Indian Journal of BiotechnologyVol 2 October 2003, pp 558-563

Nitrogen Stress Induces Magnaporthe grisea (Hebert) Barr to Secrete Protoplast-Disrupting Proteins

R Rathour*, B M Singh and P PlahaBiotechnology Centre, H P Agricultural University, Palampur 176062, India

Received 18 July 2002; accepted 28 January 2003

Experiments were conducted to determine the effect of nitrogen stress on the gene expression of Magnaportlzegrisea (Hebert) Barr, a casual agent of blast disease of rice and other graminaceous species, Concentrated culturefiltrate extracts of the fungus grown in nitrogen deficient medium exhibited strong disruptive activity against theprotoplasts of different plant species including rice. However, the same activity was not observed in culture filtrateextracts of the fungus grown in nitrogen rich medium. The results suggest that nitrogen stress induces the fungus tosecrete protoplast-disrupting factors. The nitrogen stress elicited protoplast-disrupting activity was found to beconserved among different host-limited forms of the pathogen. The microscopic events preceding the protoplastdeath and the rapidity of the response evinced plasma membrane to be the site of action of protoplast-disruptingfactors. Preliminary characterization of culture filtrate extracts suggested the heat labile proteins of 20.4 to 22.4 kDato be the prime candidates for protoplast-disrupting factors.

Keywords: Magnaporthe grisea, nitrogen stress, protoplast

IntroductionA number of physical and chemical cues are known

to induce infection related morphogenesis andexpression of pathogenicity related genes in a rangeof fungal pathogens. For example, physical contactwith a hydrophobic surface, be it the leaf cuticle or anartificial cellophane membrane, is known to stimulategermination and appressorium formation in spores ofMagnaporthe grisea (Hebert) Barr, a casual agent ofblast disease of rice and other graminaceous species(Hamer et al, 1988; Lee & Dean, 1994). Similarly,among various chemical stimuli, nitrogen limitation inthe immediate environment of many fungi has beenreported to elicit the expression of a number ofpathogenicity related genes (Snoeijers et al, 2000).Talbot et al (1997), while probing the RNA gel blotsof blast infected rice leaves with cDNA probesprepared from poly (At RNA of M. grisea grownaxenically in nitrogen deficient medium,demonstrated that nitrogen starvation elicited theexpression of a number of genes, which wereexpressed during the growth of the fungus in riceleaves. They also showed that nitrogen starvation

*Author for correspondence:Tel: 01894-230371; Fax: 91-01894-230511Evmail: pplaha@hillagric.org

elicited the production of secreted proteins that causedsenescence of rice .leaves. However, these studiescould not establish the action site of the senescenceinducing compounds. In the present study. an attempthas been made to characterize and identify the site ofaction of phytotoxic compounds secreted by M. griseafollowing nitrogen stress. For investigating the actionof compounds secreted by fungus at cellular level,isolated protoplasts of different plant species wereused in the study.

Materials and Methods

Plant Material and FungusRice genotypes, Fukunishiki (Pi-z) and Caloro (Pi-

K), finger millet tEleusine coracana) cultivar VL 17and an unknown genotype of Podophyllumhexandrum were used as plant material. Two single-soore isolates of Magnaporthe grisea, HPU-J andHPU-2 infecting rice and finger millet, respectivelywere used for the preparation of culture filtrateextracts. Isolate HPU-1 was tested virulent on Caloroand avirulent on Fukunishiki, VL 17 and P.hexandrum; whereas, isolate HPU-2 was virulent onfinger millet and avirulent on both the rice cultivarsand P. hexandrum (data not shown).

RATHOUR et al: NITROGEN STRESS INDUCES PROTOPLAST DISRUPTING PROTEINS

Preparation of Culture Filtrate Extracts (CFEs)CFEs were prepared following Talbot et at (1997)

with minor modifications. After initial growth for 4days in complete medium, fungal mycelium wasextracted through three layers of muslin cloth andrinsed thrice in sterilized distilled water. Themycelium was then divided into equal fractions andtransferred to flasks containing 100 ml of eithernitrogen (NaN03) containing minimal medium (MM)or nitrogen deficient minimal medium (MM-N).Cultures were grown for 48 hrs at 25±I°C with gentleshaking at 80 rpm min-I. Mycelium was removedfrom liquid cultures by filtration through three layersof muslin cloth. Then culture filtrates were filtersterilized through 0.45 11mmembrane filter and storedovernight at -20°C. These were then lyophilized tol/50th of the original volume and dialyzed against 10litre of distilled water for 24 hrs using a dialysistubing with 12,000 Da exclusion limit. The innerdialysate was again' lyophilized and dissolved in0.6 M mannitol solution to lI100th .of the originalvolume of culture filtrate. Media-only control CFEwas also produced using the same procedure asdescribed for culture filtrates.

Isolation of ProtoplastsSeeds of rice cultivars Fukunishiki and Caloro and

finger millet cultivar VL 17 were surface sterilized in0.1% HgCb for 5 min and rinsed thrice in sterilizeddistilled water. The seeds were then plated on solidMS (Murashige & Skoog, 1962) basal medium andincubated at 25±I°C under 18/6 hrs light/dark cycleuntil seedlings grow into 2-leaf stage. The in vitrogrown seedlings were then used to isolate leafmesophyll protoplasts following the procedure ofNomura & Kawasaki (1992). The same procedurewas used for isolating protoplasts from P. hexandrumexcept that the protoplast was isolated from callusexplants. The final concentration of protoplasts wasadjusted to 5.0 x 105mrlof mannitol.

Determination of Reaction of Protoplasts to CFEsA 200 ,ul aliquot of the culture-filtrate extract

obtained after fungal growth in nitrogen deficientmedium' [CFE(MM-N)], or the culture filtrate extractobtained after growth in nitrogen containing minimalmedium [CFE(MM)] was mixed with 200 ,LII ofprotoplast suspension of each species in 1.5 mlEppendorf tubes. A 200 ,ul aliquot of media-only CFE(adjusted to 0.6 M with mannitol) mixed with anequal volume of protoplast suspension served as

559

control. Incubations were carried out in the dark at25±I°C with gentle shaking at 60 strokes min-I. Dataon protoplast viability were recorded at regularintervals starting 30 min after incubation using Evansblue exclusion dye. About 20 ,ul of Evans blue (0.5%)prepared in 0.6 M mannitol solution was mixed with20 ,ul of treated protoplast suspension on a clean glassslide and observed under a microscope after placing acover slip. Both blue stained and disrupted protoplasts(recognized as aggregates of cytoplasm) were countedas dead, while those shining were counted as live inall the estimates. In each experiment, 3 samples wereobserved per treatment and 200 protoplasts counted ineach sample. Per cent reduction in protoplast viabilityrelative to control was calculated by the followingformula:

Reduction in viability (%) = [(C-T)/C] x 100

where C and T represent protoplast viability in controland treatment, respectively.

Analytical ProceduresProtein concentration of CFE of each isolate was

estimated by the method of Bradford (1976) usingbovine serum albumin as a standard. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of CFEs was performed on 12 per cent slabgel using discontinuous buffer system (Laemmli,1970). The gels were stained with Coomassie blue R-250 or were silver stained using commercial silverstaining kit ( Bio Rad).

Results and DiscussionSignificant changes in the protoplast were observed

after 30 min of treatment with CFEs(MM-N) (Fig. I).Cytoplasmic aggregation to one side in the protoplastswas the earliest noticeable response in most of thetreated protoplasts. Within 10-15 min, there was anapparent disruption of plasma membrane and most ofthe protoplasts had their contents protruding out of themembrane. After 1 hr of treatment with CFE(MM-N)of rice strain (HPU-l) of M. grisea, 76.1, 64.2, 86.9and 43.7% reduction of viability was observed in theprotoplasts of Fukunishiki, Caloro, VL 17 and P.hexandrum, respectively as compared to 7.0, 4.8, 6.2and 5.9% recorded in CFE(MM) treated protoplasrs(Table 1). With further increase in the time ofincubation, there was a gradual decrease in theviability of CFE(MM-N) treated protoplasts, whilesignificantly less damage was recorded during thisperiod in CFE(MM) treated protoplasts.

560 INDIAN J BIOTECHNOL, OCTOBER 2003

Table 1- Effect of culture filtrate extract (CFE) of Magnaporthe grisea rice strain (HPU-l) on protoplasts of different plant species

Reduction of protoplast viability (%)

Plant Species Ihr 2 hrs 4 hrs

CFE(MM-N) CFE(MM) CFE(MM-N) CFE(MM) CFE(MM-N) CFE(MM)1. Rice cvs.

i) Fukunishiki (I) 76.1±3.7" 7.0±2.0 83.5±3.9 9.2±4.1 87.4±1.8 14.2±3.1ii) Caloro (C) 64.2±4.2 4.8±1.3 79.7±1.0 9.3±1.6 87.5±3.1 17.0±2.7

2. Eleusine coracana cv.VL 17 86.9±4.3 6.2±1.4 91.7±3.1 12.2±2.0 97.8±1.8 20.4±3.43. Podophyllum hexandrum 43.7±2.8 5.9±1.7 46.5±2.6 6.5±1.0 52.9±2.2 11.8±2.1

"Data represent mean±standard deviation of three observations each consisting of 200 counted protoplasts. CFE(MM-N) = culture filtrateextract obtained after growth of the fungus in nitrogen deficient medium; CFE(MM) = culture filtrate extract obtained after growth of thefungus in minimal medium.

Fig. l'-Sequence of events occurring after the treatment of rice protoplasts with culture filtrate extracts obtained after the growth ofMagnaporthe grisea in nitrogen deficient medium [CFEs(MM-N)]. A, Healthy untreated protoplast; B, Protoplast exhibiting aggregationof cytoplasmic contents to one side; C, Protoplast showing protrusion of cytoplasmic contents out of the membrane (note the membraneintegrity of protoplast has been damaged); D, Empty protoplast sac following the loss of cytoplasmic contents; E, Dead protoplast stainedblue due to entry of dye into the cytoplasm (Magnification 1000X).

A considerable reduction in the viability was alsorecorded in the protoplasts of Fukunishiki, Caloro, VL17 and P. hexandrum after treatment with CFE(MM-N) of finger millet strain (HPU-2) compared totreatment with CFE(MM) of the fungus (Table 2).The CFE(MM-N) induced protoplast damageinvolved the same sequence of microscopic events as

illustrated in Fig. 1 for the CFE(MM-N) of rice strain.Since, the CFEs(MM-N) of rice and finger milletlimited forms of M. grisea exhibited strongerprotoplast disrupting activity as compared toCFEs(MM), the results suggest that the nitrogenstarvation induces the fungus to secrete factor(s) withprotoplast disrupting activity. The fact that the

RATHOUR et al: NITROGEN STRESS INDUCES PROTOPLAST DISRUPTING PROTEINS 561

Table 2- Effect of culture filtrate extract of Magnaporthe grisea finger millet strain (HPU-2) on protoplasts of different plant species

Reduction of protoplast viability (%)

Plant Species 1 hr 2 hrs 4 hrs

CFE(MM-N) CFE(MM) CFE(MM-N) CFE(MM) CFE(MM-N) CFE(MM)1. Rice cvs.

i) Fukunishiki (I) 34.6±3.2a 7.3±1.5 32.1±2.4 6.7±0.8 39.4±2.2 13.0±2.7ii) Caloro (C) 31.9±3.1 7.7±2.2 34.6±1.6 6.8±1.6 46.1±2.2 12.9±4.0

2. Eleusine coracana cv.VL 17 42.6±2.3 8.8±2.1 44.7±2.0 9.0±1.5 58.8±3.7 20.6±4.23. Podophyllum hexandrum 21.6±1.5 5.7±2.0 23.1±3.8 5.1±1.2 29.8±4.3 12.5±2.3

"Data represent mean±standard deviation of three observations each consisting of 200 counted protoplasts. CFE(MM-N) = culture filtrateextract obtained after growth of the fungus in nitrogen deficient medium; CFE(MM) = culture filtrate extract obtained after growth of thefungus in minimal medium.

Table 3 - Effect of temperature on the activity of CFE(MM-N)of Magnaporthe grisea

Temperature Reduction of protoplast viability(0C) (%)"

20 63.4 ± 5.8b

40 67.4 ± 3.860 17.6 ± 3.080 4.6 ± 2.1100 7.1 ± 1.2

aCFE(MM-N) of M. grisea strain HPU-l and protoplasts of ricecv. Caloro were used in this experimentbData recorded after 1 hr of treatment and represent mean ±standard deviation of three observations, each consisting of 200counted protoplasts.

protoplast disrupting activity of CFEs(MM-N) of riceand finger millet infecting strains exhibited similarspecies-spectrum suggests that the activity isconserved among the different host-limited forms ofthe pathogen.

Experiments conducted to determine the effect ofheat on CFE(MM-N) of HPU-l revealed that theprotoplast disrupting activity of CFE(MM-N) was lostconsiderably above 60°C, whereas temperatures lessthan 40°C had no significant effect on the activity(Table 3). The heat labile nature of the protoplastdisrupting activity of CFE(MM-N) indicates thepresence of certain proteinaceous factor(s) responsiblefor this activity. Therefore, the concentration ofproteins in CFEs of both host-limited isolates of M.grisea was determined (Table 4). The concentrationof secreted protein was significantly higher inCFEs(MM-N) than in CFEs(MM) of both the host-limited forms. SDS-PAGE analysis of the TCAprecipitable proteins from CFEs(MM-N) andCFEs(MM) of both the host limited forms revealeddifferences in their protein profiles (Fig. 2). Theprotein with apparent molecular weight of 51.2 kDawas overexpressed under nitrogen starved conditions,

Table 4-Concentration of secreted proteins in culture filtrateextracts of Magnaporthe grisea

Isolateno.

Host species Secreted protein (ug/rnl)"

HPU-lHPU-2

CFEs(MM-N) a

304.4 ± 2.56287.87 ± 8.2

CFEs(MM)b

178.16 ± 1.47180.03 ± 1.21

Oryza sativaEleusinecoracana

"Culture filtrate extracts obtained after growth of the fungus for48 hrs in nitrogen starved mediumbCulture filtrate extracts obtained after growth of the fungus for48 hrs in minimal mediumCDetermined by the Bradford method (Bradford, 1976)

while the proteins in molecular weight range of 20.4to 22.4 kDa were detected only in the protein profilesof CFEs(MM-N) of both the host-limited forms andwere conspicuously absent in CFEs(MM) proteinprofiles. As 20.4 to 22.4 kDa proteins are expresseddifferentially under nitrogen starvation of the fungus,they are likely to be associated with the protoplast-disrupting activity of CFEs(MM-N).

In vitro nitrogen starvation is known to elicit theexpression of pathogenicity genes in a number ofbacterial and fungal plant pathogens. Avirulence geneAvr9 of Cladosporium fulvum is induced both ill

planta and in vitro during nitrogen starvation(Snoeijers et al, 1999). An essential Path gene,CgDN3 exhibits strong induction in Colletotrichumgloeosporoides under nitrogen limiting conditions(Stephnson et al, 1998). In addition, Lau and Hamer(1996) has reported a hydrophobin coding gene, mpg Iof M. grisea to be expressed under in vitro nitrogenstarvation. In all the above cases, the regulation ofpathogenicity genes has to be mediated by nitrogen-responsive transcription regulators that showhomology with AREA and NIT2 class of transcriptionregulators of Aspergillus nidulans and Neurospora

562 INDIAN J BIOTECHNOL, OCTOBER 2003

M 1 2 2 1 M kD

9769

51.251.2 43

-2922.4 22.4J

-20.4 20.4

14.3

kD97-- ~~ •.,0<,_ .._ .•••.'"'.

69--'~~'~

Fig. 2-SDS-PAGE protein profiles of cultural filtrate extracts of Magnaporthe grisea. A, Protein profiles of CFEs(MM-N) (culturefiltrate extracts obtained after the growth of the fungus in nitrogen deficient medium) Lane I: CFE(MM-N) of rice strain of M. grisea:Lane 2: CFE(MM-N) of fingermillet strain of M. grisea; B, Protein profiles of CFEs(MM) (culture filtrate extracts obtained after thegrowth of the fungus in minimal medium) Lane 1: CFE(MM) of rice strain of M. grisea; Lane 2: CFE(MM) of fingermillet strain of M.grisea. Molecular weight markers given in kilodaltons in left and right side of panel A and B, respectively.

crassa, respectively. Two nitrogen regulatory areA-like genes, nprl and npr2 have also been detected inM. grisea and mutation in either of these genesresulted in the loss of pathogenicity (Lau & Hamer,1996). The nitrogen stress induced secretion ofprotoplast-disrupting factors observed during presentstudy also supports the previously proposed role ofnitrogen regulating mechanisms in the expression ofpathogenicity genes of M. grisea.

Earlier, Talbot et al (1997) reported that nitrogenstarvation of M. grisea causes the fungus to secretehigh molecular weight protein(s) (>12kDa), whichinduces senescence and desiccation in cut rice leavesafter 48 hrs of treatment. The senescence inducingactivity was found to be conserved among the M.grisea isolates infecting rice, finger millet, barley,weeping lovegrass and crabgrass. On the basis ofpreliminary experiments, they concluded thatsenescence inducing compound(s) were phytotoxicand not cytotoxic. In the present study, however,following the similar method of preparing culturefiltrates, it was demonstrated that nitrogen starvationof M. grisea causes the fungus to secrete highmolecular weight proteins that possess strongprotoplast-disrupting activity against graminaceousspecies. The events preceding the CFEs(MM-N)induced protoplast-disruption (Fig. 1) and rapidity ofthe response following treatment clearly suggest theplasma membrane to be the site of action ofprotoplast -disrupting factors.

The fact that nitrogen starvation elicits the fungusto secrete protoplast-disrupting factors seemsparadoxical since nitrogen application has long beenknown to increase the severity of rice blast disease infield (Ou, 1985). However, the exact physiologicalmechanism of nitrogen-induced enhancement ofblast severity is less understood. Otani (1952)reported that nitrogen plays its role by increasing theconcentration of soluble nitrogen, particularly aminoacids and amines, in the cells of plants receivingapplication of nitrogen. The most importantparameters, which contribute to overall blastseverity, are the penetration ratio (number of lesionsper successful penetration) and lesion size (Bastiaanset al, 1994). The soluble nitrogen that accumulatesin plant cells following nitrogen application mayserve as a suitable nutrient for the fungal growth andthus affect both the above parameters resulting inmuch more lesions that are of greater size. However,the disease promoting effect of nitrogen, assuggested above, will come into play only after thefungus breaches the cell plasma membrane to reachcell cytoplasm. While traversing its path from cuticleto host cell cytoplasm, the fungus may encounternitrogen starvation which may act as a cue for theproduction of pathogenicity determinants includingprotoplast-disrupting factors reported in the presentstudy. Production of such factors will be to theadvantage of the pathogen and facilitate its waythrough the host cell membrane into the cytoplasm to

RATHOUR et aI: NITROGEN STRESS INDUCES PROTOPLAST DISRUPTING PROTEINS

cause leakage of nutrients required for the growth ofthe pathogen.

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