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7 Biochemical and Ultrastructural Aspects of Southern

Downloaded by COLUMBIA UNIV on November 29, 2017 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

Corn Leaf Blight Disease PETER GREGORY, ELIZABETH D. EARLE, and VERNON E. GRACEN Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY 14853

The study of southern corn leaf blight disease is of major economic and scientific value. The economic importance of this fungal disease was dramatically emphasized in 1970 when it reached nearly epidemic proportions in the U.S.A., resulting in severe losses for corn growers. Scientifically, research on the disease may result in novel and major gains in plant pathology, plant breeding and higher plant genetics. The main aims of this paper are to critically describe the present state of biochemical and ultrastructural knowledge about the mechanism of the disease, to highlight the most important gaps in our knowledge and to discuss some of the ways in which these gaps can be filled. General Characteristics of Southern Corn Leaf Blight Disease The causal organism of southern corn leaf blight disease is the fungus Helminthosporium maydis Race Τ (HmT). This is one of several members of the genus Helminthosporium to produce host -specific toxins. HmT and the toxin(s) from HmT (HmT toxin) only affect corn which contains the Texas (T) source of male sterile cytoplasm. Plants containing the non-male sterile (N) or the C or S types of male sterile cytoplasms are resistant to the fungus and insensitive to HmT toxin (1,2). Nuclear genes which restore Τ cytoplasm to male fertility (Rf genes) may slightly modify the disease reaction and toxin response of corn varieties containing the Τ cytoplasm (3,4). Nuclear genes other than Rf genes can affect the HmT disease reaction (5,6,7) with some inbred lines exhibiting a higher level of resistance in Τ cytoplasm than others. In all cases, however, each inbred is more susceptible in Τ than in Ν cytoplasm. Toxin sensitivity of different inbred lines in Τ cytoplasm also varies but there is no apparent correlation between nuclear control of toxin sensitivity and the degree of disease resistance (8). It is of considerable interest that another race of H. maydis, Race 0, produces a toxin but shows no specificity for Τ cytoplasm ( 9. ) . Η. maydis Race Τ might essentially represent a Race 0 90 Hedin; Host Plant Resistance to Pests ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

7.

GREGORY

ET

AL.

Southern

Corn

Leaf Blight

i s o l a t e that has accumulated a d d i t i o n a l s p e c i f i c t o x i n production ( 9 ,10). The s p e c i f i c a l l y damage Τ cytoplasm i n the gives southern corn l e a f b l i g h t disease model system i n p l a n t pathology.


E f f e c t s of HmT

Disease

91

genes f o r cytoplasmf a c t that HmT t o x i n can absence of the fungus (11) great p o t e n t i a l as a

Toxin on Τ Cytoplasm

Treatment with HmT t o x i n produces morphological, p h y s i o l o g i c a l , u l t r a s t r u c t u r a l and biochemical changes i n Τ cytoplasm m a t e r i a l . Table I l i s t s these e f f e c t s , seen i n systems ranging i n complexity from whole p l a n t s to s u b c e l l u l a r f r a c t i o n s . The e f f e c t s l i s t e d are s p e c i f i c f o r Τ cytoplasm; Ν cytoplasm m a t e r i a l i s e i t h e r u n a l t e r e d by any t o x i n c o n c e n t r a t i o n t e s t e d or i s a f f e c t e d l e s s than Τ cytoplasm at a given c o n c e n t r a t i o n . Non s p e c i f i c e f f e c t s (e.g. carbohydrate leakage (12)) are not i n c l u d e d i n Table I. I t can be seen i n Table I that the timing, s e n s i t i v i t y and degree o f s p e c i f i c i t y of the d i f f e r e n t e f f e c t s vary considerably. The r e l a t i v e importance o f the r e p o r t e d e f f e c t s i s d i f f i c u l t to evaluate. Some e f f e c t s , such as i n h i b i t i o n of r o o t growth and changes i n i s o l a t e d mitochondria, have been observed by many workers; others have been documented only by work from a s i n g l e l a b o r a t o r y or even by a s i n g l e p u b l i c a t i o n . Comparison of work i n d i f f e r e n t l a b o r a t o r i e s i s d i f f i c u l t because no standard pro cedures f o r p r e p a r a t i o n and assay of HmT t o x i n e x i s t . Many d i f f e r e n t schemes f o r t o x i n production and p u r i f i c a t i o n are i n use (Table I I ) . Toxin a c t i v i t y i n the i n i t i a l t o x i n source i s i n f l u e n c e d by c u l t u r e c o n d i t i o n s and the f u n g a l i s o l a t e used, but the i n i t i a l t o x i n a c t i v i t y i s r a r e l y r e p o r t e d . The e f f e c t i v e n e s s of the p u r i f i c a t i o n procedures, the l e v e l of p u r i t y a t t a i n e d and the amount of n o n s p e c i f i c m a t e r i a l remaining are u s u a l l y d i f f i c u l t to assess. The use of d i f f e r e n t corn l i n e s i n d i f f e r e n t s t u d i e s or even as the source of Ν and Τ cytoplasm i n a g i v e n experiment (17) causes f u r t h e r c o m p l i c a t i o n s . In s p i t e o f problems i n i n t e r p r e t a t i o n and comparison, s t u d i e s of t o x i n e f f e c t s on Τ cytoplasm serve s e v e r a l purposes. 1) They provide convenient c r i t e r i a f o r i d e n t i f y i n g Τ cytoplasm and Τ cytoplasm components i n many d i f f e r e n t systems, a t the f i e l d l e v e l , i n seed or p o l l e n populations or i n c e l l c u l t u r e s . Usually, only Τ cytoplasm m a t e r i a l i s s e n s i t i v e to t o x i n . Other male s t e r i l e and non-male s t e r i l e cytoplasms are r e s i s t a n t . Pro cedures that a l t e r the usual response of Ν and Τ cytoplasm to t o x i n may y i e l d m a t e r i a l which w i l l f u r t h e r understanding of the d i f f e r e n c e between the two cytoplasms. Because c a l l u s from Τ cytoplasm seeds u s u a l l y f a i l s to grow on n u t r i e n t medium c o n t a i n ing t o x i n , the t o x i n r e s i s t a n t c a l l u s o c c a s i o n a l l y seen i n Τ cytoplasm c u l t u r e s i s easy to i s o l a t e f o r f u r t h e r a n a l y s i s (25, 29). Biochemical and genetic a n a l y s i s o f such t o x i n - r e s i s t a n t T c a l l u s i s now i n progress i n our l a b o r a t o r y and i n s e v e r a l other laboratories. S i m i l a r l y f a i l u r e of Τ cytoplasm p r o t o p l a s t s to !

Hedin; Host Plant Resistance to Pests ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

T

92

HOST P L A N T RESISTANCE T O PESTS

Table I .

E f f e c t s of H. maydis Race Τ t o x i n on Τ cytoplasm material

Toxin a p p l i c a t i o n ( i f not i n ambient s o l u t i o n

Effect

Timing

References

P L A N T S sprayed


injected stem

into

p i p e t t e d onto l e a f who r 1 , imma ture t i s s u e , punctured w i t h needle

l e s i o n formation

3-6 days

(13)

chlorosis, necrosis of leaves

2-4 days

c h l o r o t i c streaks

2-5 days

(10,14,15)

24-48hours

(1,15,16, 17,18)

(13,4(0

S E E D L I N G S i n h i b i t i o n of r o o t growth

30 min

(il)

i n h i b i t i o n of u r a n y l uptake i n t o vacuoles of r o o t c e l l s

21-28 hours

(19)

s t i m u l a t i o n of e l e c t r o l y t e leakage

1-4 hours

(iZ)

u l t r a s t r u c t u r a l damage to mitochondria i n root c e l l s

15-120 min

(20)

90 min f a i l u r e of root mito chondria to respond to deoxyglucose treatment d e p o l a r i z a t i o n of mem brane p o t e n t i a l d i f ference of root c e l l s D E T A C H E D l e a f f l o a t e d on toxin

2-5 min

L E A V E S - L E A F

water soaking

(21)

(22)

D I S C S 3-4

days

(1)

i n j e c t e d i n t o l e a f / l e s i o n formation

3 days

(23)

a p p l i e d to p u n c t u r e / l e s i o n formation

12 hours 48 hours

(24)

3 days

(18)

cut end i n t o x i n

chlorophyll retention i n dark


(11)

7.

Southern

GREGORY E T A L .

Table I .

Corn

Leaf Blight

93

Disease

(cont.)

Toxin a p p l i c a t i o n ( i f not i n ambient s o l u t i o n )

Timing

Effect

a

References

3 days

leaf piece floated on t o x i n

chlorophyll i n dark

leaf

i n h i b i t i o n o f dark C0 fixation

9-10 hours

i n h i b i t i o n of l i g h t C0 fixation

9 hours

(18)

i n h i b i t i o n of l i g h t CO f i x a t i o n

15-60 min

(26)

stimulation of elec t r o l y t e leakage

7-23 hours

(27)

discs

retention

(25) (15,18)

2

leaf

discs


2


2


3 hours

(19)

vacuum i n f i l t r a s t i m u l a t i o n o f e l e c t r o - 2 hours t i o n of l e a f pieces l y t e leakage

(12)

epidermal peels f l o a t e d on t o x i n

inhibition ο f l i g h t stimulated Κ uptake by guard c e l l s

3 hours

(26)


i n h i b i t i o n of trans piration

3 hours

(19)

5-60 min

(26)

I S 0 L A T E D

R O O T S

s t i m u l a t i o n of e l e c t r o l y t e leakage i n h i b i t i o n of uptake t o x i n present during a e r a t i o n of roots

8 6

Rb

i n h i b i t i o n of develop ment o f augmented R b uptake

2 hours (12) (10 min i n 1 expt)

(Z)

90 min 2 hours

(28,47)

28 days

(25,29)

86

C A L L U S i n agar medium

i n h i b i t i o n o f growth

P O L L E N (from Τ cytoplasm p l a n t s w i t h TRf genes) i n agar medium

i n h i b i t i o n of germin a t i o n and p o l l e n tube growth

2 hours


(30)

HOST

94

Table I .

PLANT

RESISTANCE T O PESTS

(cont.) Timing

References

i n h i b i t i o n of volume increase

1 day

(31)

collapse

1-3 days

(31.32)

u l t r a s t r u c t u r a l damage to mitochondria

5-60 min

(20.33)

Effect



P R O T O P L A S T S

I S O L A T E D

M I T O C H O N D R I A

uncoupling o f o x i d a t i v e phosphorylation s t i m u l a t i o n o f NADH oxidation

(34-39) (15,23,34 35,37,38 40,41)

i n h i b i t i o n of malatepyruvate o x i d a t i o n

(15,34,35 37,38,40 41)

i n h i b i t i o n o f a-ketoglutarate oxidation

(37,41)

p a r t i a l i n h i b i t i o n of succinate oxidation ( i n sucrose o r KC1 assay media)

(15_,34,35 38)

stimulation of succinate o x i d a t i o n ( i n mannitol or sucrose assay media)

(41,42)

s t i m u l a t i o n of ATPase activity

(38,41)

a c t i v a t i o n o f cytochrome oxidase

(41)

a c t i v a t i o n of s u c c i n a t e cytochrome C reductase 32 Ρ acinhibition of cumulation

(41)

increase i n l i g h t trans mit tance ( s w e l l i n g )

(17,38) (17,23,34 43)


7.

GREGORY E T A L .

Table I .

Southern

Corn Leaf

Blight

95

Disease

(cont.)


Effect

Timing

3

References

(33,43)

u l t r a s t r u c t u r a l changes (swelling, disruption of inner membranes) M I C R O S O M E S +


i n h i b i t i o n of K stimulated ATPase a

30-60 min

(44,45)

are noted approximate time from exposure to t o x i n u n t i l e f f e c t s and/or measured e f f e c t s seen w i t h i n seconds o r a few minutes


HOST P L A N T RESISTANCE T O PESTS

96 Table I I .

Toxin Preparations Used i n P h y s i o l o g i c a l , Biochemical and U l t r a s t r u c t u r a l Studies Processing

i n f e c t e d leaves

b o i l i n g water e x t r a c t i o n

(30)

methanol e x t r a c t i o n , d i a l y s i s

(11)

methanol e x t r a c t i o n

(14)

methanol, e t h y l acetate ex traction

(12)

methanol, e t h y l acetate ex t r a c t i o n , chromatography (chloroform-methanol), e t h y l acetate e l u t i o n

(37,39,40, 41,44,45)

H. maydis race Τ mycelium + agar c u l ture medium Downloaded by COLUMBIA UNIV on November 29, 2017 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

References

I n i t i a l Toxin Source

(abridged)

H. maydis race Τ mycelium + c u l t u r e filtrate

chloroform e x t r a c t i o n o f ground mycelium and c u l ture f i l t r a t e

H. maydis race Τ c u l ture f i l t r a t e

none

(18)

(.3,11,13,16, 46)

dialysis desalting,

(1,34) ultrafiltration

(17,23,26, 43,70)

desalting, u l t r a f i l t r a t i o n , p a r t i a l Sephadex p u r i f i c a t i o n

(17)

f r e e z e d r y i n g , methanol ex traction

(19)

e t h y l acetate e x t r a c t i o n

(29)

methanol-water, chloroform extraction methanol-water, butanol extraction chloroform

extraction

(8,15,38)

(28,47)

(7,20,31, 32,33)


7.

GREGORY E T A L .

Southern

Corn

Leaf Blight

97

Disease

1

s u r v i v e t o x i n treatment may permit d e t e c t i o n of 'T p r o t o p l a s t s whose response to t o x i n has been a l t e r e d by genetic manipulations l i k e p r o t o p l a s t f u s i o n or o r g a n e l l e t r a n s f e r . 2) They provide a v a r i e t y o f assays f o r HmT t o x i n a c t i v i t y . S e n s i t i v e bioassays are e s s e n t i a l both f o r rigorous s t u d i e s o f t o x i n a c t i o n and f o r t o x i n p u r i f i c a t i o n procedures. E v a l u a t i o n of a s i n g l e t o x i n p r e p a r a t i o n i n assays i n v o l v i n g succinate, NADH or m a l a t e - d i c h l o r o phenol indophenol (DCPIP) r e s p i r a t i o n by i s o l a t e d mitochondria and dark C 0 f i x a t i o n by l e a f d i s c s showed a q u a n t i t a t i v e i n h i b i t i o n by the t o x i n (15). Moreover, these assays are 5-10X as s e n s i t i v e to t o x i n as the s e e d l i n g root growth assay and 20-100X as s e n s i t i v e as l e a f l e s i o n assays (15). I n h i b i t i o n of p r o t o p l a s t s u r v i v a l i s a l s o 5-10X as s e n s i t i v e to t o x i n as s e e d l i n g root growth (32). E l e c t r o l y t e leakage i s one o f the l e a s t s e n s i t i v e bioassays (18, 19). 3) They provide information about the mode of t o x i n a c t i o n i n Τ cytoplasm c e l l s . The slower e f f e c t s l i s t e d i n Table I are probably secondary e f f e c t s , f a r removed from the i n i t i a l i n t e r a c t i o n of t o x i n with Τ cytoplasm. Primary e f f e c t s should be r a p i d , h i g h l y s p e c i f i c f o r Τ cytoplasm and a t l e a s t as s e n s i t i v e to t o x i n as l a t e r e f f e c t s . I t should a l s o be p o s s i b l e to r e l a t e the primary e f f e c t s to the observed cytoplasmic i n h e r i t a n c e o f t o x i n s e n s i t i v i t y . Both mitochondria and plasma membranes have been i m p l i c a t e d as primary s i t e s o f e a r l y t o x i n a c t i o n .


2

Evidence f o r the Plasma Membrane as a S i t e of Toxin A c t i o n Some p h y s i o l o g i c a l s t u d i e s suggest that plasma membranes and mi tochondrial membranes are a f f e c t e d by HmT t o x i n . Evidence f o r plasma membrane e f f e c t s i s based on observations t h a t a f t e r t o x i n treatment e l e c t r o l y t e leakage occurs (12,17,19,27), Κ or R b uptake i s i n h i b i t e d (7,28,47), l i g h t - s t i m u l a t e d K+ movement i n t o guard c e l l s i s i n h i b i t e d (26), p a r t i a l d e p o l a r i z a t i o n o f the plasma membrane p o t e n t i a l d i f f e r e n c e occurs (22), and K*" stimu l a t e d ATPase from corn root microsomes i s i n h i b i t e d (44,45). This l a t t e r p o i n t i s s i g n i f i c a n t because microsomal p r e p a r a t i o n s from oat roots contain fragments o f plasma membranes ( 4 8 ) . Because some o f these e f f e c t s occur w i t h i n minutes a f t e r t o x i n treatment (12,22) and because of the t o x i n e f f e c t on micro somal K stimulated ATPase, i t has been suggested that HmT t o x i n acts d i r e c t l y on plasma membranes. However the importance o f some of the observations i m p l i c a t i n g plasma membranes i s ques t i o n a b l e because of the weak s p e c i f i c i t y f o r Τ cytoplasm (28,47), the p r e l i m i n a r y nature o f the r e p o r t (22), the i n a b i l i t y o f other workers to repeat the microsomal ATPase r e s u l t s (49), and the high l e v e l s o f crude t o x i n o f t e n needed. There i s the f u r t h e r problem o f i n t e r p r e t i n g cytoplasmic i n h e r i t a n c e o f d i f f e r e n t i a l membrane s e n s i t i v i t y . When a h i g h l y a c t i v e , Τ c y t o p l a s m - s p e c i f i c chloroform e x t r a c t a b l e t o x i n i s used, no u l t r a s t r u c t u r a l damage to plasma membranes of Τ p r o t o p l a s t s i s seen a t times when m i t o c h o n d r i a l d i s r u p t i o n i s +

86

+



98

HOST P L A N T


apparent (20,33). Although crude HmT c u l t u r e f i l t r a t e caused plasma membrane damage i n Ν and Τ corn (50), the l a t t e r u l t r a s t r u c t u r a l e f f e c t s could be due to the presence o f contaminants i n HmT crude f i l t r a t e . I t seems p o s s i b l e that some o f the observed plasma membrane e f f e c t s may r e s u l t from p r i o r m i t o c h o n d r i a l i n a c t i v a t i o n . We cannot e l i m i n a t e the p o s s i b i l i t y that r a p i d and s p e c i f i c e f f e c t s on plasma membranes occur but are below the l e v e l o f u l t r a s t r u c t u r a l r e s o l u t i o n , although we a l s o have p r e l i m i n a r y f r e e z e - e t c h data that f a i l s to d e t e c t plasma membrane damage with chloroform-extracted t o x i n (50). At present, i t appears that s p e c i f i c i t y of HmT t o x i n f o r Τ cytoplasm r e s i d e s i n i t s i n t e r a c t i o n with mitochondria. I d e n t i f i c a t i o n of the d i f f e r e n c e s between Ν and Τ mitochondria which l i m i t the e f f e c t of t o x i n to Τ mitochondria and the mechanism o f a c t i o n o f HmT t o x i n on Τ mitochondria i s o f c o n s i d e r a b l e i n t e r e s t . Evidence f o r Mitochondria as a S i t e o f Toxin A c t i o n The cytoplasmic i n h e r i t a n c e o f t o x i n s e n s i t i v i t y suggests that an a l t e r e d m i t o c h o n d r i a l o r c h l o r o p l a s t genome i s i n v o l v e d . A c h l o r o p l a s t s i t e seems u n l i k e l y both because no e f f e c t of t o x i n on enzyme a c t i v i t i e s i n i s o l a t e d c h l o r o p l a s t l a m e l l a e has been observed (26) and because non-green t i s s u e s l i k e roots and c a l l u s are very s e n s i t i v e to t o x i n . Several l i n e s o f evidence p o i n t to the mitochondria as an important and p o s s i b l y primary s i t e of HmT t o x i n a c t i o n . E f f e c t s of HmT Toxin on I s o l a t e d Mitochondria. Evidence f o r an e f f e c t o f HmT t o x i n on m i t o c h o n d r i a l physiology comes from s e v e r a l r e p o r t s o f toxin-induced a l t e r a t i o n i n r e s p i r a t i o n r a t e s with s e v e r a l s u b s t r a t e s and uncoupling o f o x i d a t i v e phosphoryla t i o n i n mitochondria i s o l a t e d from corn c o n t a i n i n g Τ cytoplasm (Table I, Table I I I ) . Evidence f o r HmT toxin-induced s t r u c t u r a l changes comes from l i g h t transmittance and u l t r a s t r u c t u r a l s t u d i e s which have shown that i s o l a t e d Τ mitochondria s w e l l a f t e r t o x i n treatment (Table I ) . Toxin treatment o f i s o l a t e d mitochondria a l s o r e s u l t s i n damage to c r i s t a e and l o s s of matrix d e n s i t y (Table I ) . Outer m i t o c h o n d r i a l membranes appear to be u l t r a s t r u c t u r a l l y u n a f f e c t e d by HmT t o x i n . These m u l t i p l e e f f e c t s of HmT t o x i n are very r a p i d (Table I ) . Mitochondria i s o l a t e d from Τ corn w i t h nuclear genes f o r f e r t i l i t y r e s t o r a t i o n (TRf) a r e a l s o s e n s i t i v e to t o x i n (23,37), but a longer time i s r e q u i r e d f o r complete i n h i b i t i o n o f malate o x i d a t i o n than when Τ mitochondria are used. The HmT t o x i n e f f e c t s are s p e c i f i c f o r Τ (and TRf) mitochondria. Even very high concentrations of t o x i n have no e f f e c t s on mitochondria i s o l a t e d from p l a n t s with Ν cytoplasm (Table I , Table I I I ) . However, the removal o f the outer membrane of Ν mitochondria r e s u l t s i n sen s i t i v i t y o f the inner membranes to t o x i n as shown by i n h i b i t i o n of malate-DCPIP r e s p i r a t i o n (70).


7.

GREGORY E T A L .

Table I I I .

Southern

Corn

Leaf Blight

99

Disease

E f f e c t of HmT Toxin on R e s p i r a t o r y A c t i v i t i e s of Root Mitochondria from T-cytoplasm and N-cytoplasm Corn Rate of r e s p i r a t i o n (nmol 02/min/mg p r o t e i n )

Substrate

W64A(T)


NADH

Malate + pyruvate

State 3

243

273

State 4

79

88

+ Toxin

407*

88

State 3

80

98

State 4

20

21

+ Toxin Succinate

W64A(N)

21

0*

State 3

175

193

State 4

86

69

+ Toxin

150*

69

* A d d i t i o n of ADP o r the uncoupler 2,4-dinitrophenol (40 μΜ) d i d not s t i m u l a t e the r a t e o f oxygen uptake by Τ mitochondria a f t e r treatment with HmT t o x i n . Ν mitochondria remained well-coupled i n the presence of HmT t o x i n .

Oxygen uptake was measured with a Clark-type oxygen e l e c trode (Yellow Springs Instrument Company) i n 3 ml of a medium c o n t a i n i n g 0.4 M sucrose, 10 mM KC1, 2.5 mM M g C l , 4 mM Κ Η Ρ 0 , 20 mM HEPES, pH 7.4, χ mg/ml bovine serum albumin and 0.4 mg of mitochondria i s o l a t e d from roots of 3-day-old W64A(T) or W64A(N) s e e d l i n g s . Other a d d i t i o n s , as i n d i c a t e d , were 1.5 ymol NADH, 30 ymol malate plus 30 ymol pyruvate, 30 ymol s u c c i n a t e , and 33 yg HmT t o x i n (from a p r e p a r a t i o n which caused 50% i n h i b i t i o n of r o o t growth o f W64A(T) seedlings a t a c o n c e n t r a t i o n o f 0.65 yg/ ml). State 3 rates were measured a f t e r a d d i t i o n of 150 to 300 nmol of ADP, and State 4 r a t e s a f t e r subsequent exhaustion of the added ADP. HmT t o x i n was added during State 4 r e s p i r a t i o n . 2


2

4


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H O S T P L A N T RESISTANCE T O

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E f f e c t s of HmT Toxin on Mitochondria i n s i t u . Although the evidence f o r r a p i d , s e n s i t i v e and s p e c i f i c e f f e c t s of HmT t o x i n on i s o l a t e d Τ mitochondria i s c l e a r , the argument f o r mitochondria as the primary s i t e of t o x i n a c t i o n i n i n t a c t c e l l s has been weakened by a f r e q u e n t l y c i t e d r e p o r t that r e s p i r a t i o n and ATP content of s e e d l i n g t i s s u e are not s p e c i f i c a l l y a f f e c t e d by t o x i n i n time periods during which some i n h i b i t i o n of root growth can be detec ted (17). Unfortunately the method f o r measuring r e s p i r a t i o n was not given and the t i s s u e s assayed were s e e d l i n g shoots, f o r which no r a p i d growth i n h i b i t i o n by t o x i n was described. Toxin penetra t i o n i n t o the i n t a c t 2.5 cm detached shoots used may have been a l i m i t i n g f a c t o r i n the experiment. Recent u l t r a s t r u c t u r a l s t u d i e s (20,33) by our group should r e s o l v e the apparent c o n t r a d i c t i o n between t o x i n e f f e c t s on i s o l a t e d mitochondria and mitochondria i n s i t u . A f t e r t o x i n t r e a t ment of roots and mesophyll p r o t o p l a s t s of Τ corn, the f i r s t u l t r a s t r u c t u r a l e f f e c t s observed were changes i n the mitochondria s i m i l a r to those seen i n t o x i n - t r e a t e d i s o l a t e d mitochondria. F i f t e e n minutes exposure of roots to t o x i n r e s u l t e d i n some damage to the mitochondria of c e l l s i n the root cap and elongation zone. By two hours, many root mitochondria showed s w e l l i n g , r e d u c t i o n i n c r i s t a e and l o s s of matrix d e n s i t y (Figure 1 ) . HmT t o x i n had no e f f e c t on mitochondria i n Ν roots (Figure 1). Mitochondria w i t h i n mesophyll p r o t o p l a s t s from Τ corn were a f f e c t e d even more r a p i d l y and at lower t o x i n concentrations probably because pene t r a t i o n problems were minimized (33). Some p r o t o p l a s t mitochon d r i a were damaged w i t h i n 5 minutes and by 30 minutes, almost a l l of the p r o t o p l a s t s were a f f e c t e d . P r o t o p l a s t s t r e a t e d with t o x i n f o r 30 minutes and then washed thoroughly and c u l t u r e d a l l c o l l a p s e d w i t h i n a few days, l i k e p r o t o p l a s t s continuously exposed to t o x i n . The r a p i d t o x i n damage to the p r o t o p l a s t s was apparent l y i r r e v e r s i b l e (32). Toxin p a r t i a l l y p u r i f i e d by chloroform e x t r a c t i o n had no e f f e c t on mitochondria i n Ν p r o t o p l a s t s , even when high concentrations were used (33). Plasma membranes and other components of Ν and Τ p r o t o p l a s t s looked normal a f t e r 60 minutes exposure to chloroform e x t r a c t a b l e t o x i n . Physiological and biochemical studies of p r o t o p l a s t s c o n t a i n i n g mitochondria damaged by b r i e f t o x i n treatments have not yet been done; however, i t seems l i k e l y that r a p i d t o x i n e f f e c t s on mitochondrial func t i o n s i n v i v o w i l l be detected, perhaps even before u l t r a s t r u c t u r a l damage i s apparent. R e v e r s i b i l i t y o f Toxin A c t i o n There i s some evidence which suggests that HmT t o x i n a c t i o n i s r e v e r s i b l e . Arntzen e t a l . (17) i n a study on Τ corn seedlings showed that roots t r e a t e d with t o x i n f o r one hour and then r e t u r n ed to a t o x i n - f r e e medium grew more slowly than the c o n t r o l f o r approximately 6 hours, then s t a r t e d to grow f a s t e r , and by 10 hours were growing as v i g o r o u s l y as the c o n t r o l s . The degree to


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Figure 1. Ultrastructural effects of chhrοform-extractable HmT toxin on mitochondria in the region of elongation of susceptible (W64A(T)) and resistant (W64A(N)) corn roots. Experimental conditions are exactly as described in Ref. 20. Treatments were (A) W64A(T), treated for 2 hr with chloroform-extractable toxin; (B) W64A(T), no toxin added; (C) W64A(N), treated ior 2 hr with chloroform-extractable toxin; (D) W64A(N), no toxin added. The calibration bar represents 0.1 μ in each case.



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w h i c h t h e r o o t s grew a f t e r t h e t o x i n t r e a t m e n t was d e p e n d e n t o n t o x i n c o n c e n t r a t i o n ( 1 7 ) . H a l l o i n e£ a l (12) t r e a t e d l e a v e s o f Τ c o r n w i t h t o x i n f o r 3 h o u r s a n d t h e n washed t h e l e a v e s f o r 1 h o u r i n t o x i n - f r e e medium. The t o x i n - t r e a t e d l e a v e s showed much h i g h e r r a t e s o f e l e c t r o l y t e l e a k a g e t h a n t h e u n t r e a t e d controls. I n t e r p r e t a t i o n of t h i s l a t t e r data i s d i f f i c u l t be c a u s e i t was n o t s t a t e d w h e t h e r t h e i n c r e a s e d r a t e was d i f f e r e n t f r o m t h e r a t e s o b t a i n e d f o r l e a v e s c o n t i n u o u s l y grown i n t o x i n . R e c e n t e x p e r i m e n t s i n o u r l a b o r a t o r y (32) showed t h a t w a s h i n g o f Τ c o r n p r o t o p l a s t s , f o l l o w i n g 30 m i n u t e t r e a t m e n t s w i t h chloroform-extractable t o x i n , d i d not prevent p r o t o p l a s t c o l lapse. I n t h e same s e t o f e x p e r i m e n t s T - c o r n p r o t o p l a s t c o l l a p s e was p r e v e n t e d b y w a s h i n g t h e p r o t o p l a s t s a f t e r 15 m i n u t e t o x i n t r e a t m e n t s . Two l i n e s o f e v i d e n c e s u g g e s t t h a t t h e e f f e c t o f HmT t o x i n o n o x i d a t i v e p h o s p h o r y l a t i o n i n i s o l a t e d Τ m i t o c h o n d r i a i s r e v e r s i b l e . F i r s t l y , w i t h NADH a s t h e s u b s t r a t e , t h e d e g r e e o f t o x i n - i n d u c e d i n h i b i t i o n o f m i t o c h o n d r i a l ATP formation increases with increasing toxin concentration; the h y p e r b o l i c nature o f the curves suggests r e v e r s i b i l i t y o f t o x i n binding (38). Secondly, t o x i n - t r e a t e d Τ mitochondria recovered most o f t h e i r p h o s p h o r y l a t i n g c a p a c i t y when t o x i n was removed by w a s h i n g t h e m i t o c h o n d r i a w i t h t o x i n - f r e e medium ( 3 8 ) . The d a t a o n r o o t s , p r o t o p l a s t s , a n d i s o l a t e d m i t o c h o n d r i a s u g g e s t t h a t HmT t o x i n does n o t b i n d f i r m l y t o t o x i n - s e n s i t i v e s i t e s i n Τ c e l l s , but r a t h e r t h a t an e q u i l i b r i u m e x i s t s between bound a n d unbound t o x i n . Speculations Mitochondria

o n t h e M e c h a n i s m o f T o x i n - I n d u c e d Damage t o Τ

The mechanism b y w h i c h HmT t o x i n damages Τ m i t o c h o n d r i a i s unknown. A l t h o u g h HmT t o x i n i n d u c e s m u l t i p l e e f f e c t s i n Τ m i t o c h o n d r i a (Table I , Table I I I ) i t i s t h e o r e t i c a l l y p o s s i b l e that a l l of the observed e f f e c t s r e s u l t from a s i n g l e , p r i m a r y e f f e c t caused by a s i n g l e type of t o x i n molecule. A l i k e l y primary e f f e c t i s a t o x i n - i n d u c e d i n c r e a s e i n t h e i o n p e r m e a b i l i t y o f t h e i n n e r mem branes o f Τ mitochondria. Toxin-induced i o n leakage might occur i n one o f t h r e e ways. F i r s t l y , t h e t o x i n c o u l d a c t a s a n i n n e r m i t o c h o n d r i a l mem b r a n e d i s r u p t a n t a s s u g g e s t e d b y Gengenbach elt a l (35) o n t h e basis of u l t r a s t r u c t u r a l data. Such a c t i o n w o u l d r e s u l t i n e x t e n s i v e s t r u c t u r a l damage, i n a d d i t i o n t o c a u s i n g i n c r e a s e d i o n p e r m e a b i l i t y , a n d s h o u l d l e a d t o l e a k a g e o f i n n e r membrane m a t r i x components s u c h a s m a l a t e d e h y d r o g e n a s e . However, r e c e n t e x p e r i ments i n our l a b o r a t o r y (42) h a v e shown t h a t t o x i n t r e a t m e n t does not induce leakage o f malate dehydrogenase from Τ m i t o c h o n d r i a ( T a b l e I V ) . One c r i t i c i s m o f the l a t t e r e x p e r i m e n t i s t h a t o n



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a d d i t i o n of t o x i n , malate dehydrogenase could have leaked from the Τ m i t o c h o n d r i a l inner membrane but may have been h e l d w i t h i n the mitochondria by the outer membrane. Secondly, the t o x i n may a c t as a c l a s s i c a l uncoupler and promote the passage of protons through the Τ m i t o c h o n d r i a l inner membrane by a c t i n g as a l i p o p h i l i c weak a c i d . A t h i r d p o s s i b i l i t y i s that the t o x i n i s an ionophore and induces increased p e r m e a b i l i t y of Τ m i t o c h o n d r i a l inner membranes to one or more types of c a t i o n by complexing w i t h the appropriate c a t i o n t o form a l i p i d s o l u b l e complex which can pass through the inner membrane. Whichever mode of a c t i o n i s c o r r e c t , we w i l l be faced with e x p l a i n i n g why Ν mitochondria are completely r e s i s t a n t to HmT t o x i n . At present we do not know whether the inner or the outer m i t o c h o n d r i a l membrane mediates r e s i s t a n c e . A p o s s i b l e r o l e of the outer m i t o c h o n d r i a l membrane i n r e s i s t a n c e was suggested by Watrud £t a l (70) because removal of the outer membrane from Ν mitochondria y i e l d e d a t o x i n - s e n s i t i v e inner membrane p r e p a r a t i o n as judged by t o x i n i n h i b i t i o n of the malate-DCPIP r e a c t i o n . I n t e r p r e t a t i o n of t h i s l a t t e r experiment should be cautious be cause the technique used to prepare the inner membranes i n v o l v e d osmotic s w e l l i n g and s h r i n k i n g of mitochondria i n combination with h i g h speed discontinuous sucrose gradient c e n t r i f u g a t i o n . The l a t t e r procedure could w e l l have damaged the i n n e r membranes and no p h y s i o l o g i c a l data were presented to the contrary (70). Consequently, i t i s p o s s i b l e that t o x i n s e n s i t i v i t y of the inner membranes of Ν mitochondria (70) was due to a b n o r m a l i t i e s brought about by the inner membrane i s o l a t i o n technique r a t h e r than the removal of the outer m i t o c h o n d r i a l membrane per se. Much c a r e f u l work on whole mitochondria and on subm i t o c h o n d r i a l f r a c t i o n s i s needed before we can d i s c o v e r the nature of HmT t o x i n a c t i o n on Τ mitochondria and the l a c k of t o x i n a c t i o n on Ν mitochondria. T h i s work i s a f o c a l p o i n t of our present experiments because i t i s l i k e l y to r e v e a l the biochemical b a s i s of southern corn l e a f b l i g h t d i s e a s e .

Comparative Biochemistry of Mitochondria from Ν and Τ Cytoplasms The A c t i o n of Chemicals (Other than HmT Toxin) on Ν and Τ Mitochondria. S e v e r a l chemicals known to induce one or more responses s i m i l a r to those of HmT t o x i n have been a p p l i e d to Ν and Τ mitochondria. Q u a n t i t a t i v e d i s t i n c t i o n s between Ν and Τ mitochondria have a r i s e n from these experiments. The f o l l o w i n g chemicals were used: Valinomycin. Valinomycin (VAL) s t i m u l a t e s NADH o x i d a t i o n and s w e l l i n g i n mitochondria under c e r t a i n r e a c t i o n c o n d i t i o n s by a c t -


104

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Table IV.


E f f e c t o f HmT Toxin on the Release o f Malate Dehydro genase (MDH) from Τ Mitochondria

Treatment

MDH a c t i v i t y (nmol NADH oxidized/min) Supernatant

None

+ HmT Toxin Downloaded by COLUMBIA UNIV on November 29, 2017 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

PLANT

+ T r i t o n X-100

MDH a c t i v i t y r e l e a s e d i n t o supernatant % of t o t a l a c t i v i t y Mitochondria

190

3,200

6%

150

2,400

6%

150

3,600

4%

190

3,600

5%

3,600

50

99%

3,500

20

99%

Mitochondria from W64A(T) r o o t s (about 0.5 mg p r o t e i n ) were suspended i n 3 ml o f 0.4 M sucrose, 10 mM KC1, 2.5 mM M g C l , 4 mM KH P0 , 20 mM HEPES, pH 7.4 (Medium A) c o n t a i n i n g 1.5 ymol NADH and 300 nmole ADP. As i n d i c a t e d , HmT t o x i n (33 yg) o r the d e t e r gent T r i t o n X-100 (to a c o n c e n t r a t i o n o f 0.15%) were added. A f t e r i n c u b a t i o n f o r 5 min a t room temperature the samples were cent r i f u g e d a t 18,000 xg f o r 18 min a t 25°. Each p e l l e t was sus pended i n 0.5 ml o f M e t ï u m A, and s o l u b i l i z e d with T r i t o n X-100 (0.15%) to r e l e a s e the t o t a l enzyme a c t i v i t y . Appropriate a l i q u o t s of the supernatants and p e l l e t s were assayed f o r MDH a c t i v i t y i n 3 ml o f 0.4 M sucrose, 20 mM HEPES, 0.25 mM NADH, 1 mM oxaloacetate, 1 mM KCN, pH 7.4. The o x i d a t i o n of NADH was monitored by the change i n absorbance a t 340 nm. 2

2

4



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ing as a Κ - s p e c i f i c ionophore (51). VAL induced s i m i l a r respons es i n Ν and Τ mitochondria with respect to o x i d a t i o n o f exogenous NADH and m i t o c h o n d r i a l s w e l l i n g , although the l a t t e r was more marked i n Ν mitochondria a t low VAL concentrations (38,43). Gramicidin D. Gramicidin (GRAM) causes s w e l l i n g i n KC1 o r NaCl media, uncouples o x i d a t i v e phosphorylation, and stimulates NADH o x i d a t i o n i n KC1 o r sucrose media (52). GRAM caused more s t i m u l a t i o n o f NADH o x i d a t i o n by Τ mitochondria than by Ν mito chondria, although GRAM-induced s w e l l i n g of Ν mitochondria was always s l i g h t l y greater than that i n Τ mitochondria (43). 2,4-Dinitrophenol (DNP) uncouples o x i d a t i v e phosphoryla t i o n . DNP caused dramatic s t i m u l a t i o n o f NADH r e s p i r a t i o n i n both Ν and Τ mitochondria. However, a t high l e v e l s o f DNP, the s t i m u l a t i o n was s i g n i f i c a n t l y higher f o r Τ mitochondria than f o r Ν mitochondria (43). Bednarski e t a l . (38) could not detect s i g n i f i c a n t d i f f e r e n c e s between Τ and Ν mitochondria i n s e n s i t i v i t y to DNP. Sodium azide i s a terminal oxidase i n h i b i t o r . The degree of i n h i b i t i o n of NADH o x i d a t i o n by sodium azide was the same f o r Ν and Τ mitochondria (43). N i g e r i c i n (NIG) plus Κ causes uncoupling i n mitochondria. Τ mitochondria were m o r e s e n s i t i v e than were Ν mitochondria to uncoupling by NIG plus Κ (38). D e c e n y l s u c c i n i c A c i d . D e c e n y l s u c c i n i c a c i d (DSA) causes m i t o c h o n d r i a l s w e l l i n g by mediating membrane d i s r u p t i o n . DSA t r e a t ments a l s o s t i m u l a t e exogenous NADH o x i d a t i o n and r e s u l t i n l o s s of o x i d a t i v e phosphorylation (53). DSA s t i m u l a t e d NADH o x i d a t i o n a t low concentrations but caused i n h i b i t i o n a t higher concentra t i o n s . The Ν and Τ mitochondria responded s i m i l a r l y to DSA with respect to NADH o x i d a t i o n . However, DSA caused c o n s i d e r a b l y more s w e l l i n g i n Ν mitochondria than i n Τ mitochondria (43). Calcium ( i n the presence and absence o f phosphate). Calcium i n the absence o f i n o r g a n i c phosphate stimulated NADH o x i d a t i o n i n Ν mitochondria somewhat more than i n Τ mitochondria. Calcium plus phosphate induced s i m i l a r s t i m u l a t i o n s o f NADH o x i d a t i o n i n the Ν and Τ mitochondria (43). D i g i t o n i n . D i g i t o n i n i s a saponin which can d i s r u p t b i o l o g i c a l membranes. A t low concentrations d i g i t o n i n i s s e l e c t i v e f o r s t e r o i d - r i c h membranes. Evidence f o r t h i s s e l e c t i v i t y comes from the f i n d i n g o f Schnaitman et^ al^. (54) that the outer membrane o f r a t l i v e r mitochondria i s more d i g i t o n i n - s e n s i t i v e than the inner membrane. These authors suggested that as d i g i t o n i n i s known to combine with c h o l e s t e r o l (a major membrane s t e r o i d i n animals) on a 1:1 b a s i s , i t was p o s s i b l e that the outer m i t o c h o n d r i a l mem branes were higher i n c h o l e s t e r o l than the inner membrane. Sub sequent s t u d i e s with guinea p i g l i v e r mitochondria showed that the s t e r o i d content o f the outer m i t o c h o n d r i a l membrane was s i x times more concentrated, on a p r o t e i n b a s i s , than i n the inner membrane (55) . Gregory e_t aJU (56) have r e c e n t l y s t u d i e d the r e s p i r a t o r y e f f e c t s o f low concentrations o f d i g i t o n i n on N, C, +


HOST P L A N T RESISTANCE TO


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and Τ mitochondria (Figure 2 ) . The Τ mitochondria were dramati c a l l y more d i g i t o n i n - s e n s i t i v e than the Ν o r C mitochondria, as i n d i c a t e d by i n h i b i t i o n o f malate and s u c c i n a t e r e s p i r a t i o n and uncoupling o f o x i d a t i v e phosphorylation (Figure 2 ) . There were a l s o d i f f e r e n c e s between the Ν and C mitochondria with r e s p e c t to d i g i t o n i n s e n s i t i v i t y (Figure 2 ) . These data were thought (56) to r e f l e c t d i f f e r e n c e s i n membrane s t e r o i d composition between the N, Τ and C mitochondria. I t was noted (56) that the d i g i t o n i n e f f e c t s were somewhat s i m i l a r to those o f HmT t o x i n on Τ mitochon dria. The data o u t l i n e d above showed that compounds other than HmT toxin can produce d i f f e r e n t i a l e f f e c t s on v a r i o u s inner mem brane f u n c t i o n s i n Ν and Τ mitochondria and thus s t r o n g l y sup ports the concept that those mitochondria d i f f e r with r e s p e c t to membrane s t r u c t u r e .

0

2

4

6

.8

1.0

1.2

14

16

18

20

Digitonin Concentration (mg digitonin/mg mitochondrial protein) Figure 2. Effects of digitonin on respira tion and oxidative phosphorylation in mito chondria isolated from W64A(N), W64A(T), and W64A(C) roots. Experimental condi tions are found in Ref. 56.


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Composition o f Ν and Τ Mitochondria.

N u c l e i c A c i d s . Levings and P r i n g (57) have shown that Ν and Τ cytoplasms d i f f e r with respect to m i t o c h o n d r i a l DNA (mt DNA). In these experiments, mt DNA was i s o l a t e d from the Ν and Τ cytoplasms of s e v e r a l corn l i n e s . The mt DNA's were subjected to r e s t r i c t i o n enzyme fragment a n a l y s i s using each of the r e s t r i c t i o n endonucleases Eco RI (52,58) , H i n D I I I (57.,5j0 , Bam I (57), and Sal I (57). Each of these enzymes cut DNA a t d i f f e r e n t sequence s p e c i f i c s i t e s and each y i e l d e d d i s t i n c t i o n s between the mt DNA from Ν and Τ cytoplasms (57). In a d d i t i o n , i t was shown (57) that the male s t e r i l e cytoplasms other than T, namely S, C and EP, d i f f e r e d with r e s p e c t to mt DNA. As these d i f f e r e n c e s may have been due to contaminating DNA from n u c l e a r , b a c t e r i a l or v i r a l sources, Levings and P r i n g (58) were c a r e f u l to confirm the i d e n t i t y of t h e i r mt DNA preparations by using buoyant d e n s i t y determinations i n n e u t r a l cesium c h l o r i d e and showing that t h e i r mt DNA p r e p a r a t i o n s appeared as a s i n g l e band with a buoyant d e n s i t y of 1.706 g/cm which i s c h a r a c t e r i s t i c of higher p l a n t mt DNA (59). In a d d i t i o n , the upper and lower DNA bands obtained from cesium c h l o r i d e - e t h i d i u m bromide g r a d i e n t s were i n agreement with the expected value f o r mt DNA. There was no i n d i c a t i o n of n u c l e a r DNA contamination because the upper and lower DNA bands on cesium c h l o r i d e - e t h i d i u m bromide gradients were i n d i s t i n g u i s h a b l e from each other and, i n any case, r e s t r i c t i o n enzyme fragment a n a l y s i s of the complex nuclear DNA does not y i e l d d i s t i n c t bands which c o u l d be confused with mt DNA bands (58). The p o s s i b i l i t y of contamination o f the mt DNA prepara t i o n s by v i r a l DNA was not r u l e d out (58). Previous work i n v o l v i n g buoyant d e n s i t y a n a l y s i s o f mt DNA's on n e u t r a l cesium c h l o r i d e had f a i l e d to demonstrate d i f f e r e n c e s between Ν and Τ cytoplasms (60). D i f f e r e n c e s between the mt DNA o f suscep t i b l e and r e s i s t a n t c e l l s are a p r e r e q u i s i t e f o r t h e o r i e s i n v o l v i n g Τ mitochondria as primary f a c t o r s i n c y t o p l a s m i c a l l y i n h e r i t e d s u s c e p t i b i l i t y to southern corn l e a f b l i g h t d i s e a s e . Whether or not the observed mt DNA d i f f e r e n c e s are a s s o c i a t e d with d i f f e r e n c e s i n disease s u s c e p t i b i l i t y remains to be seen. 3

Proteins 1. Subunit f i n g e r p r i n t s o f t o t a l m i t o c h o n d r i a l p r o t e i n . The use of high r e s o l u t i o n two-dimensional e l e c t r o p h o r e s i s i n our l a b o r a t o r y has f a i l e d to show q u a l i t a t i v e d i f f e r e n c e s between N, C and Τ mitochondria with r e s p e c t to t o t a l p r o t e i n subunits (61). The a n a l y t i c a l technique, developed by O ' F a r r e l l (62), i n v o l v e s s e p a r a t i o n of m i t o c h o n d r i a l p r o t e i n according to i s o e l e c t r i c p o i n t by i s o e l e c t r i c f o c u s i n g i n the f i r s t dimension, followed by s e p a r a t i o n according to molecular weight by sodium dodecyl s u l f a t e e l e c t r o p h o r e s i s i n the second dimension. The


108

HOST P L A N T RESISTANCE T O

r e s u l t i s a " f i n g e r p r i n t " of the mitochondrial p r o t e i n subunits. The technique i s h i g h l y r e p r o d u c i b l e so that each spot on one separation can be matched with a corresponding spot on a d i f ferent separation. In a d d i t i o n the technique can r e s o l v e p r o t e i n s d i f f e r i n g i n a s i n g l e charge (62). In our experiments, v i s u a l i z a t i o n of the p r o t e i n f i n g e r p r i n t s was by means of s t a i n ing r a t h e r than by autoradiography which i s more s e n s i t i v e . I t i s therefore very p o s s i b l e that one or more p r o t e i n s were un detected i n our experiments and that q u a n t i t a t i v e d i f f e r e n c e s e x i s t between the p r o t e i n s of N, C and Τ mitochondria. Future experiments i n our laboratory i n v o l v i n g a combination of auto radiography and two-dimensional e l e c t r o p h o r e s i s of mitochondrial p r o t e i n s which are l a b e l l e d with e i t h e r C or S , w i l l f a c i l i t a t e b e t t e r s e n s i t i v i t y and q u a n t i f i c a t i o n of the system. 2. Cytochromes. P r i n g (63) has reported that Τ mitochon d r i a contain a f u l l complement of cytochromes a + a$ b and c as detected by d i f f e r e n c e spectra at 25°C. I t has a l s o been shown that Τ cytoplasm contains 7-12% more cytochrome b than does Ν (63) and that TRf mitochondria contain s l i g h t l y more c y t o chrome a + a 3 than do Ν mitochondria (64) i n each of f i v e inbred lines. Small d i f f e r e n c e s i n cytochrome b and c content of Ν and TRf mitochondria were noted, but these d i f f e r e n c e s were not ap parent f o r a l l of the inbred l i n e s t e s t e d (64). S t e r o i d s . Q u a n t i t a t i v e and q u a l i t a t i v e determinations of m i t o c h o n d r i a l s t e r o i d s i n our l a b o r a t o r y have y i e l d e d no r e p r o d u c i b l e d i f f e r e n c e s between Ν and Τ cytoplasms (65). Lack of r e p r o d u c i b i l i t y may be a f u n c t i o n of v a r y i n g p u r i t y among the d i f f e r e n t mitochondrial preparations. The experiments i n v o l v e d e x t r a c t i o n of a crude s t e r o i d p r e p a r a t i o n from the i s o l a t e d mito chondria, p u r i f i c a t i o n of the s t e r o i d s by s i l i c i c a c i d column chromatography, followed by GLC of the f r e e s t e r o i d s . Three major s t e r o i d s were found i n the Ν and the Τ mito chondria: campesterol, s t i g m a s t e r o l and s i t o s t e r o l . The i d e n t i t y of these compounds was confirmed by GLC-mass spectroscopy i n c o l l a b o r a t i o n with Dr. S. F. Osman, ARS-USDA, Eastern Regional Research Center, Wyndmoor, Pa. A minor s t e r o i d was a l s o found, but the i d e n t i t y of t h i s compound i s not known at present. The f a c t that Ν and Τ mitochondria are d i f f e r e n t i a l l y s e n s i t i v e to d i g i t o n i n (56) as explained p r e v i o u s l y , suggests that f u r t h e r experiments i n v o l v i n g f r a c t i o n a t i o n , p u r i f i c a t i o n , and s t e r o i d analyses of inner and outer m i t o c h o n d r i a l membranes may y i e l d d i f f e r e n c e s between these mitochondria. I t i s of i n t e r e s t that s t e r o l d i f f e r e n c e s have been found between Ν and Τ t a s s e l s during development (66). llf


PESTS

3 5

9

Studies on the Molecular S p e c i f i c Toxin

Structure of H. maydis Race Τ Host

The exact molecular s t r u c t u r e of HmT

t o x i n i s not yet known



7.

GREGORY

E T

AL.

Southern

Corn

Leaf

Blight

Disease

109

and there i s disagreement as to which c l a s s of compounds HmT t o x i n belongs. Our current information on HmT t o x i n s t r u c t u r e c o n s i s t s of p u b l i s h e d data by Karr e_t a l . ( 2 4 ) and a recent personal com munication from J . M. Daly of the U n i v e r s i t y o f Nebraska (67). Karr e t a l . ( 2 4 ) i s o l a t e d f i v e t o x i c compounds from crude HmT culture f i l t r a t e . I t was p o s t u l a t e d that the t o x i c compounds were terpenoids, e x i s t i n g f r e e or as g l y c o s i d e s , ranging i n molecular weight from 350 to 600 and a chemical assay f o r HmT t o x i n was developed (68). This work i n v o l v e d f r a c t i o n a t i o n and p u r i f i c a t i o n of the t o x i c components i n combination with a l e a f puncture b i o assay f o r t o x i n a c t i v i t y i n each f r a c t i o n . The f r a c t i o n s which e x h i b i t e d a c t i v i t y i n the bioassay were analysed f o r chemical s t r u c t u r e by chemical t e s t s , chromatography, mass spectroscopy and nuclear magnetic resonance a n a l y s i s . The chemical assay f o r HmT t o x i n was a g e n e r a l i z e d t e s t f o r terpenoids using s u l f u r i c a c i d - a c e t i c anhydride reagent. The involvement of the f i v e t e r penoid compounds i n HmT t o x i n s p e c i f i c i t y i s open to q u e s t i o n . A subsequent comparison (69) of the chemical assay d e s c r i b e d by Karr ejt a l . (68) to measure the f i v e terpenoid t o x i n components i n p a r t i a l l y p u r i f i e d preparations r e v e a l e d that both HmT and HmO (which does not produce a h o s t - s p e c i f i c toxin) produce compounds s i m i l a r to the toxins i d e n t i f i e d by Karr e t a l . ( 2 4 ) . Consequently i t i s p o s s i b l e that the f i v e terpenoid compounds c h a r a c t e r i z e d by Karr ejt S L L . ( 2 4 ) are not a s s o c i a t e d w i t h the h o s t - s p e c i f i c a c t i v i t y of HmT t o x i n . I t i s i n t e r e s t i n g , however, that when Watrud et a l . (70) t r e a t e d Τ mitochondria w i t h a mixture of toxins I and I I (see r e f e r e n c e 2JL f ° terminology), the c h a r a c t e r i s t i c r e sponses were e l i c i t e d . Another problem i n the work of Karr e t a l . ( 2 4 ) d e r i v e s from the use o f the i n s e n s i t i v e l e a f puncture b i o assay i n the assessment of t o x i c a c t i v i t y of the v a r i o u s f r a c t i o n s (15,69). I t i s p o s s i b l e that one or more of the discarded f r a c tions contained non-terpenoid h o s t - s p e c i f i c t o x i n a c t i v i t y . r

A non-terpenoid s t r u c t u r e f o r HmT t o x i n has been proposed r e c e n t l y by Kono and Daly (67). Some d e t a i l s of the s t r u c t u r e have not been f i n a l i z e d , but a great deal of u s e f u l i n f o r m a t i o n has been gained. The molecule has an e m p i r i c a l formula of 2 9 5 0 ° 9 (542 daltons) and i s extremely a c t i v e a g a i n s t Τ corn, causing complete n e c r o s i s of the f i r s t true l e a f a t l e v e l s as low as 5 to 10 ng. The t o x i c i t y of the molecule was s p e c i f i c f o r Τ corn as no t o x i c e f f e c t was observed on Ν corn or on other p l a n t s p e c i e s . The use of a very s e n s i t i v e t o x i n bioassay, i n which dark C 0 f i x a t i o n by corn l e a f d i s c s was measured (18) insured that no b i o l o g i c a l l y a c t i v e t o x i n f r a c t i o n s were discarded p r i o r to s t r u c t u r a l a n a l y s i s . In aqueous s o l v e n t s the m a t e r i a l chromatographed as a s i n g l e d i f f u s e spot, but i n mixtures of methanol and non-polar organic compounds a major and two minor components with host s p e c i f i c i t y were observed. These a d d i t i o n a l compounds were probably isomeric o r homologous because a c e t y l a t i o n of t o x i n r e s u l t e d i n the formation of a t e t r a a c e t a t e of e m p i r i c a l formula C37H58O13 (710 daltons) which a l s o r e s o l v e d i n t o a major and two C

H

2


HOST P L A N T

110


minor components. The IR, NMR and UV s p e c t r a of two of the a c e t y l a t e d d e r i v a t i v e s could be d i s t i n g u i s h e d only by the e x t i n c t i o n c o e f f i c i e n t i n the UV. There were i n s u f f i c i e n t amounts o f the t h i r d component to make comparisons. C and proton NMR of the major a c e t y l a t e d d e r i v a t i v e support the e x i s t e n c e of four hydroxyl groups and f i v e carbonyl groups i n the o r i g i n a l t o x i n . On the b a s i s of IR, C and proton decoupling NMR and mass s p e c t r a of the component i s o l a t e d i n the l a r g e s t y i e l d , two p o s s i b l e s t r u c t u r e s have been proposed f o r HmT t o x i n (Figure 3). Work to determine the exact s t r u c t u r e of the molecule i s s t i l l i n pro gress. Whether the s i n g l e p u r i f i e d t o x i n molecule of Kono and Daly (67) mimics the m u l t i p l e m i t o c h o n d r i a l e f f e c t s induced by crude t o x i c f i l t r a t e and c h l o r o f o r m - t o x i n i s a c r i t i c a l and ex c i t i n g question. 1 3


1 3

Concluding Remarks Mechanism o f Southern Corn Leaf B l i g h t Disease. In our o p i n i o n present data suggests that a major component (and perhaps the primary component) of southern corn l e a f b l i g h t disease i s the HmT toxin-induced damage of mitochondria i n s u s c e p t i b l e (T c y t o plasm) corn. This o p i n i o n i s based on two major pieces of exper imental evidence. F i r s t l y , s e v e r a l groups have shown that i s o l a t e d s u s c e p t i b l e Τ mitochondria (but not the r e s i s t a n t mito chondria) are s e v e r e l y damaged by HmT t o x i n . The p h y s i o l o g i c a l m a n i f e s t a t i o n of t h i s damage, should the damage a l s o occur i n s i t u , would l e a d to the death of corn c e l l s which c o n t a i n Τ cytoplasm. Secondly, u l t r a s t r u c t u r a l s t u d i e s by our group have shown that HmT t o x i n can damage Τ mitochondria i n s i t u and that t h i s damage i s s i m i l a r to that observed i n t o x i n - t r e a t e d , i s o l a t e d Τ mito chondria. However, i t i s of extreme importance to e l u c i d a t e whether or not HmT t o x i n a f f e c t s Τ m i t o c h o n d r i a l physiology i n v i v o , because a t present there i s no c o n c l u s i v e evidence i n t h i s area. S e n s i t i v e measurements o f i n v i v o r e s p i r a t i o n and phos p h o r y l a t i o n i n t o x i n - t r e a t e d corn p r o t o p l a s t s are i n progress i n our l a b o r a t o r y and may have great relevance to the nature of southern corn l e a f b l i g h t d i s e a s e . The cytoplasmic i n h e r i t a n c e of s u s c e p t i b i l i t y to the disease may a l s o be c o n s i s t e n t w i t h the mitochondria (which c o n t a i n DNA) being of primary importance. Should the t r a n s f e r of r e s i s t a n t (N) mitochondria to s u s c e p t i b l e (T) p r o t o p l a s t s r e s u l t i n a c o n f e r r i n g of r e s i s t a n c e to these p r o t o p l a s t s , there w i l l be l i t t l e doubt that mitochondria are a major f a c t o r i n the r e s i s t a n c e of corn to southern corn l e a f b l i g h t d i s e a s e . This experiment i s i n progress i n our l a b o r a t o r y . The Use of the Southern Corn Leaf B l i g h t System i n Studies on M i t o c h o n d r i a l Genetics i n Higher P l a n t s . L i t t l e i s known about the m i t o c h o n d r i a l genetics of higher p l a n t s . Although asexual genetic manipulation such as p r o t o p l a s t f u s i o n and o r g a n e l l e t r a n s f e r can b r i n g together cytoplasmic components from d i v e r s e c e l l types, few m i t o c h o n d r i a l markers are a v a i l a b l e i n higher



7.

GREGORY

E TA L .

OH

α.

O H

Corn Leaf

Ο

Ο

Ο

Ο

Blight

O

H

Ο

111

Disease

O H

Ο

Ο

/WWWWWWA OH

Figure 3.

Southern

O H

O H

Ο

Ο

O H

Ο

Two possible structures for HmT toxin recently proposed by Kono and Daly

(67)



112

HOST PLANT RESISTANCE TO PESTS

plants. Because HmT toxin rapidly and specifically destroys both Τ cytoplasm protoplasts and Τ mitochondria within protoplasts (20,31,32,33) the toxin will be a useful probe for following mitochondrial transfers. The response of protoplasts and mito chondria to HmT toxin after fusion of Ν and Τ cytoplasm proto plasts, or after transfer of isolated Ν mitochondria to Τ proto plasts, may answer several important questions: can "foreign" mitochondria be transferred to a different cytoplasm?; can such mitochondria survive, function, replicate, and alter the survival of host protoplasts?; do mitochondria carry all the genetic material that determines cytoplasmic response to toxin? Tech niques developed using HmT toxin as a probe for successful genetic manipulations with mitochondria may facilitate a new experimental approach to the study of mitochondrial genetics in higher plants. Literature Cited 1. Hooker, A.L., Smith, D.R., Lim, S.M., Beckett, J.B., Plant Dis. Rep. (1970) 54, 708. 2. Smith, D.R., Hooker, A.L., Lim, S.M., Beckett, J.B., Crop Sci. (1971) 11, 772. 3. Gracen, V.E., Grogan, C.O., Plant Dis. Rep. (1972) 56, 432. 4. Berquist, R.R., Peverly, G., Plant Dis. Rep. (1972) 56, 112. 5. Hallauer, A.R., Martinson, C.A., Agronomy J. (1975) 67, 497. 6. Lim, S.M., Plant Dis. Rep. (1974) 58, 811. 7. Caunter, I.G., Ph.D. thesis, 1977, Cornell University, "A Genetic Study of Nuclear Controlled Resistance to Southern and Yellow Leaf Blights in Maize" (Zea mays L.). 8. Payne, G.A., Yoder, O.C., Phytopathology (1977)67,in press. 9. Lim, S.M., Hooker, A.L., Genetics (1971) 69, 115. 10. Yoder, O.C., Gracen, V.E., Phytopathology (1975) 65, 273. 11. Lim, S.M., Hooker, A.L., Phytopathology (1972) 62, 968. 12. Halloin, J.M., Comstock, J.C., Martinson, C.A., Tipton, C.L., Phytopathology (1973)63,640. 13. Gracen, V.E., Forster, M.J., Sayre, K.D., Grogan, C.O., Plant Dis. Rep. (1971) 55, 469. 14. Turner, M.T., Martinson, C.A., Plant Dis. Rep. (1972) 56, 29. 15. Yoder, O.C., Payne, G.A. Gregory, P., Gracen, V.E., Physiol. Plant Path. (1977) 10, 237. 16. Wheeler, H., Williams, A.S., Young, L.D., Plant Dis. Rep. (1971) 55, 667. 17. Arntzen, C.J., Koeppe, D.E., Miller, R.J., Peverly, J.H., Physiol. Plant Path. (1973) 3, 79. 18. Bhullar, B.S., Daly, J.M., Rehfeld, D.W., Plant Physiol. (1975) 56, 1. 19. Wheeler, H., Ammon, V.D., Phytopathology (1977)67,325. 20. Aldrich, H.C., Gracen, V.E., York, D., Earle, E.D., Yoder, O.C., Tissue and Cell (1977) 9, 167. 21. Koeppe, D.E., Malone, C.P., Miller, R.J., Plant Physiol. (supp.) (1973) 51, 10.



113 7. GREGORY ET AL. Southern Corn Leaf Blight Disease 22. Mertz, S.M. Jr., Arntzen, C.J., Plant Physiol. (supp.) (1973) 51, 16. 23. Watrud, L.S., Hooker, A.L., Koeppe, D.E., Phytopathology (1975) 65, 178. 24. Karr, A.L. Jr., Karr, D.B., Strobel, G.A., Plant Physiol. (1974) 53, 250. 25. Earle, E.D., unpublished. 26. Arntzen, C.J., Haugh, M.F., Bobick, S., Plant Physiol. (1973) 52, 569. 27. Gracen, V.E., Grogan, C.O., Forster, M.J., Can. J. Bot. (1972) 50, 2167. 28. Frick, H., Bauman, L.F., Nicholson, R.L., Hodges, T.K., Plant Physiol. (1977) 59, 103. 29. Gengenbach, B.G., Green, C.E., Crop Sci. (1975) 15, 645. 30. Laughnan, J.R., Gabay, S.J., Crop Sci. (1973) 13, 681. 31. Pelcher, L.E., Kao, K.N., Gamborg, O.L., Yoder, O.C., Gracen, V.E., Can. J. Bot. (1975) 53, 427. 32. Earle, E.D., Gracen, V.E., Yoder, O.C., Gemmill, K.P., submitted to Plant Physiol. 33. York, D.W., unpublished. 34. Miller, R.J., Koeppe, D.E., Science (1971) 173, 67. 35. Gengenbach, B.G., Miller, R.J., Koeppe, D.E., Arntzen, C.J., Can. J. Bot. (1973) 51, 2119. 36. Krueger, W.A., Josephson, L.M., Hilty, J.W., Phytopathology (1974) 64, 735. 37. Barratt, D.H.P., Flavell, R.B., Theoretical and Applied Genetics (1975) 45, 315. 38. Bednarski, M.A., Izawa, S., Scheffer, R.P., Plant Physiol. (1977) 59, 540. 39. Peterson, P.Α., Flavell, R.B., Barratt, D.H.P., Theoreti cal and Applied Genetics (1975) 45, 309. 40. Flavell, R., Physiol. Plant Path. (1975)6,107. 41. Peterson, P.Α., Flavell, R.B., Barratt, D.H.P., Plant Dis. Rep. (1974) 58, 777. 42. Matthews, D.E., unpublished. 43. Gengenbach, B., Koeppe, D.E., Miller, R.J., Physiol. Plant. (1973) 29, 103. 44. Tipton, C.L., Mondal, M.H., Uhlig, J., Biochem. Biophys. Res. Comm. (1973) 51, 725. 45. Tipton, C.L., Mondal, M.H., Benson, M.J., Physiol. Plant Path. (1975)7,277. 46. Gracen, V.E., Forster, M.J., Grogan, C.O., Plant Dis. Rep. (1971) 55, 938. 47. Frick, H., Nicholson, R.L., Hodges, T.K., Bauman, L.F., Plant Physiol. (1976) 57, 171. 48. Hodges, T.K., Leonard, R.T., Bracker, C.E., Keenan, T.W., Proc. Nat. Acad. Sci. U.S.A. (1972) 69, 3307. 49. Scheffer, R.P. IN Biochemistry & Cytology of Plant-Parasite Interaction [K. Tomiyama, J.M. Daly, I. Uritani, H. Oku and S. Ouchi, eds], p. 118, Elsevier Scientific Publishing Company, 1976.



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Acknowledgements The experiments performed in our laboratory were supported in part by grant 75002 from the Rockefeller Foundation. We gratefully acknowledge the help of Dr. D.E. Matthews, H. Pham, and D.W. York in the preparation of this paper.


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