PRINT VIP Host Parasite Interaction

8/3/2019 PRINT VIP Host Parasite Interaction

1/17

R E V I E W

HOST-PATHOGEN INTERACTIONS IN PLANTS

Plants, When Exposed to Oligosaccharides of Fungal Origin, Defend

Them selves by Accum ulating Antibiotics

P E T E R A L B E R S H E I M an d B A R B A R A S . VA L E N T

From the Department o f Chemistry, University of Colorado, Boulder, Colorado 8030 2

KEY WO RDS soybeans phytoalexins

oligosaccharides 9 elicitors cell wallsplant pathogens

P lan t s a re exposed to a t t ack by an immense a r rayof mic roorgan i sms , ye t a re r e s is t an t to a lmos t a llo f these po ten t i a l pes t s. M any p lan t s r e spond toan invas ion by a pa thogen ic o r nonpa thogen icmic roorgan i sm, whe the r a fungus , a bac te r ium ora v i rus , by accumu la t ing phy toa lex ins , l ow mo lec -u la r we igh t compounds which inh ib i t t he g rowthof mic roorgan i sms . Phy toa lex ins a re p robab ly a l sotox ic to h ighe r an ima l and p lan t ce ll s (38 ) . T heprodu c t ion o f phy toa lex ins appea r s to be a wide -sp read mech an i sm by which p lan t s a t temp t to

de fend themse lves aga ins t mic robes and , pe rhaps ,aga ins t o the r pes t s (6 , 16 , 17 , 28 , 32 , 36 , 37 , 38 ,63) . Molecu les o f mic rob ia l o r ig in which t r igge rphy toa lex in accumula t ion in p l an t s have beencal led e l ic i tors (33) . Plants recognize and respondto e l ic i to r s a s fo re ign molecu les . H ow ever, p l an t sare unl ikely to have suff ic ient genet ic mater ia l tocode fo r un ique r ecogn i t ion sys t ems fo r eve rybacter ia l species and s t ra in and every fungal raceand v i rus tha t p l an t s a re exposed to and r e spondto defensively. Thus, e l ic i tors are l ikely to bemolecu les p resen t in many d i ff e ren t mic robes and ,in fact , the e l ic i tor to be descr ibed in this paper isa s t ructural poly sacchar ide o f the mycel ia l wal ls of

many fung i (11 ) .P H Y T O A L E X I N S -- G E N E R A LA N T I B I O T I C S

There a re abou t 100 known phy toa lex ins d i s t r ib -u t e d a m o n g a n e q u a l ly i m p r e s si v e n u m b e r o f p l an t

species . I t cannot yet be said , a l though i t i s

probable , that a l l p lants have the abi l i ty to synthe-s ize phytoalexins; but i t can be said that the abi l i tyto produce phytoalexins is a character is t ic ofp lan t s tha t i s w idespread . Mos t p l an t s appea r top roduce seve ral s t ruc tu ral ly r e l a t ed p hy toa lex ins .G r e e n b e a n s (Phaseolus vulgaris)a n d p o t a t o e s(Solanum tuberosum)a re each capab le o f accu-mula t ing more than ha l f a dozen d i ff e ren t bu ts t ruc tu ra l ly r e l a t ed phy toa lex ins . The phy toa lex -ins p rodu ced by d i ff e ren t mem bers o f a p l an tfamily, a l though seldom ident ical , are s t ructural lyrela ted. Indeed, the s t ructural re la t ionships be-tween phy toa lex ins have been used to s tudy thesys temat ic r e la t ionsh ips amo ng h igh e r p l an t s (30 ) .

Al l phy toa lex ins a re l i poph i l i c compounds . Thefour phytoalexins presented in Fig. 1 are i l lus t ra-t ive o f the m os t d ive r se types o f phy toa lex ins . Themos t s tud ied phy toa lex in o f soybeans i s g lyceo ll in(Fig. 1) (12) . L yn e et a l. (42) have chara cter izedtwo add i t iona l soybean phy toa lex ins wh ich a res t ruc tu ra l i somers o f g lyceo l l in and which a ppea rto have s imilar ant ibiot ic character is t ics . In thisman uscr ipt , g lyceol l in will be a gener ic term refer-r ing to the g lyceo l lin i somers wh ich c o -chrom ato -g raph on th in - l aye r ch romatography p la t e s . Thesyn thesi s o f g lyceo l l in , a ph eny lp ropan o id de r iva -t ive , i s p robab ly in i t i a t ed f rom pheny la l an ine v iathe r eac tion ca ta lyzed by pheny la l an ine am mo nia -

lyase (F ig . 2 ) , bu t , a s ye t , no b iosyn the t i c pa thwayfor the p roduc t ion o f a phy toa lex in has beencomple te ly desc r ibed .

The m echan i sm b y which phy toa lex ins s top thegrowth o f cel ls i s no t u nder s too d . Som e ev idencesugges t s tha t phy toa lex ins a l t e r p l a sma mem-

J. CELLBIOLOGY ~) Th e Ro ckefeller Un iversity P ress 9 0021-9525/78/0901-062751.00 627


2/17

~ H

G LY C E O L L I NA PTEROCARPAN FROM

SOYBEANS (Glycine max)

OH

CAPSIDIOLA $ESOUITERPENE FROM

PEPPERS (Capsicum frutescens)

HH 3 C ~ ~ C H 3

0~ COOH

ORCHINOLA PHENANTHRENE FROM

ORCHIDS (Or chidaceae)

WYERONE ACIOA POLYACETYLENE FROM

BROAD BEA NS (Vic ia faba)

FIGURE 1 Structu rally diverse phy toalexin s.

3 o o . o o . ? S C o A

1 ,,- 2 ,- --,,- ~ ,

PHENYL- CINNAMICALANINE ACID p-COUMARIC p-COUMAROYL oA

ACID fI . PHENYLALANINE AMMONIA-LYASE m/

2 CINNAMIC ACID 4-HYDROXYLASE - ~ /3 p-COUMARATE CoA LIGASE

HGLYCEOLLIN

FIGURE 2 Proposed pathway for the biosynthesis of glyceollin, a phytoalexin of soybeans.

b ranes (46 , 52 , 62 ) ; i t has a l so been sugges tedtha t phy toa lex ins inh ib i t ox ida t ive phosphory la -t ion (46) . On the o the r hand , some phy toa lex ins ,b e i n g f u r a n o c o u m a r i n s o r r e l a t e d c o m p o u n d s( 1 4 ) , ar e k n o w n t o c r os s -l in k D N A w h e n e x p o s e dto u l t r av io le t l igh t (44 ) . I t may be tha t a ll o r mo s tp h y t o a l e x i n s h a v e a c o m m o n m o d e o f a c t i o n .Add i t iona l r e sea rch on the mechan i sm o f ac tionof phy toa lex ins i s needed .

Assay ing the d egree o f tox ic i ty o f a ph y toa lex in

can be compl ica ted by the ab i l i t y o f the t e s to rgan i sms to me tabo l i ze and the reby de tox i fyphy toa lex ins (8 , 53 , 54 ) . In add i t ion , many phy-toa lex ins appea r to be s t a t i c agen t s r a the r thantox ic agen t s . G lyceo l l in , the so ybean phy toa lex in ,appears to s top the growth of cel ls wi thout ki l l ingthem. Glyceo l l in inh ib i t s t he g rowth , i n v i t ro , o ft h e s o y b e a n p a t h o g e nPhytophthora m egaspermavar. sojae (Pms) , t he causa l agen t o f roo t and s t emro t . S teven Thomas , i n ou r l abora to ry, has found

6 2 8 THE JOURNAL OF CELL BIOLOGY 9 VOLUME 78, 197 8


3/17

t ha t g lyc eo l l i n w i l l a l so s top the g rowth o f t h reeG r a m - n e g a t i v e b a c t e r i a ,P s e u d o m o n a s g l y c i n e a ,Rh izob ium t r i fo l i i ,a n d R h i z o b i u m j a p o n i c u m ,o ft h e G r a m - p o s i t i v e b a c t e r i u m ,Baci l lus subt i l i s ,an do f b a k e r ' s y e a s t ,S a c c h a r o m y c e s c e r e vi s ia e .I n t e r -

e s t ing ly, i t r equ i r e s a bou t 25 /~g /ml o f g lyceo l l into inh ib i t by 50% and 100 /xg /ml to i nh ib i t by1 0 0 % t h e g r o w t h o f a l l o f t h e s e d i f f e r e n t o rg a -n i sms (F ig . 3 ) . The f ac t t ha t i t r equ i r e s t he samec o n c e n t r a t i o n o f g l y c e o l li n t o i n h i b i t t h e g r o w t h o fa v a r i e t y o f c e ll t y p e s s u g g e s ts t h a t t h e m e c h a n i s mof ac t ion o f g lyceo l l i n i s a chem ica l ly ca t a lyzedr e a c t i o n ( s u ch a s t h e p h o t o s e n s i t i z e d f u r a n o c o u -m a r i n s o r s u c h a s d i s r u p t i o n o f m e m b r a n e s ) r a t h e rt h a n a n e n z y m e c a t a l y z e d re a c t i o n . T h e r e s u l t sp r e s e n t e d i n F i g . 3 s u p p o r t t h e h y p o t h e s i s t h a t ap l a n t ' s p h y t o a l e x i n s c a n p o t e n t i a l l y p r o t e c t t h ep l a n t f r o m a b r o a d s p e c t r u m o f m i c r o o rg a n i s m s .

P h y t o a l e x i n s a r e e i t h e r n o t d e t e c t a b l e o r a r e

d e t e c t a b l e i n o n l y v e r y l o w c o n c e n t r a t i o n s i nh e a l t h y p l a n t s . P h y t o a l e x i n s h av e b e e n f r e q u e n t l yre fe r r ed to a s s t r e s s me tabo l i t e s , fo r ce l l s wh icha r e i n j u r e d o r e x p o s e d t o t o x ic a g e n t s o f t e n a c c u -m u l a t e p h y t o a l e x i n s . H o w e v e r, c h a l l e n g e o fp l a n t s b y m i c r o o rg a n i s m s u n i f o r m l y r e s u l t s i n t h ea c c u m u l a t i o n o f l a rg e a m o u n t s o f p h y t o a l e x i n s .G l y c e o l l i n i s a c c u m u l a t e d i n l a rg e a m o u n t s b ys o y b e a n t i s s u e s i n r e s p o n s e t o i n f e c t i o n b y P m s ,t h e s o y b e a n p a t h o g e n , a s w e l l a s i n r e s p o n s e t oc h a l l e n g e b y a w i d e v a r i e t y o f n o n p a t h o g e n i cm i c r o o rg a n i s m s .

E L I C IT O R S O F P H Y T O A L E X I N

A C C U M U L AT I O N - - A S S AY SP l a n t s r e c o g n i z e a n d r e s p o n d t o t h e p r e s e n c e o fm i c r o b e s b y a c c u m u l a t i n g p h y t o a l e x i n s . T h e r e -f o r e , p l a n t s m u s t b e r e c o g n i z i n g m o l e c u l e s s y n t h e -s i zed by these mic ro bes . The f i r s t c lues t o t hen a t u r e o f t h e m o l e c u l e s w h i c h t r i g g e r th e a c c u -m u l a t i o n o f p h y t o a l e x i n s c a m e f r o m s t u d i e s o fp a t h o g e n s g r o w n i n c u l tu r e . T h e c u l t u r e m e d i u m ,f r e e o f t h e p a t h o g e n w h i c h h a d g r o w n i n t h emed ium, was shown , i n s eve ra l i n s t ances , t o e l i c i tp h y t o a l e x i n a c c u m u l a t i o n i n t h e p a t h o g e n ' s h o s t(2 -4 , 22 , 23 , 33 , 35 , 48 , 49 , 59 , 64 ) . A des i r e t ok n o w t h e c h e m i c a l n a t u r e o f a n e l i c i t o r p r o m p t e dt h e p u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f t h e e l i c i to rp r e s e n t i n t h e c u l t u r e f i l t ra t e s o f P m s , t h e f u n g a lp a t h o g e n w h i c h c a u s e s r o o t a n d s t e m r o t i n s o y -beans (5 ) . To pu r i fy th i s b io log ica l ly ac t ive mole -c u l e , i t w a s n e c e s s a r y t o d e v e l o p q u a n t i t a t i v ea s s a y s f o r t h e b i o l o g i c a l a c t i v it y. A r t h u r Ay e r s

E ~ o0.~

~ . O B . S U B T I L IS : ~ 1 9p g / m !\ \ ' , , , , ~ p G LV C IN E A : ~ a 4 , g / m ,

O'D'~t50.; ~ ~ O S. CEREVISI~: 2 5 /~g/ml

0,1

0 1 0 2 0 3 0 4 0 5 0 1 0 0

G LY C E O L L I N ~ g / m l

FmoxE 3 Similar concentrations of glyceollin are re-quired to inhibit the growth ofBacillus subtilis, Pseu-domonus glycinia,and Saccharomyces cerevisiae.

u n d e r t o o k t h i s c h a l l e n g e i n t h e a u t h o r s ' l a b o r a -t o r y.

Soy bean t i s sues accum ula t e g lyceo l l i n i n r e -sponse to t he e l i c i t o r s p re se n t i n t he f lu id o f Pmscu l tu re s . Th i s r e sponse i s t he bas i s o f t h ree b io log -i ca l a s says tha t we re de ve lo ped . The f i r s t a s sayd e v e l o p e d b y Ay e r s w a s a m o d i f i c a t i o n o f t h ec o t y l e d o n a s s a y f i r s t u s e d b y F r a n k a n d P a x t o n( 2 3 ) . F o r t h i s a s s a y, a s e c t i o n , a p p r o x i m a t e l y 1m m t h i c k a n d 6 m m i s d i a m e t e r , i s c u t f r o m t h el o w e r s u r f ac e o f c o t y l e d o n s o b t a i n e d f r o m 8 - d a y -o l d s o y b e a n s e e d l i n g s . T h e s a m p l e t o b e a s s a y e dfo r i t s ab i l i t y t o e l i c i t t he a ccum ula t ion o f t hes o y b e a n p h y t o a l e x i n , g l y c e o l l i n , i s p l a c e d i n a100-p.1 d rop on the cu t su r f aces o f each o f a s e t o f1 0 c o t y l e d o n s . T h e t r e a t e d c o t y l e d o n s a r e i n c u -b a t e d a t 2 6~ o n m o i s t fi l t er p a p e r in c o v e r e dP e t r i d is h e s . T h e w o u n d d r o p l e t s o n t h e c o t y l e -d o n s t u r n r e d ( F i g . 4 ) d u e t o t h e p r e s e n c e o f a nu n i d e n t i f ie d c o m p o u n d c a l l e d PA k ( 2 3 ) . T h e r e dc o l o r i s u s u a l l y r o u g h l y p r o p o r t i o n a l t o t h ea m o u n t o f g l y c e ol l i n i n t he w o u n d d r o p l e t . A f t e r20 h , each se t o f 10 co ty l edons i s t r ans fe r r ed wi thf o r c e p s t o 2 0 m i o f d i s t i l le d w a t e r c o n t a i n e d i n a5 0 - m l E r l e n m e y e r f l a s k . T h is p r o c e d u r e r i n s e s o fft h e d r o p l e t s o f s a m p l e f l u i d t h a t a r e r e t a i n e d o nt h e w o u n d e d s u r f a ce . S i n c e g l y c e o l li n a b s o r b sl i g ht a t 2 8 5 n m , t h e a b s o r b a n c e a t 2 8 5 n m o f th ew o u n d d r o p l e t s o l u t i o n i s a m e a s u r e o f e l ic i t o rac t iv i ty. The con t r ib u t ion o f g lyceo l l i n t o t hea b s o r b a n c e a t 2 8 5 n m o f w o u n d d r o p l e t s o l u ti o n sw a s d e t e r m i n e d b y i s o l a t i n g t h e g l y c e o ll i n f r o mt h e w o u n d d r o p l e t s o l u t i o n s ( F ig . 5 ) ( 5 ) . T h e d a t ao f F i g . 5 d e m o n s t r a t e t h a t t h e r e i s a l i n e a r r e l a -t i o n s h i p b e t w e e n t h e a m o u n t o f g l y c e o l li n in t h ew o u n d d r o p l e t s o l u t i o n s a n d t h e a b s o r b a n c e o ft h e d r o p l e t s a t 28 5 n m .

P. ALBERSHEIM AND B. S. VALENTHost-Pathogen Interactions in Plants 6 2 9


4/17

FIouRE 4 The cotyledon assay. A section is cut from the lower surface of cotyledons obtained from 8-day-old soybean seedlings. The sample to be assayed for elicitor activity is placed in a droplet on the cutsurface. Elicitor causes the colorless phytoalexin, glyceollin, to accumulate in the wound droplets. Anunidentified red pigment also accumulates in the wound droplets.

5 o

4o

z

30

t9

o 2 0

I0

0 I I I I0 0.5 1.0 1.5 2.0

ELICITOR A C T I V I T Y ( A 2 8 5nm}

FIGURE 5 The cotyledon assay. A linear relationshipexists between the amount of glyceollin present in thewound droplets and the absorbance of the droplets at285 nm (5). (Fig. 5 reprinted by copyright permissionfrom: 1976, Plant Physiology, 57: 753.)

The hypocotyls of 5-day-old soybean seedlingsare used in a second biological assay of elicitoractivity (5). This assay is a quantitative form ofthe semi-quantitative assay used by Klarman andGerdema nn (34) and by Keen (31). The soybean

seedlings are r emoved from the soil and rinsed intap water. The seedlings are then mounted onhorizontal stainless steel needles (1 mm in diam-eter, 11 cm in length) by piercing the hypocotyls 5mm below the cotyledons (Fig. 6). The resultingvertical slit wounds in the hypocotyls are about 5mm in length. A set of 10 seedlings is mounted oneach needle, and the seedlings are s uspended withtheir roots in water to within 2 cm of the wounds.A 20-/zl drop of an elicitor preparation or atriturated Pms preparation is applied to eachhypocotyl wound. Each set of 10 seedlings isremoved from its needle after incubat ion in aclosed chambe r at 26~ for 24 h at 100% humid-ity. A 14-mm segment of hypocotyl, centered onthe wound, is cut from each seedling, and theglyceollin is extracted and purified from each setof segments. The amount of glyceollin present inspots which are eluted from thin-layer chromatog-raphy plates is determined by the absorbance at285 nm. The data of Fig. 7 demonstrate that, atlow elicitor concentrations, there is a linear rela-tionship betwee n the amou nt of elicitor applied toeach hypocotyl and the amou nt of glyceollin ex-tracted from each hypocotyl (5). It requires


5/17

F m u ~ 6 The hypocotyi assay. 5-day-old hypocotyiseedlings are mo unted on stainless steel needles. E licitorpreparations are placed in the resulting vertical sli twounds. Glyceollin, which accumulates in the tissuesurrounding the wound, is extracted, purified, and quan-titated.

hypoc o ty l t o r e su l t i n su ff i c i en t g lyceo l l i n accu -m u l a t i o n t o i n h i b i t c o m p l e t e l y t h e g r o w t h o f P m sin v i t ro .

E l i c i to r s i so l a t ed f rom the cu l tu re f l u ids o f t h reed i ff e ren t r aces o f Pms a l l have equ iva l en t ab i l i t i e sto e l i c i t g lyceo l l i n accum ula t ion in t he hypoco ty l so f t h e s o y b e a n c u l t i v a r H a r o s o y 6 3 ( F i g . 7 ) . T h i s

o b s e r v a t i o n , a n d a d d i t i o n a l d a t a t o b e d e s c r i b e dl a t e r , d e m o n s t r a t e c o n c l u s iv e l y t h a t t h e a b i l i ty tos t i m u l a t e p h y t o a l e x i n p r o d u c t i o n i s n o t i n i ts e l fs u f fi c ie n t t o d e t e r m i n e w h e t h e r a p a r t i c u l a r r a c eo f a p a t h o g e n c a n i n f e c t a p a r t i c u l a r c u l t i v a r o f i t shos t s .

G l y c e o l l i n is a p h e n y l p r o p a n o i d - d e r i v e d p h y t o -a l ex in (12 ) . Th e re fo r e , e l i c i t a t i on o f g lyceo l l i naccumula t ion migh t a l so r e su l t i n t he inc reaseda c t iv i t y o f t h e e n z y m e s i n v o l v e d i n g e n e r a l p h e n -y l p r o p a n o i d m e t a b o l i s m . T h e f i rs t su c h e n z y m e i n

t h e s y n t h e s i s o f p h e n y l p r o p a n o i d s i s p h e n y l a l a n i n ea m m o n i a - l y a s e ( F i g . 2 ). A t h i r d a s s a y fo r e l i c it o r sinvo lves the s t imu la t ion o f t he ac t iv i ty o f pheny l -a l a n i n e a m m o n i a - l y a s e a n d t h e a c c u m u l a t i o n o fg lyceo l l i n i n suspens ion -cu l tu red soyb ean ce l l s( 2 0 ) . T h i s a ss a y d o e s n o t d e p e n d o n w o u n d i n g o fthe soybean t i s sues .

T h e l e v e l o f p h e n y l a l a n i n e a m m o n i a - l y a s e i n -c reases r ap id ly (F ig . 8 ) 3 h a f t e r exposu re o f t hesuspen s ion -cu l tu r ed ce l ls t o t he e l i c i t o r (20 ) . Thei n c r e a s e i n a c t iv i t y o f t h e p h e n y l a l a n i n e a m m o n i a -lyase in soybean ce l l suspens ion cu l tu re s t r ea t edwi th e l i c i t o r s i s fo l lowed by the ac cum ula t ion o fg l y c e o ll i n ( F i g . 8 ). F u r t h e r m o r e , t h e u p t a k e o f

n i t r a t e f r o m t h e m e d i u m a n d t h e i n c r e a s e i n f r e s hw e i g h t o f t h e c e l ls a r e g r e a t l y r e d u c e d o r s t o p p e di n r e s p o n s e t o t h e e x o g e n o u s l y s u p p l i e d e l i c i t o r(F ig . 9 ) . These r e su l t s i nd ica t e t ha t l a rge changesi n t h e m e t a b o l i s m o f c e ll s u s p e n s io n c u l t u r e s a r ei n d u c e d b y t h e a c t i o n o f th e P m s e l i c i to r.

T h e a b i l i ty o f th e P m s e l i c i to r t o i n c r e a s e p h e n -y l a l a n i n e a m m o n i a - l y a s e a c t i v i t y i n s u s p e n s i o n -c u l t u r e d s o y b e a n c e l l s i s d e p e n d e n t o n t h e a m o u n to f e li c i t o r a d d e d ( F i g . 1 0 ) . M a x i m u m s t i m u l a t i o no f t h e e n z y m e a c t iv i t y r e s u l ts f r o m t h e a d d i t i o n o f1 p .g o f f r ac t ion I Pms e l i c i t o r (6 , 7 ) . A t t he t im et h e s e e x p e r i m e n t s w e r e c a r r i e d o u t , f r a c t i o n Ie l i c i to r w a s t h e m o s t p u r i f i ed e l i c i t o r p r e p a r a t i o n

a v a i l a b l e . E l i c i t o r m o l e c u l e s a c c o u n t f o r o n l ya b o u t 1 0 % o f th e m o l e c u l e s p r e s e n t i n fr a c t io nIV, and tha t i s why the spec i f i c ac t iv i ty o f t hef r ac t ion IV e l i c i t o r (F ig . 10 ) i s lower than tha t o ff r ac t ion I e l i c i t o r. S ince the ave rage molecu la rwe igh t o f f r ac t ion I e l i c i t o r i s abou t 105, concen-t r a t ions a s l ow as 10 nM e l i c i t o r r e su l t i n max i -m u m s t i m u l a t i o n o f p h e n y l a l a n i n e a m m o n i a - l y a s eac t iv i ty i n t hese susp ens ion -c u l tu red ce l l s .

T h e s h a p e o f t h e c u r v e s i n F i g . 1 0 d e s e r v ef u r t h e r c o m m e n t . T h e P m s e l i c i t o r i s a c a r b o h y -d ra t e , a s w i l l be d esc r ibed l a t e r i n t h i s a r t i c l e . Thee l i c i to r r e c e p t o r s o f p l a n t s a r e l i k e ly t o b e p r o -t e i n - c a r b o h y d r a t e - b i n d i n g p r o t e in s . C a r b o h y-

d r a t e - b i n d i n g p r o t e i n s a r e , b y d e f i n i t io n , l e c t in s .T h e a b i l i ty o f le c t in s to b i n d t o c a r b o h y d r a t e s o nthe ce l l su r f ace o f an ima l ce l l s i s we l l known (39 ,40 ) . W ha t i s pa r t i cu l a r ly i n t e re s t ing fo r t h i s d i scus -s ion abou t l ec t in s i s t he sh ape o f t he l ec t in concen-t r a t i o n c u rv e s o b t a i n e d w h e n m e a s u r i n g t h e a b i l i t y

P. ALBERSHEIMAND B. S. VALENTHost-Pathogen Interactions in Plants 631


6/17

~g OF 40

GLYCEOLLIN

ISOLATED 30

FROM

EACH 20

HYPOCOTYL

10

&

24-HOUR HYPOCOTYL ASSAY / D

FRACTION I

R a c e = o

R a c e 2 ~ &

0.1 0.2 0.4 0.8 1.6

~g OF FRACTION I ELICITOR APPLIED PER HYPOCOTYL

( 1/ug ~ 10 11 mo les )

FIGURE 7 The hypocotyl assay. The am ou nt of glyceollin extracted from hypocotyls and pu rified byth in- layer chromatography i s de termined by the absorbance a t 285 nm. A l inear re la t ionship exis ts , a tthese low elicitor concentrations, between the amount of elicitor applied and the amount of glyceollinextracted.

| |.oc' ! !

1i

10 20 30 40

HOURS

~ o o

, o o

o ~

,o o i | |

FIGURE 8 Cel l-susp ensio n assay. Elicito rs stimulatethe ac t ivi ty of p henyla lanine ammonia- lyase and theaccu mula tion of glyceollin in suspe nsion-cu ltured soy-bea n cells. Sterile Pm s elicitor was a dded at zero ho urs(20). (Fig. 8 reprinted by copyright permission from:1976;Plant Physiology,57: 777. )

o f l e c t i n s t o b i n d t o a n i m a l c e ll s u r fa c e s . T h eshape o f t he se cu rv es i s e s s en t i a l l y i den t i c a l t o t heshape o f t he e l i c i t o r a c t i v i t y cu r ve s i l l u s t r a t ed i nF i g . 1 0 . A f i n e e x a m p l e o f a l e c t i n - b i n d i n g c u r v ew i t h t h i s s h a p e i s d e s c r i b e d b y N o v o g r o d s k y a n d

A s h w e l l ( 4 5 ) w h o e x a m i n e d t h e a b i l i t y o f a l e c t i n ,t h e m a m m a l i a n h e p a t i c - b i n d i n g p r o t e i n , t o s t im u -l a t e m i t o g e n e s i s i n t h y m u s - d e r i v e d l y m p h o c y t e s .

S o y b e a n t i s s u e s r e s p o n d t o e x t r e m e l y s m a l la m o u n t s o f P m s e l i c i to r. A s l i tt l e a s 1 0 -1 2 m o l o fa n e l i c i t o r app l i ed t o a s i ng l e hy poco ty l o r co ty l e -

d o n s t i m u l a t e s q u a n t i t i e s o f g l y c e o l li n s u f fi c i e n t toi n h i b i t t h e g r o w t h o f P m s a n d o t h e r m i c r o o rg a -n i sms i n v i t r o . I t i s impre s s ive t o obse rve t hee f fe c t s o n t h e g r o w i n g s u s p e n s i o n - c u l t u r e d so y -b e a n c e l l s c a u s e d b y t h e a d d i t i o n o f s u b m i c r o m o -l a r q u a n t i t i e s o f t h e e l i c i t o r. T h e s e c e l l s r e s p o n dt o t h e s m a l l a m o u n t o f e l i c i t o r , w h i c h i s a c a r b o -h y d r a t e , e v e n t h o u g h t h e c e l l s a r e g r o w i n g i n t h ep r e s e n c e o f 5 0 m M s u c ro s e , a n o t h e r c a r b o h y -d r a t e . C l e a r l y, t h e m e t a b o l i s m o f t h e p l a n t c e l l swh ich a r e exp osed t o e l i c i to r s is d r am a t i ca l l ya l t e r e d a t e l i c i to r c o n c e n t r a t i o n s w h i c h a r e e q u i v -a l e n t t o t h e c o n c e n t r a t i o n s o f h o r m o n e s r e q u i r e df o r e f fe c t iv e m e t a b o l i c r e g u l a t i o n .

T H E C H E M I C A L N A T U R E O F T H E P M SE L I C I T O R

The P ros e l i c i t o r was f i r s t i so l a t ed and chemica l l yc h a r a c t e r i z e d f r o m o l d c u l t u r e s o f P m s . T h e e l ic i -t o r m a y h a v e b e e n r e l e a s e d i n t o t h e c u l t u r e f l u i db y a u t o l y s is ( 6 , 7 ) . I t w a s l a t e r d e m o n s t r a t e d t h a te l i c it o r - a c ti v e m o l e c u l e s w i t h t h e s a m e c h e m i c a la n d b i o l o g i c a l p r o p e r t i e s a s t h e e l i c it o r m o l e c u l e si s o l a t e d f r o m P m s c u l t u r e f l u i d c a n b e i s o l a t e df r o m m y c e l i a l w a l l s o f P m s ( 6 ) . T h e c u l t u r e f l u ida n d m y c e l i a l w a l l e l i c i t o r s a r e h e a t s t a b l e ; t h e

e l i c i t o r p r e p a r a t i o n s c a n b e a u t o c l a v e d f o r 3 h a t1 2 1 ~ w i t h o u t l o s s o f a c ti v i ty. T h e e l i c i to r m o l e -c u l e s a r e h e t e r o g e n e o u s i n s i z e , r a n g i n g f r o m am o l w t o f - 5 , 0 0 0 t o - 2 0 0 , 0 0 0 . T h e e l ic i to r l a ck si o n i z a b l e g r o u p s , a s t h e e l i c i to r d o e s n o t b i n d t oa n i o n a n d c a t i o n e x c h a n g e c o l u m n s u n d e r a v a r i-

6 3 2 T H E J O U RN A L O F C E L L B I O L O G Y- V O L U M E 78 , 1 9 7 8


7/17

Za ~

o L

Z1 , n ,

Z

n -z ~

~ w

b ~

2

1

0 / . _~

I0

8

6

-" v

b

4

2 5

i | 2 . ~1 .5

I

71 5 8 7 8AG E OF C0LT0ae IDAYS]

FIGURE 9 Cell-suspension ass ay . Pms elicitor wasadded (arrows) to 5 1/2-day-old suspension-culturedsoybean cells. The elicitor stimulates the activity ofphenylalanine ammonia-lyase (panel a) , inhibits thegrowth of the cells (panel b), and inhibits the uptake ofnitrate from the culture medium (panel c) (20). (Fig. 9reprinted by copyright permission from: 1976, PlantPhysiology,57:777.)

ety of experimental conditions. The elicitor isstable at room temperature between pH 2 and 10.The elicitor is not digested by proteases. All ofthese properties r uled out the possibility thatelicitors are proteins or nucleic acids. On the o therhand, these are the expected properties of elici-tors, if elicitors are neutral polysaccharides. In-deed, all of the Pros-produced elicitor-activemolecules examined have been found to be

glucans (5-7, and unpublished results of theauthors). The best method for obtaining largeamounts of Pms elicitor is partial acid hydrolysisof the mycelial walls (unpublished results of theauthors). The series of polysaccharides and oligo-

saccharides so obtained are extremely active aselicitors of soybea n phytoalexins.Methylation analysis of the Pms elicitors has

demonstrated that the elicitors are largely 3-1inkedpolymers with glucosyl branches to C-6 of aboutone of every three of the backbone glucosylresidues (7, and unpublished results of the au-thors). Approximately 75% of the glycosidic link-ages and 90% of the glycosyl residues in theelicitor-active glucan are hydrolyzed and releasedby an exo-B-1,3-glucanase isolated from E u g l e n agracilis (10), indicating that the Pms mycelial wallglucan is predominantly a B-linked polymer. Op-tical rotation and NMR studies have confirmed

that the glucan is B-linked (unpublishe d results ofthe authors). In fact, this elicitor-active glucan,which constitutes as much as 65% of the Pmsmycelial walls, has been d emonstra ted to be chem-ically indistinguis hable from w hat is known a boutthe structural B-glucans of the mycelial walls ofother Phy toph tho ra species (11).

The exo-fl-l,3-glucanase isolated from E. gra-

] !Lu i

i!i "

. , , * , H , , * , * J , , , ,, I * J , * . * ,

0 . 1 1 1 0 t 0 0

E L I C I T O R C O N C E N T R AT I O N ( . q J jkesose per ml)

FIguRE 10 Cell-suspension assay. The ability of thePros elicitor to stimulate phenylalanine ammonia-lyaseactivity n suspension-cultured soybean cells is dependent

on the amount of elicitor added. The open circlesrepresent fraction I Pros elicitor and the closed circlesfraction IV Pms elicitor. A more detailed explanation ofthese fractions is given in the text and in references6 and 7 (20). (Fig . 10 reprinted by copyright per-mission from: 1976, Plant Physiology,57:778.)

P. ALBERSHEIM AND B. S. VALENT Host-Pathog en Interactions in Plants 633


8/17

cilis h a s b e e n v a l u a b l e i n t h e c h a r a c t e r i z a t i o n o fthe Pm s e l i c i t o r (7 , and un pub l i sh ed r e su l t s o f t heau tho r s ) . Tha t po r t ion o f t he e l i c i t o r, wh ich i sr e l e a s e d f r o m t h e w a l l s b y a q u e o u s e x t r a c t io n a t121~ i s he t e ro gene ous in s i ze , w i th an ave rage

m o l w t o f - 1 0 0 , 0 0 0 . T h eE. grac i l i s

e n z y m eh y d r o l y z e s g l u c a n s f r o m t h e n o n r e d u c i n g e n d a n di s c a p a b l e o f h y d r o l y z i n g t h e g l y c o s id i c b o n d o ft h r e e - l i n k e d g l u c o sy l r e si d u e s t h a t h a v e o t h e r g l u -c o s y l r e s i d u e s a t t a c h e d t o C - 6 . T h e p r o d u c t o ft h e e x o g l u c a n a s e - d e g r a d e d m y c e l i a l w a l l - r e l e a s e de l i c i t o r is s ti l l he t e r ogen eous in s i ze , bu t has ana v e r a g e m o l w t o f - 1 0 , 0 0 0 ( F i g . 11 ) . T h is h ig h l yb r a n c h e d g l u e an f r a g m e n t r e t a i n s a s m u c h a c t i v i tya s th e u n d e g r a d e d e l i c i t o r. T h e p r e d o m i n a n t g l y -cos id i c l i nkages r ema in ing a f t e r ex t ens ive exog lu -canase t r ea tmen t a r e 3 -1 inked , 3 ,6 -1 inked , andte rm ina l g lucosy l l i nkages in a r a t i o o f 1 :1 :1 .S ign i f i can t amoun t s o f 4 -1 inked and 6 -1 inked g lu -

cosy i r e s idues a re a l so p re sen t . The f ac t t ha t t heexog lucanase r educes the ave rage s i ze o f t he e l i c i -t o r- ac t ive molec u le s by a f ac to r o f 10 i s ev idencetha t i t is t he g lucan cha ins t ha t posses s t he e l i c i t o rac t iv i ty.

T h e g l u c an n a t u r e o f t h e P m s e l i c it o r h a s b e e nconc lus ive ly e s t ab l i she d by expos ing the e l i c i t o r-a c t i ve o l i g o s a c c h a r i d e s , p r o d u c e d b y p a r t i a l a c i dhydro lys i s o f myce l i a l wa l l s , t o t he ac t ion o f ah i g h ly p u r i f i e d / ~ - e x o g l u c a n a s e o b t a i n e d f r o m t h ece l l wa l l s o f suspens ion -cu l tu red soybean ce l l s .T h i s e n z y m e c o n v e r t s t h e o l i g o s a c c h a r id e s t o g l u -cose wh i l e abo l i sh ing the e l i c i t o r ac t iv i ty (K . C l inea n d P. A l b e r s h e i m , u n p u b l i s h e d r e su l t s ).

120

"~ 100

> 8o

~ 6o

~ 4o

2o

o

B I D - G E L A-O.SM Vo Vi

~-1,3-EXOGLUCANASE IGESTEDELICITOR

10 20 30 40

FRACTION

FmURE 11 A /3-1,3-exoglucanase isolate d fromE u-glena gracilisreduces by 10-fold the average molecularweight of the mycelial wall-released Pms elicitor. V0 isthe void volume and V the included volume of thecolumn (7) . (Fig. 11 reprin ted by copyright permissionfrom: 1976,Plant Physiology,57:770.)

.1 6

.14

.12

O0 .08

.0 6

.0 4

.0 2

P e r i o d a t e t r e a t m e n t o f t h e w a l l - r e l e a s e d e l i c i to rh a s c o n f i r m e d t h e p o l y s a c c h a r i d e n a tu r e o f t h ea c t iv e c o m p o n e n t a n d s u g g e s t s a n e s s e n t i a l ro l e o fa b r a n c h e d o l i g o s a c c h a ri d e h a v in g t e r m i n a l g l y c o -s y l r e s i d u e s . E x p o s i n g t h e e l i c it o r to p e r i o d a t e

e l imina te s a lmos t a l l o f t he e l i c i t o r ac t iv i ty (F ig .1 2 ) . O n t h e o t h e r h a n d , a c o n s i d e r a b l e p o r t i o n o fthe e l i c i t o r ac t iv i ty i s r ega in ed i f t he pe r ioda te -d e g r a d e d p o l y m e r s a r e r e d u c e d w i t h s o d i u m b o -r o h y d r i d e a n d t h e n s u b j e c t e d t o m i l d a c id i c h y -d ro lys i s . S ince the 3 - and 3 ,6 -1 inked g lucosy lr e s i d u e s a r e r e s i s ta n t t o p e r i o d a t e d e g r a d a t i o n , i ts e e m s l i k e l y t h a t t h e p e r i o d a t e h a s d e s t r o y e d t h ee l i c i t o r ac t iv i ty by mo d i fy ing the t e rm ina l g lucosy lr e s i d u e s o r t h e q u a n t i t a ti v e l y m i n o r b u t p e r i o d a t e -suscep t ib l e 4 - and /o r 6 -1 inked g lucosy l r e s idues .H o w e v e r, r e c o v e r y o f e l i c i t o r a c t i v i t y, b y m i l da c i d h y d r o l y s i s o f t h e p e r i o d a t e - i n a c t i v a t e d e l i ci -t o r , p o i n t s t o p e r i o d a t e a t t a c k o n t h e t e r m i n a l

g lucosy l r e s idues a s t he cause fo r p e r iod a te i nac t i -v a t i o n o f t h e e l i c i t o r. T h e d e g r a d a t i o n o f 4 - o r 6 -l i nked g lucosy l r e s idues wou ld l ead to sp l i t t i ng o ft h e g l u c a n c h a i n , w h i l e p e r i o d a t e d e s t r u c t i o n o ft e r m i n a l g l u c o sy l r e si d u e s f o l l o w e d b y m i l d a c i dh y d r o l y s i s c o u l d l e a d t o t h e e x p o s u r e o f n e wt e r m i n a l g l u c o s y l r e si d u e s w h i c h m i g h t p r o v i d ethe p rope r s t ruc tu re o f an ac t ive e l i c i t o r.

The r equ i r em en t fo r e l i c i t o r ac t iv i ty o f ab r a n c h e d o l i g o s a c c h a r id e is s u p p o r t e d b y t h e o b -se rva t ion tha t 3 -1 inked f l -g lucans wh ich l ackbranches to C-6 o r have on ly a s ing le C-6

FRACT ION I \

A F T E R M I L D A C I DO HYDROLYSIS

- - A F T E R _ ~ P E R I O D AT E-- -

.1 .2 .3 .4 .5

Jug APPLIED PER COTYLEDON

FIGURE 12 Peri oda te treat me nt eliminates almost allof the activity (closed circles) of the Pms elicitor (trian-gles). A considerable port ion of the elic itor activity isrecovered if the perioda te-treated elicitor is reduced withsodium borohydride and then subjected to mild acidichydrolysis (open circles).

6 3 4 T H E J O U R N A L O F C E L L B I O L O G Y " V O L U M E 7 8 , 1 9 7 8


9/17

b r a n c h e d g l u c o s y l r e s i d u e , s u c h a s l a m i n a r i n , h a v el i t t l e o r no e l i c i t o r ac t iv i ty ( l e s s t han one thou-%s a n d t h o f t h e P m s e li c i t o r ) . I n d e e d , a s e r ie s o f r ,c o m m e r c i a l l y a v a i l a b le p o l y s a c c h a r i d e s , o l i g o s a c- -c h a r i d e s , m e t h y l g l y c o s i d e s, a n d s i m p l e s u g a r s

h a v e b e e n t e s t e d f o r e l i c i t o r ac t i v it y, a n d , b e s i d e sl a m i n a r i n , t h e o n l y c o m m e r c i a l l y a v a i l a b l e p r o d -u c t f o u n d w i t h d e t e c t a b l e e l i c i t o r a c t iv i t y w asn i g e r a n , a m y c e l i a l w a l l g lu c a n f r o m t h e f u n g u s ,Aspergi l lus n iger(20 ) .

A m a j o r g o a l o f o u r r e s e a r c h i s t h e d e t e r m i n a -t i o n o f t h e d e t a i l e d m o l e c u l a r s t r u c t u r e o f t h eac t ive - s i t e o f t he P ms e l i c i t o r. I t i s expe c ted tha tth i s goa l w i l l be ach ieved by the i so l a t ion ands t r u c t u r a l c h a r a c t e r i z a ti o n o f t h e s m a l l e s t p o s s i b l ee l i c i t o r- ac t ive o l igosac cha r ide wh ich can be de -r ived f rom the g lucan e l i c i t o r. A r e l a t ive ly sma l le l i c i to r - a c t i v e o l i g o s a c c h a r i d e h a s b e e n p r o d u c e dby pa r t i a l ac id hydro lys i s o f P ros myce l i a l wa l l s .

T h e s e r i e s o f o li g o s a c c h a r i d e s o b t a i n e d b y t h i sp a r t i a l h y d r o l y s i s h a v e b e e n p a r t i a l l y r e s o l v e d ,f i r s t , by low- reso lu t ion (F ig . 13 ) and then by h igh -r e s o l u t i o n ( F i g. 1 4 ) B i o - G e l P - 2 g e l p e r m e a t i o nc h r o m a t o g r a p h y ( B i o - R a d L a b o r a t o r i e s , R i c h -m o n d , C a l i f . ). O l i g o s a c c h a r i d e s c o n t a i n i n g a s f e was n ine g lucosy l r e s idues s t i l l r e t a in e l i c i t o r ac t iv -i t y. G l u c o s e i s t h e o n l y d e t e c t e d c o m p o n e n t o ft h e s e o l i g o s a c c h a r i d e s.

T h e s m a l l e s t e l i c i t o r -a c t i v e o li g o s a c c h a r i d e - co n -t a in ing f r ac t ion f rom the h igh r e so lu t ion P -2 co l -u m n ( F i g . 1 4 ) h a s b e e n s u b d i v i d e d b y h i g h p r e s -s u r e l i q ui d c h r o m a t o g r a p h y ( F i g . 1 5 ) i n t o a t l ea s tf ive o l igosacc ha r ide f r ac t ions . Two o f t he f ive

o l i g o s a c c h a r i d e f r a c t i o n s o b t a i n e d b y h i g h p r e s -

"--O3. O

g

2 ,0

1.O

OO

B I O ' G E L P - 2C H R O M AT O G R A P H Y

2 0 0 4 0 0 6 0 0

E L U T I O N V O L U M E ( M L )

Elicitor-active oligosaccharides are pro-mtrRE 13duced by partial acid hydrolysis of Pms mycelial walls.The series of oligosaccharides obtained are partiallyresolved by low-resolution gel permeation chromatogra-phy. The smallest elicitor-active oligosaccharides areincluded in the shaded column fractions.

B I O - G E L P - 2CHROMA1OGRAPHy

5 0 7 0 9 0 11 0

E L U T I O N V O LU M E ( M L )

The smallest elicitor-active oligosaccha-mt~RE 14rides (shaded fraction illustrated in Fig. 13) are furtherresolved by high-resolution gel perm eation chromatog-raphy. The smallest elicitor-active oligosaccharides areincluded in the shaded column fractions.

| H I G H - P R E S S U R E LIQUID

CHROMATOGRAM CF 1=-2E LI (~TOR - AC 'n V E

E L U T O N V O L U M E D

FIGURE 15 The smallest elicitor-active oligosaccha-tides from the high resolution gel permeation column

(shaded fraction illustrated in Fig. 14) are further re-solved by high-pressure liquid chromatography on acommercially available "carb ohyd rate" column usingacetonitrile-water as a solvent. The refractive indexcurve is propor tional to the carbohy drate content of thecolumn effluent. O f the four largest carbohydrate peaks,the two last eluting from the column contain activeelicitors.

s u r e l i q u i d c h r o m a t o g r a p h y c a n b e d e g r a d e d b yt r e a t m e n t o f t h e m i x t u r e o f o l i g o s a c c h a r i d e s w i t hthe E. gracilis e x o - f l - l , 3 - g l u c a n a s e . T h e r e a p p e a r sto be l i t t l e l o s s o f e l i c i t o r ac t iv i ty a f t e r t r e a tm en tw i t h t h e e x o g l u c a n a s e . O f t h e t h r e e o l i g o s a c c h a -r i d e f r a c t io n s r e m a i n i n g a f t e r e x o g l u c a n a s e t r e a t -me n t , two have e l i c i t o r ac t iv i ty. The pu re s t e l i c i-t o r - a c t i v e o l i g o s a c c h a r i d e f r a c t i o n o b t a i n e d b yh i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y s ti l l c o n t a i n sa t l e a s t t w o d i s t i nc t o l i g o s a c c h a r id e s . M e t h y l a t i o nana lys i s o f t h i s pu res t e l i c i t o r- ac t ive o l igosaccha -

P. ALBERSHEIMAND B. S. VALENTHosvP athogen Interactions in Plants 635


10/17

ride indicates an approximate composition of two6-1inked, two 3-1inked, two 3,6-1inked, and threeterminal glucosyl residues. Indir ect evidence sug-gests to us that the 4-1inked glucosyi residuesdetected in the sample are part of a contaminating

inactive oligosaccharide. One possible, very ten-tative structure of this elicitor-active oligosaccha-ride is illustrated in Fig. 16 (unpu blished results ofthe authors). Further purification is required be-fore a definitive structure of an elicitor-activeoligosaccharide can be obtaine d.

ELICITORS LACK RACE SPECIFICITY

The three Pms races (races 1, 2, and 3) aredistinguished by their differing abilities to infectvarious soybean cultivars. However, the elicitorsof phytoalexin acc umulation are not the specificitydetermining factors in the Pros-soybean system.The elicitor obtained from each of the three Pms

races purifies in exactly the same ma nner , and allthe discernible structural features of the elicitorsfrom the three races are identical (7). The activi-ties of the elicitors purified from the three Pmsraces were carefully examined using the threeseparate bioassays: the cotyledon assay (Fig. 17)(5), the hypocotyl assay (Fig. 18) (5), and the cellsuspension-culture assay (20). All three assaysgive the same results, that is, the activities of theelicitors from different Pms races are identical (7).This is true for low levels of applied elicitor as wellas for levels of elicitor which stimulate maximumglyceollin accumula tion (Figs. 7, 17, and 18).Therefore, the three races of Pms are equally

O O

H

FIGURE 16 One possible, very tentative structure of anelicitor-active oligosaccharide is illustrated. Further pu-rification of the elicitor-active oligosaccharides is re-quired before a definitive structure can be obtained.

E

zo

.t ->t -

O

ul

3 .0

2 s

1s RACE 1 9RACE 2 oR A C E 3 9

I |

0 0.5 1.0

p g E L I C I T O R A P P L I E D P ER C O T Y L E D O N

FIGURE 17 The elicitors purified from three differentPms races have an identical ability to stimulate glyceollinaccumulation in soybean cotyledons.

150

>,I, -0

00..

z 1 0 0 ,

&_z

,,J

o 5 0

1 2 3 4

J ug E L I C I T O R A P P L I E DPER H Y P O C O T Y L

FmuRE 18 The elicitors purified from three differentPms races have an identical ability to stimulate glyceoblin accumulation in soybean hypocotyls.

effective at stimulating phytoalexin accumulationin the tissues of susceptible and resistant cultivarsof their host. The results contradict the earlierreport of Keen (31) which claimed that the Pmselicitors are race specific.

The results of another type of experiment sup-

63 6 THE JOURNAL OF CELL BIOLOGY " VOLUME 78 , 19 78


11/17

p o r t o u r c o n c l u s io n t h a t e l i c it o r s a r e n o t r e s p o n s i -b l e fo r r ace - spec i f i c r e s i s t ance in t he Pm s-soyb eans y s t e m . S o y b e a n h y p o c o t y l s a c c u m u l a t e g l y c e o ll i nw h e n i n o c u l a t e d w i t h l i v in g m y c e l i a o f P m s . T h erespon se , wh ich i s cha rac t e r i s t i c o f na tu ra l i n fec -

t i ons wi th e i the r an in fec t ive o r a non in fec t ive r aceo f Pms , i s r e t a ined wi th th i s i nocu la t ion t ech -n i q u e . W e h a v e c o m p a r e d t h e r e l a t iv e e ff e c t iv e -nes s o f l i ve myce l i a and pu r i f i ed e l i c i t o r in s t imu-l a t ing g lyceo l l in accum ula t ion (6 ) . The r e su l t i st h e f o l l o w i n g : T h e o n s e t a n d t h e r a t e o f g l y c eo l l i naccum ula t ion in s eed l ings inocu la t ed wi th in fec t ive( c o m p a t i b l e ) m y c e l i a i s i n d i st i n g u i s h a b le f r o m t h eo n s e t a n d r a t e o f g l y c e o l l i n a c c u m u l a t i o n i n s e e d -l i ng s i n o c u l a t e d w i t h e i t h e r n o n i n f e c t i v e ( i n c o m -p a t i b l e ) m y c e l i a o r p u r i f i e d e l i c i t o r ( F i g . 1 9 ) .T h e s e r e s u l ts d e m o n s t r a t e t h a t d i f f e r e n c e s i n t h er a t e s o f g l y c e o l li n a c c u m u l a t i o n i n r e s p o n s e t od i f f e r e n t ra c e s o f P m s a r e n o t l i k e l y t o a c c o u n t f o r

t h e r e s i st a n c e o r s u s c e p t ib i l i ty o f v a r i o u s s o y b e a ncu l t i va r s t o t he Pm s r aces .T h e a v a i l a b l e e v id e n c e d o e s i n d i c a t e t h a t e l i ci -

t o r s have a ro l e i n r e s i s t ance even though they a re

1 0 0

8 OU

g,I z

o 6 o

E

Z

,. J

9J 4 0oW

2 0

00

9 I N C O M P A T I B L E PM S

9 C O M P AT I B L E P M S

O E L I C I T O R

D

I J I

3 6 9 1 2 1 5

H O U R S

FiGum~ 19 The o nset and the rate of glyceollin accu-mulation in the hypocotyls of soybean seedlings inocu-lated with the mycelia of an infective (compatible) raceof Pms is indistinguishable from the onset and rate ofglyceollin accumulation in seedlings inoculated e itherwith a noninfective (incompatible) race of Pms or withpurified Pms elicitor (6). (Fig. 19 re printed by copyrightpermission from: 1976,Plant Physiology,57:764.)

n o t d e t e r m i n a n t s o f r a c e s p e c i f i c it y. T h e e l i c i to ri s o l a t ed f r o m P m s i s c a p a b l e o f p r o t e c t i n g s o y b e a nh y p o c o t y i s fr o m i n f e c ti o n b y a n o r m a l l y i n f e c ti v er a c e o f P m s i f t h e e l i c it o r is a p p l i e d t o t h eh y p o c o t y l s 6 h b e f o r e i n o c u l a t i o n w i th P m s ( 6 ,

a n d u n p u b l i s h e d r e s u l t s o f t h e a u t h o r s ) . T h e e l ic i -t o r c a n n o t p r o t e c t s o y b e a n t i s s u e w h e n a p p l i e ds imul t an eous ly wi th an in fec t ive r ace o f Pms (F ig .19 ) .

E L I C IT O R S A R E W I D E S P R E A DI N N AT U R E

S o y b e a n p l a n t s h a v e e v o l v e d t h e a b i l i t y to r e c o g -n ize and r e spo nd to t he s t ruc tu ra l f l -g lucan o fPhy toph tho ra my ce l i a l wa l l s . S imi l a r f l -g lucansa re found in t he wa l l s o f a w ide r ange o f fung i(11 ) . On e fu ngus con ta in ing such /3 -glucans i sb r e w e r ' s y e a s t ,S. cerevis iae , a n o n p a t h o g e n o fp l a n t s . A n e l i c i t o r h a s n o w b e e n p u r i f i e d fr o m a

c o m m e r c i a l l y a v a i l a b l e e x t r a c t o f b r e w e r ' s y e a s t( D i f c o L a b o r a t o r i e s , D e t r o i t , M i c h . ) ( 2 5 ) . T h e8 0 % e t h a n o l i n s o l u b l e f ra c t i o n o f t h e y e a s t e x t r a c tcon ta ins a v e ry ac t ive e l i c i t o r o f g lyceo l l i n accu -m u l a t i o n i n s o y b e a n s . M o s t o f t h e p o l y s a c c h a r i d ei n t hi s 8 0 % e t h a n o l i n s o l u b l e f r a c t i o n i s m a n n a n .H o w e v e r, y e a s t e x t r a c t d o e s c o n t a i n s m a l lam oun t s o f t he /3 -g lucan . The g lucan can bea l m o s t c o m p l e t e l y s e p a r a t e d f r o m t h e m a n n a n b yb i n d i n g th e m a n n a n t o a n a f f i n i ty c o l u m n c o n s i s t-i n g o f c o n c a n a v a l i n A c o v a l e n t l y a t t a c h e d t o S e p h -a r o s e . T h e g l u c a n c a n b e s e p a r a t e d f r o m g l y c o p r o -t e i ns b y b i n d i n g t h e p r o t e i n s t o s u l f o p r o p y l - S e p h -a d e x . B o t h t h e p u r i f i e d m a n n a n a n d p u r i f i e d

g l u c a n r e m a i n c o n t a m i n a t e d b y s m a l l a m o u n t s( = 2 % ) o f a r a b i n o g a l a c t a n . R i b o s e - c o n t a i n i n gp o l y m e r s , w h i c h c o n t a m i n a t e t h e 8 0 % e t h a n o li n s o lu b l e fr a c t i o n , a r e r e m o v e d o n a d i e t h y l -a m i n o e t h y l - c e l l u l o s e c o l u m n . T h e e l i c i t o r ac t i v it yo f th e c r u d e 8 0 % e t h a n o l p r e c i p i t a t e o f y e a s te x t r a c t r e si d e s i n t h e g l u c a n c o m p o n e n t ( F i g . 2 0 ) .T h e s m a l l a m o u n t o f r e s i d u a l a c t i vi t y r e m a i n i n gi n t h e m a n n a n f r a c t i o n c a n b e a t t r i b u t e d t o t h eo b s e r v e d c o n t a m i n a t i o n o f t h i s f r a c t i o n b y t h eg l u c a n ( 2 5 ). T h e g l u c a n is c o m p o s e d o f t h e s a m eg lucosy l l i nkages found in t he Pm s e l i c i t o r (F ig .2 1 ) .

T h e s a m e q u a n t i t i e s o f th e y e a s t a n d P m s

e l i c it o r s a r e r e q u i r e d f o r 5 0 % o f m a x i m u m s t i m u -l a t i on o f g l y c e o l l i n a c c u m u l a t i o n i n t h e c o t y l e d o n s( F i g . 22 A ) a n d h y p o c o t y l s ( F ig . 2 2 B ) o f s o y b e a ns e e d l i n g s ( F i g . 2 2 ) . A l t h o u g h t h e y e a s t e l i c i t o rf a il s t o s t i m u l a t e t h e s a m e m a x i m u m l e v e l o fg l y c e o ll i n a c c u m u l a t i o n i n t h e s o y b e a n t i s s u e s a s

P. ALBERSHEIMAND B. S. VALENTHost-Pathogen Interactions in Plants 637


12/17

E

1 .2

Documents

PRINT VIP Host Parasite Interaction