staples 1981; Staples et al

1983; Allen et al. 1991a).There has been considerable controversy as to whatstimulates

P. graminis tritici

to dierentiate appressoriaso precisely and eciently over stomata. Because of theinconsistent and often very poor results obtained with

Planta (1997) 202: 163±170

Role of topography sensing for infection-structure dierentiationin cereal rust fungi

Nick D. Read

, Lesley J. Kellock

, Tony J. Collins

, Alan M. Gundlach

Molecular Signalling Group, Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building,Edinburgh EH9 3JH, UK

Institute of Ecology and Resource Management, University of Edinburgh, School of Agriculture Building, Edinburgh EH9 3JG, UK

Department of Electrical Engineering, University of Edinburgh, Edinburgh EH9 3JL, UKReceived: 17 October 1996 /Accepted: 5 December 1996

Correspondence to

: N.D. Read; Tel.: 44 (131) 650 5335;Fax: 44 (131) 650 5392 E-mail: nick.read@ed.ac.uk

induction on arti®cial substrata bearing topographicalfeatures, some workers have suggested a role forchemical signalling in appressorium induction in

P. graminis tritici

and related cereal rusts. However, thesigni®cance of chemicals inducing rust appressoria undernatural conditions is not clear (Hoch and Staples 1991;Read et al. 1992). Nevertheless, there is evidence that thechemical environment associated with stomata mayprovide inductive stimuli for appressorium formation(Grambow and Reisener 1976

;

Grambow 1977, 1978;Grambow and Reidel 1977; Grambow and Grambow1978).In this study, we found that 91±99% of germ tubes of the cereal rusts

P. graminis tritici

(wheat stem rust) and

P. hordei

(barley brown rust) dierentiate appressoriaupon encountering host stomata. Using arti®cial micro-fabricated substrata we have shown, for the ®rst time,that a reproducibly high percentage (83±86%) of germtubes of these rusts can be induced to dierentiateappressoria in response to topographical signals alone.Germ tubes of these species dierentiated over closelyspaced, multiple ridges and grooves, but not to asigni®cant extent over single ridges. The inductivetopographies commonly initiated a cascade of dieren-tiation events which resulted in the successive formationof appressoria, infection pegs, vesicles, infection hyphaeand, very occasionally, haustorial mother cells. With theexception of appressoria, these infection structures areusually only formed inside the leaf. It is suggested thatthe inductive topographical signals in vivo are associatedwith the close spacing of cell junctions adjacent to thestomata of cereal leaves.

Materials and methods

Fungal and plant material.

The fungal material used was

Pucciniahordei

, race octal 1653 (obtained from the Welsh Plant BreedingStation, Aberystwyth, UK) and

P. graminis tritici

, isolate 84 fromthe National Institute for Agricultural Botany (Cambridge, UK).For experimentation, uredospores of

P. hordei

and

P. graministritici

were harvested from infected plants (Feekes growth stage 12,Tottman and Makepeace, 1979) of the susceptible barley (

Hordeumvulgare

L.) cultivar Golden Promise and susceptible spring wheat(

Triticum aestivum

L.) cultivar Armada, respectively.

Analysis of appressorium dierentiation in vivo.

Dierentiationin vivo was investigated on host leaf segments. Leaves of eachcereal were taken from four-week-old plants and 60-mm-longsections from each leaf excised. The leaves were inoculated with3 mg of fresh uredospores in a settling tower which was 23 cmdiameter and 76 cm high. The spores were blown from a rolledsheet of paper into a hole at the top of the settling tower using acompressed-air can (Kenro; H.A. West, Edinburgh, UK). Thespore density on the leaf surface was 100±150 spores

)

. Theinoculated leaf segments were placed in Petri dishes, adaxial sideuppermost with their cut ends under wet ®lter paper, and incubatedat 22

C for 24 h. Percentage appressorium formation in vivo wasquanti®ed as the percentage of those germlings which hadencountered stomata and formed appressoria over those stomata.

Design and manufacture of microfabricated silicon wafers.

Preciselyde®ned microtopographies were etched in 76-mm-diameter siliconwafers by a process of reduction projection photolithography andreactive ion beam etching (Hoch et al. 1996).The X-Y dimensions of the microtopography in the silicon werede®ned as a `mask' in anti-re¯ective chromium on a 120-mm glassplate. The pattern of the mask was designed using CADENCECAD software. These data ware used by an electron beam patterngenerator to write the pattern onto a layer of electron beam resistcovering the chromium-coated plate (Compugraphics Internation-al, Glenrothes, UK). The pattern was written in the mask resistwith a 1-

m electron beam spot and developed using standard resistsolvents. The exposed chromium layer was then removed with ferricchloride and the remains of the resist removed with appropriatesolvents.The Z-dimension of the microtopography was de®ned by theprecise thickness of a layer of SiO

on the surface of the siliconwafer which was created by thermal oxidisation in steam (burningH

in O

at 950

C). The de®nition of the Z-dimension of themicrotopography relies on the fact that SiO

is preferentially etchedover the silicon (at a SiO

: silicon etch ratio of >12:1). Precisecontrol of the depth of the microtopography was achieved byetching the exposed SiO

completely. Wafers with topographies0.116 and 2.4

m deep had oxidisation times of 30 min and 48 h,respectively.Preparation of the wafers for de®nition of the X-Y dimensionsof the topographical pattern involved spin coating the oxidisedwafers with positive HIPR6512 photoresist (Microelectronic Ma-terials, Livingstone, UK). Adhesion of the resist to the wafer wasimproved by vacuum-baking the wafer in hexamethyldisilazeneprior to coating. The solvent in which the resist was dissolved wasthen removed by baking at 90

C on a hot plate.Using a Reduction Direct Step On wafer machine (EatonOptimetrix, Anheim, Calif. USA), a 10

reduced image of themask was printed onto the resist with 436-nm illumination from ashort-arc Hg lamp. The exposed resist was then removed bydeveloping it in dilute methyl ammonium hydroxide which reactswith the indene carboxylic acid created within the light-exposedregions of the photoresist. The X-Y dimensions result from etchingthe exposed SiO

only.The SiO

was etched at a rate of approximately 1

)

using radio-frequency plasma in a mixture of CHF

and He in aPK2440 reactor (Plasmatherm, Kresson, N. J., USA). Once all theexposed SiO

was etched, the remains of the photoresist wasremoved by low-pressure O

plasma and then immersion in fumingHNO

.The microfabricated silicon wafers used had 2.0-

m-widetroughs etched to depths of 0.116, 0.223, 0.55, 0.72, 1.20, 1.50,2.00 and 2.40

m. The spacings of these troughs were 1.5, 2.5 or50

Preparation of polystyrene replicas of silicon wafers.

The microto-pographies used to analyse appressorium induction were negativereplicas of the microfabricated silicon wafers (Fig. 1).Wafer replicas were made by heat-pressing polystyrene onto themicrofabricated silicon wafer using a method adapted from thatdescribed by Hoch et al. (1996). For this purpose, 12 mm

12 mmpieces of Petri dish (Philip Harris, Clydebank, UK) were used. Twowafers were placed `microfabricated side' up on a glass microscopeslide (BlueStar; Chance Proper, Warley, UK) and each wafer had apiece of Petri dish placed on top of it followed by a secondmicroscope slide. Five microscope slides were sandwiched betweentwo ceramic kitchen tiles with a weight on top (totalweight



450 g) and then heated at 200

C for 50 min. Whilsthot, the wafers were separated from the polystyrene replicas byplunging into cool distilled water. Control substrata (¯at surfaces)were prepared in the same way except the silicon wafers were notincluded in the sandwich.Wafers were regularly cleaned by ®rst soaking them inchloroform for 4±5 h to remove pieces of polystyrene then washedbrie¯y in clean chloroform and allowed to dry, `microfabricatedside' up, on ®lter paper. The wafers were then left under a mixtureof 50 ml concentrated H

and 25 ml of 30% H

overnightafter which the wafers were thoroughly washed in distilled waterbefore being dried, `microfabricated side' down, on ®lter paper.164 N.D. Read et al.: Topography sensing in cereal rusts

Inoculation and incubation of sporelings on wafer replicas.

Polysty-rene replicas of silicon wafers were arranged on glass microscopeslides sitting on the surface of 0.4% tap water agar, and inoculatedwith approximately 3 mg of fresh uredospores to give a sporedensity of 100±150 spores

)

. Ater allowing 3 min for thespores to settle onto the substratum, the lids of the Petri dishes werecoated with a ®ne spray of tap water before replacement, and thedishes sealed with Para®lm (American Can Co., Greenwich, Conn.,USA) to ensure the retention of 100% relative humidity. The disheswere then incubated in the dark for 3 h at 18

C, then 22

C for45 h with a daily 16-h photoperiod.

Quantitation of appressorium dierentiation on wafer replicas.

After48 h, germlings growing on wafer replicas were ®xed for 1 h inlactophenol vapour by placing drops of lactophenol (BDH, Dorset,UK) on the agar surface adjacent to the replicas. The germlingswere then stained for 5 min with 3% Uvitex BHT (Ceiba Geigy,Manchester, UK) in 20% glycerol. Quantitation of percentageappressorium dierentiation was done by imaging these prepara-tions with a Polyvar ¯uorescence microscope (Reichert-Jung,Vienna, Austria) using a U1 ®lter block. Each experiment wasperformed at least twice with at least ®ve replicates per experiment;1000±1800 uredospore germlings were analysed for each datumpoint shown in Figs. 6±8.

Nuclear staining.

Dierentiated germlings were ®xed in 100%ethanol, air-dried and then stained for 25 s in 0.01

)

-diamidino-2-phenylindole (DAPI; Sigma, Dorset, UK) followed bywashing in distilled water for 1 min and subsequent counter-staining for 30 s in 0.001% Calco¯uor (Sigma). These preparationswere imaged with a Reichert-Jung Polyvar ¯uorescence microscopeusing a U1 ®lter block, and micrographs were recorded on KodakTMAX 400 35-mm ®lm.

Low-temperature scanning electron microscopy.

Fungal and plantmaterial were prepared for low-temperature scanning electronmicroscopy (LTSEM) using the EMscope SP2000 cryopreparationsystem (Beckett and Read 1986) interfaced with a Cambridge S250scanning electron microscope. Samples were mounted on standardEMscope LTSEM stubs using Tissue Tek O.C.T. compound (MilesLaboratories, Naperville, Ill., USA) as a cryoadhesive. Dependingon subsequent preparation, samples were either mounted ¯at on thestub surface or located on edge in grooves cut into the surface of the stub (Jeree and Read 1991). Mounting was performed asrapidly as possible (within 1 min) to minimise specimen desicca-tion. The stub with mounted specimens was cryo®xed by plungingit into sub-cooled nitrogen under dry argon gas. Some samples werefreeze-fractured whilst others were not. Freeze-fracturing wasperformed using either a knife or blade which had been pre-cooled.Specimens were examined either fully frozen-hydrated or partiallyfreeze-dried after warming to

)

C to

)

C on the microscopecold stage. All material was ®nally sputter-coated with gold andexamined below

)

155

C with accelerating voltages between 5 and7 kV. Scanning electron micrographs were recorded on KodakTMAX 100 120 roll ®lm.

Measurements of spacings of epidermal cell junctions.

Segments of four-week-old wheat leaves were prepared for LTSEM and randomimages, observed at a magni®cation of

1000 on the microscopescreen, were digitally captured and analysed with I-Scan (ISSgroup, Manchester, UK), a PC-based, slow-scan image acquisitionsystem. Measurements were made with the integral

line

command.Measurements were taken at 90

to the long axis of the leaf andwere placed randomly along the long axis of each stomatalcomplex.

Results

Infection-structure dierentiation in vivo.

In vivo, thegerm tubes of both

Puccinia hordei

and

P. graminis tritici

exhibited marked directional growth which was more-or-less perpendicular to epidermal cell junctions. Shortlateral branches, of variable length, were usually formedover the cell junctions (see Fig. 1 in Read et al. 1992). Onencountering a stomatal complex, a germ tube dieren-tiated an elongated appressorium which was speci®callylocated over and completely covered the stomatal poreand those regions of guard cells adjacent to the pore(Fig. 2). Of germ tubes making contact with stomata,98.6  0.5 (SE) of those of

P. hordei,

and 91.6%  3.3(SE) of those of

P. graminis tritici

, dierentiatedappressoria on barley and wheat leaves, respectively.Penetration through the stomatal pore occurred afterappressorium formation and typically resulted in asuccession of dierentiation events within the subs-tomatal cavity. The series of infection structures formedby both species were comprised of: an infection peg; anelongated, septate vesicle; infection hyphae arising fromeach end of the vesicle; and a haustorial mother cellarising at the end of each infection hypha on contactwith a mesophyll cell surrounding the substomatal cavity(Fig. 3). In susceptible host-pathogen combinations, thehaustorial mother cell commonly gave rise to anintracellular haustorium (Kellock 1994).

Fig. 1A±F.

Microtopographies on polystyrene wafer replicas used toanalyse appressorium induction. These microtopographies were in theform of negative replicas moulded in polystyrene from microfabri-cated silicon wafers possessing the positive template.

Multipleridges with a 1.5-

m spacing.

Multiple ridges with 2.5-

m spacing.

Single ridges with a 50-

m spacing.

Two ridges with a 1.5-

mspacing.

Three ridges with a 1.5-

m spacing.

Four ridges with a1.5-