inclusion bodies in cells infected with radish mosaic virus

7/29/2019 Inclusion Bodies in Cells Infected with Radish Mosaic Virus

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J. gen. Vircl. (i 97 0, x3, 51-57 5 IPrinted in Great Britain

I n c l u s i o n B o d i e s i n C e ll s I n f e c te d w i t h R a d i s h M o s a i c V i r u sBy ZLATA STEFANAC AND N. LJUBESICBotanical Institute, University o f Zagreb, Yugoslaviaand Rudjer Bo~kovib Institute, Zagreb, Yugoslavia

(Accepted 25 M a y ~97I)SUMMARY

Turnip cells infected with radish mosaic virus contain characteristic largevesiculated cytoplasmic inclusion bodies, visible by light and electron microscopy.They consist of spherical aggregates of virus particles, sometimes in a crystallinearray, embedded in a membranous mass. The presence of active dictyosomes andabundant endoplasmic reticulum in the outer zone of young inclusions suggeststha t these organelles are involved in the formation of inclusion bodies.

INTRODUCTIONThere is little information about the intracellular changes induced by viruses of thecowpea mosaic virus group. Cowpea mosaic virus itself induces the formation of cyto-

plasmic inclusions of vesicular type (Agrawal, I964), but there are no electron micrographsshowing their ultrastructure. In the cytoplasm of Chenopodium amaranticolor Coste andReyn., cowpea mosaic virus forms crystalline aggregates which are frequently enclosed bymembranes (Milne, I967).Another member of the group, radish mosaic virus (RaMV), induces in certain cruci-ferous species vesiculated inclusion bodies (Z. Stefanac & D. Mamula, unpublishedresults) which, in the light microscope, resemble those induced by cowpea mosaic virus(Agrawal, I964). To get more data on the structure and formation of RaMV inclusions,we have made a comparative study by light and electron microscopy.

METHODSViruses and host plant. Most of the work was done with the HZ isolate of the European

strain of RaMV (cryptogram */* : */* : S/S: S/C1) found in turnip in Yugoslavia (Z. Stefanac &D. Mamula, unpublished results). The type strain of RaMV from California (Campbell,I964) served as comparative material for light microscopy. The viruses were maintainedin turnip (Brassica rapa L. var. rapa) which was kept in a glasshouse at 18 to 25 . For thestudy of the intracellular changes strips of epidermal tissue were taken from the region ofthe main vein of the leaf.Light microscopy. The strips of epidermis were used either living or fixed. For fixationthe strips were placed in cold acetone for 5 min., then transferred to 6o % (v/v) ethanol.

Unfixed material was stained with a 1% (w/v) aqueous solution of phloxine or withLugol's solution [3 % (w/v) aqueous solution of potassium iodine containing 2 % (w/v) ofiodine]. Fixed material was mounted in Millon's reagent [5"5 % (w/v) of mercury in conc.nitric acid] or in Sudan III (saturated alcoholic solution) and slightly warmed. Feulgenstaining was applied to fixed leaf pieces as described by Harrison, Stefanac & Roberts(197o).


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5 2 Z . S T E F A N A C A N D N . L J U B E S I CElectron microscopy. S t r ip s o f e p i d e r m a l t is s u e f r o m i n f e c te d o r u n i n f e c t e d p l a n ts w e r e

f i x e d f o r 3 0 r a in . i n ~ % ( v / v ) g l u t a r a l d e h y d e i n c a c o d y l a t e b u f f e r p H 7 "2 a n d p o s t f i x e d f o r2 h r i n ~ % ( w / v ) o s m i u m t e t r o x i d e . A f t e r f i x a t i o n , s a m p l e s o f t is s u e w e r e d e h y d r a t e d i na n e t h a n o l s e r i e s a n d e m b e d d e d i n A r a l d i t e . S e c t i o n s w e r e c u t w i t h a R e i c h e r t O m U zu l t r a m i c r o t o m e a n d e x a m i n e d i n a S i e m e n s E l m i s k o p I .

RESULTSLight microscopy

Inclusion formation and appearanceB o t h s t ra i n s o f R a M V i n d u c e d d i s t in c t iv e i n c lu s i o n b o d i e s in t h e c y t o p l a s m o f Brassicarapa L . v a r . rapa. T h e s e i n c l u s i o n s w e r e n e v e r s e e n i n t h e c e l l s f r o m h e a l t h y p l a n t s .F o l l o w i n g i n o c u l a t i o n o f t h e p l a n t s , i n c l u s i o n s a r o s e a f t e r 3 to 5 d a y s in i n o c u l a t e d

l e a v e s , a n d a f t e r 5 t o IO d a y s i n u n i n o c u l a t e d l e a v e s. T h e y w e r e s p h e r i c a l o r e l i p s o i d a lg r a n u l a r b o d i e s, s o m e t i m e s c o n t a i n i n g s m a l l v es ic le s a n d m e a s u r i n g f r o m 3 x 6 t o1 5 x 20 # m . G e n e r a l l y o n l y o n e i n c l u s i o n b o d y o c c u r r e d i n a c e l l, b u t a f e w c e l l s c o n t a i n e du p t o f i v e t o e i g h t s m a l l g r a n u l a r b o d i e s . I t s e e m e d t h a t s e v e r a l sm a l l g r a n u l a r b o d i e sa r o s e i n t h e c y t o p l a s m 2 t o 3 d a y s a f t e r in o c u l a t i o n o f th e p l a n t s , a n d t h e s e q u i c k ly a g g r e -g a t e d t o f o r m a s i n g l e l a r g e i n c l u s i o n a s f o u n d w i t h s o m e o t h e r p l a n t v i r u s i n f e c t i o n s(Sheffie ld , 193 0 .

T h e i n c lu s i o n b o d i e s b e c a m e l a r g e r w i t h t im e , a n d e v e n t u a l l y o f t e n e x c e e d e d th e n u c l e u si n s iz e , m e a s u r i n g u p t o 7 2 5 # m . O f th e s e e n l a r g e d i n c l u s i o n b o d i e s , s o m e w e r e w i t h o u tg r a n u l e s ( F i g . I a ) ; o t h e r s c o n t a i n e d f i n e g r a n u l e s ( F i g . I b ) o r r o u g h g r a n u l e s ( F i g . ~ c ) .T h e m a j o r i t y o f t h e e n l a r g e d i n c l u s i o n s c o n t a i n e d l a r g e v e s ic l es .

I n c l u s i o n b o d i e s w e r e e s p e ci a ll y n u m e r o u s t w o o r t h r e e w e e k s a f t e r i n o c u l a t i o n a n dw e r e s o m e t i m e s p r e s e n t a f t e r 4 t o 6 m o n t h s .Effect o f microchem ical reagents

A f t e r t r e a t m e n t w i t h L u g o l ' s s o l u t i o n t h e i n c l u s i o n b o d i e s b e c a m e d a r k b r o w n a n d t h en u c l e i l i g h t b r o w n ( F i g . 2 ) ; w i t h p h l o x i n e t h e i n c l u s i o n s t u r n e d r e d . T h e s e r e a c t i o n s ,e s p e c i a l ly t h e f i rs t , w e r e u s e f u l f o r q u i c k d e t e c t i o n o f t h e i n c l u s i o n b o d i e s . T h e i n c l u s i o n ss t a i n e d a c h a r a c t e r i s t i c b r i c k - r e d w i t h M i l l o n ' s r e a g e n t f o r p r o t e i n s . T h e F e u l g e n t e s t f o rD N A w a s n e g a ti v e . G l o b u l e s in t h e s u r f a c e z o n e o f r o u g h g r a n u l a r i n c l u s io n s s t a in e d r e dw i t h S u d a n I I I , w h i c h m e a n s t h a t t h e y c o n t a i n e d l i p i d s .

Electron microscopyI n t h e e l e c t r o n m i c r o s c o p e t h e p l a s ti d s a n d m i t o c h o n d r i a i n i n f e c t e d c e ll s l o o k e d n o r m a la n d w e r e d i s t r i b u t e d t h r o u g h o u t t h e c y t o p l a s m o u t s i d e t h e i n c l u s i o n s . T h e c y t o p l a s m w a se x t e n si v e ly v e s i c u la t e d ; d i c t y s o m e s a n d e n d o p l a s m i c r e t i c u l u m o c c u r r e d m o r e f r e q u e n t l yt h a n i n h e a l t h y c e l l s .General appearance o f inclusions

W h e n e x a m i n e d i n t h e e l e c t r o n m i c r o s c o p e , t h e i n c l u s i o n b o d i e s w e r e s e e n t o c o n s i s to f a b a s ic m e m b r a n o u s m a s s c o m p o s e d o f n u m e r o u s v e s i cl es o r m e m b r a n e s ( F ig . 3 , 4 )a n d c o n t a i n e d p r o t o p l a s m i c s t r u c t u r e s s u c h a s d i c t y o s o m e s , e n d o p l a s m i c r e t i c u l u m ,r i b o s o m e s a n d , o c c a s i o n a l l y , l i p i d g l o b u l e s , p l a s t i d s , m i t o c h o n d r i a a n d t h e s t r u c t u r e sk n o w n a s ' m i c r o b o d i e s ' . T h e b a s ic m e m b r a n o u s m a s s f r e q u en t l y c o n t a i n e d r o u n d a g g re -g a t e s o f s p h e r i c a l p a r t i c l e s ( F ig . 4 ) a n d s o m e t i m e s l a r g e v e s ic l e s w i t h a d i a m e t e r o f 2 # m .


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In c l u s i o n b o d i e s a s so c i a t e d w i t h ra d i sh m o sa i c v i ru s 53( F i g . 3 b ) o r m o r e . T h e s e v e s i c l e s w e r e a n a l o g o u s t o t h e v e s i c l e s v i s i b l e i n s i d e t h e i n c l u s i o n sb y l i g h t m i c r o s c o p y . T h e i n c l u s i o n b o d i e s w e r e n o t s e p a r a t e d f r o m t h e s u r r o u n d i n gc y t o p l a s m b y a n y s p ec i al c o n t i n u o u s m e m b r a n e .

T h e o u t e r z o n e o f y o u n g i n c l u si o n b o d i e s c o n t a i n e d a c t i v e d i c ty o s o m e s a n d e n d o p l a s m i cr e t i c u l u m ( F i g . 3 ). T h e i r i n n e r p a r t c o n s i s t e d o f r o u n d v e s ic l e s a n d m e m b r a n e s . I n g e n e r a l ,t h e v e s i c l e s c l o s e s t t o t h e s u r f a c e z o n e w e r e s m a l l e r t h a n t h o s e f u r t h e r i n s i d e t h e i n c l u s i o n s .

Fig. iFig. i. Inclusion bodies in the cytoplasm of living epidermal cells of Brassica rapa L. var. rapainfected with the European strain of RaMV. (a) Inclusion body (I) with big vesicles. (b) Twoslightly granular inclusions near the nuclei (N) in neighbouring cells. (c) Roughly granularinclusion attache d to th e nucleus. Note the presence of large lipid globules (arrows).

Fig. 2Fig. 2 A group of infected cells treated w ith Lug ol's solution. Inclusion bodies (I)are strained m ore intensely than the nuclei (N).


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54 Z . S T E F A N A C A N D N . L J U B E S I (~

Fig. 3Fig. 3. E lectron micrographs of par ts of R aM V inclusions. Six day old infection. (a) Dictyosom es(D) and endoplasm ic re ticulum (ER) associated with the surface zone of the inclusion body (I). (b)Section probab ly made throug h the surface zone of inclusion in tangentia l direction. Vacuole (VC);vesic les (V); mitochondrion (M).


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Inclusion bodies associated with radish mosaic virus 55

Fig. 4Fig. 4. Virus aggregates within the membran ous mass of inclusion bodies. (a) Rou nd aggregatecontaining scattered virus particles. Note the centres of crystallization (arrows). (b) A large virusmicrocrystal . Inser t shows the hexag onal arrangemen t of virus par tic les. Vacuole (VC); membran ousmass (MM): chloroplast (C) .


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56 z . STEFANAC AND N. LJUBE~ICV i r u s a g g r e g a t e s

T h e a g g r e g a t e s o f u n i f o r m l y si z e d s p h e r i c a l p a r t i c l e s ( F ig . 4 ) w e r e r a n d o m l y d i s t r i b u t e dt h r o u g h o u t t h e m e m b r a n o u s m a s s o f t h e i n cl u s io n b o d ie s , b o t h n e a r th e s u r f a c e a n df u r t h e r i n si d e. T h e i r n u m b e r r e a c h e d a m a x i m u m 2 to 3 w e ek s a f t e r th e p l a n ts w e r e i n o c u -l a te d . T h e a g g r e g a t e s w e r e s e en o n l y i n t h e i n c lu s i o n s a n d n o t i n o t h e r p a r t s o f i n fe c t e dcells.

T h e p a r t i c l e s i n t h e a g g r e g a t e s w e r e e i t h e r s c a t t e r e d ( F i g . 4 a ) o r a r r a n g e d r e g u l a r l y( F i g . 4 b ) , u s u a l ly in a h e x a g o n a l p a t t e r n . T h e c e n t r e - t o - c e n t r e d i s t a n c e b e t w e e n t h e p a r t i c l e si n c r y s t a l li n e a r r a y w a s 2 5 r im . , a n d w a s a n a l o g o u s t o t h e d i a m e t e r o f n e g a t i v e l y s t a in e dp a r t ic l e s o f R a M V . O n t h e ba s is o f t h e i r s iz e a n d t h e i r p r e s e n c e o n l y i n t h e i n f e c te d p l a n ts ,t h e s e p a r t i c l e s a r e c o n s i d e r e d t o b e r a d i s h m o s a i c v i r u s p a r t i c l e s in s i tu .

DISCUSSIONF o r m a t i o n o f in c l u s io n b o d i e s is t h e m o s t p r o m i n e n t s t r u c t u r a l c h a n g e v i si b le in t u r n i p

c e ll s i n f e c te d w i th R a M V . I n t h e l i g ht m i c r o s c o p e , i n c l u si o n b o d i e s o f R a M V a r e r a t h e rb u l k y a n d c h a r a c t er i s ti c a ll y v e s i cu l a te d . T h e y m a y b e u s e fu l f o r r a p i d d i a g n o s is o f i n f e c -t i o n w i t h R a M V b e c a u s e t h e y d if f e r f r o m t h o s e i n d u c e d b y s o m e o t h e r v i r u se s a f f e c ti n gC r u c i f e r a e , i. e. f r o m t u r n i p m o s a i c , c a u l if l o w e r m o s a i c , t u r n i p y e l lo w m o s a i c a n d t u r n i pc r i n k l e v i r u s e s ; m o r e o v e r , t h e i n c l u s i o n s i n d u c e d b y t h e s e v i r u s e s a r e a l s o c h a r a c t e r i s t i ca n d a ll d if f e r m a r k e d l y o n e f r o m a n o t h e r ( M i l i6 i6 , ~ te f a n a c & M a m u l a , I 9 69 ) .

T h e i n c l u s i o n b o d i e s o f R a M V , u n l i k e t h o s e o f o t h e r p o l y h e d r a l v ir u s e s, c o n t a i n v i r u sp a r t i c l e s a s s e m b l e d i n t h e s p h e r i c a l , s o m e t i m e s c r y s t a l l i n e a g g r e g a t e s w h i c h a r e d i s t r i b u t e dw i t h i n t h e m e m b r a n o u s m a s s o f th e i n c lu s i o ns . P a r t i c le s o f c a u l if l o w e r m o s a i c a n d d a h l i am o s a i c v i r u s e s , f o r e x a m p l e , o c c u r i n s i d e i n c l u s i o n b o d i e s , n o t , h o w e v e r , i n t h e f o r m o fc r y s t a l l in e a g g r e g a t e s , b u t i n d i v i d u a l l y e m b e d d e d i n m a t r i x ( F u j i s a w a et a l . 1967; K i t a j i m a ,L a u r i t i s & S w i f t , I 9 6 9 ). T h e a g g r e g a t e s o f p o l y h e d r a l v ir u s e s a r e u s u a l l y d i s t r i b u t e dt h r o u g h o u t t h e c y t o p l a s m ( L a w s o n & H e a r o n , ~ 9 7o ; W e i n t r a u b & R a g e t l i, ~ 97 o). H o w e v e r ,t h e p o s s i b i l i t y e x i s t s t h a t c r y s t a l l i n e v i r u s a g g r e g a t e s a r e i n c o r p o r a t e d i n s i d e i n c l u s i o nb o d i e s o f s o m e m o r e p o l y h e d r a l v i r u s e s . P a r t i c u l a r c r y s t a l l i n e a g g r e g a t e s w e r e f o u n d i nX - b o d i e s o f P e t u n i a r i n g s p o t v ir u s ( R u b i o - H u e r t o s , ~ 9 6 8) ; i n c a s e t h e y a r e b u i l t o f v i r u sp a r t i c l e s , t h e y w o u l d s h o w a c e r t a i n s i m i l a r i ty w i t h R a M V i n c l u s io n s .

T h e p r e s e n c e o f d i c t y o s o m e s a n d e n d o p l a s m i c r e ti c u l u m i n t h e s u r f a c e z o n e o f y o u n gi n c l u s i o n b o d i e s ( F i g . 3 ) a n d t h e i r a b u n d a n c e i n t h e c y t o p l a s m o f i n f e c t e d c e l l s i n d i c a t e ac e r t a i n r e l a t i o n s h i p b e t w e e n a c t i v i t y o f th e s e o r g a n e l l e s a n d v i r u s m u l t i p l i c a t i o n o ra s se m b ly . F u r t h e r m o r e , t h e y m a y b e i n v o lv e d in t h e f o r m a t i o n o f th e m e m b r a n o u s m a s so f t h e R a M V i n c lu s i on s . I n cl u s i o n b o d i e s c o n t a in i n g n u m e r o u s m e m b r a n e s d e r i v e d f r o md i c t y o s o m e s h a v e b e e n f o u n d i n c o n n e c t i o n w i t h a n u m b e r o f d i ff e r e n t v i r u s in f e c ti o n s .D i c t y o s o m e s f o r m m e m b r a n e s i n th e X - b o d i es o f P e t u n i a r i n g s p o t v i r u s ( R u b i o - H u e r t o s ,~ 96 8) a n d v e s i c u l a r s t r u c t u r e s in t h e X - b o d i e s o f s u g a r b e e t m o s a i c v i r u s ( F u j i s a w a , M a t s u i& Y a m a g u c h i , ~ 96 7). T h e y a r e a l so i n v o l v e d i n th e f o r m a t i o n o f i n c lu s i o ns o f d a h l i a m o s a i cv i ru s ( K i t a j im a et a l . I969) .

W e w i s h t o t h a n k P r o f e s s o r R . N . C a m p b e l l o f t h e U n i v e r s i t y o f C a l i f o r n ia , D a v i s , f o rp r o v i d i n g t h e t y p e c u lt u r e o f R a M V . W e a r e a l so g r a t e f u l t o th e I n s t i tu t e o f B i o lo g yU n i v e r s i t y o f Z a g r e b , f o r t h e u s e o f S i e m e n s E l m i s k o p I .


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Inclusion bodies associated with radish mosaic virus 57R E F E R E N C E S

A a R A W A L , H . ( I 9 6 4 ) . I d e n t i f i c a t i o n o f c o w p e a m o s a i c v i r u s i s o l a t e s . Mededelingen van de Landbouwhoge-school te Wageningen6 4 ~ 5 , I .C AM P BE LL , R . N . ( I 9 6 4 ) . R a d i s h m o s a i c v i r u s , a c r u c i f e r v i r u s s e r o l o g i c a l l y r e l a t e d t o s t r a i n s o f b e a n p o dm o t t l e v i r u s a n d t o s q u a s h m o s a i c v i ru s . Phytopathology 5 4 , I 4 1 8 .F U JI SA W A , 1 , M M S O I , C . ~ Y A M A a U C m , A . ( 1 9 6 7 ). I n c l u s i o n b o d i e s a s s o c i a t e d w i t h s u g a r b e e t m o s a i c .Phytopathology 5 7 , 2 I O .FUJISA~VA, . , R U BI O- HU E RT O S, M . , M A TS U I, C . & Y I AM A G U C HI , A . ( i 9 6 7 ) . I n t r a c e l l u l a r a p p e a r a n c e o f c a u l i f l o w e r

m o s a i c v i r u s p a r ti c l e s . Phytopathology 5 7 , I I 3 o .H A RR IS O N, B . D . , S TE EA N AC , Z . & RO B ER T S, I . M . ( I 9 7 0 ) . R o l e o f m i t o c h o n d r i a i n t h e f o r m a t i o n o f X - b o d i e si n c e l l s o f Nicotiana clevelandii i n f e c t e d b y t o b a c c o r a t t l e v i r u se s . Journal of General Virology 6 , I z 7 .K IT A JI M A , E . W . , L A U R I n S , J . A . & S W I FT , H . ( 1 9 6 9 ) . F i n e s t r u c t u r e o f z i n n i a l e a f t i s s u e i n f e c t e d w i t h d a h l i am o s a i c v i r u s . Virology 2 9 , z 4 o .L AW S ON , R . H. & HE AR ON , S. 0 9 7 O ) . S u b c e l l u l a r l o c a l i z a ti o n o f c h r y s a n t h e m u m a s p e r m y v i r u s i n t o b a c c oa n d c h r y s a n t h e m u m l ea f t is s u e. Virology 4 ' , 3 o .

M I LI ~I d, D . , g T EE A N AC , Z . & M A M U L A , D . ( I 9 6 9 ) . I n t r a c e l l u l a r c h a n g e s i n d u c e d b y c r u c i f e r v i r u s e s . PlantVirology, p . 5 4 , P r o c e e d i n g s o f t h e 6 t h C o n f e r e n c e o f C z e c h o s l o v a k i a n P l a n t V i r o lo g i s ts O l o m o u cI 96 7 . C z e c h o s l o v a k A c a d e m y o f S c ie n c es , P r a h a .M IL NE , R . G . ( I 9 6 7) . E l e c t r o n m i c r o s c o p y o f l e a v es i n f e c te d w i t h s o w b a n e m o s a i c v i r u s a n d o t h e r s m a l lp o l y h e d r a l v i r u s e s . Virology 3 2 , 5 8 9 .R UB IO -H UE RT OS , M . ( I 9 6 8 ) . F u r t h e r s t u d i e s o n u l t r a s t r u c t u r e o f p l a n t s i n f e c t e d w i t h Petunia r i n g s p o t v i r u s .Protoplasma 6 5 , 4 6 5 .S HE FF IE LD , F . M . L . ( I 9 3 I ) - T h e f o r m a t i o n o f i n t r a c e l l u l a r i n c l u s i o n s i n s o l a n a c e o u s h o s t s i n f e c t e d w i t ha u c u b a m o s a i c o f to m a t o . Annals of Applied Biology 4, 4 7 I .W H N TR A UB , M . & R A GE TL L ~ . W . J. ( I 9 7 0 ). E l e c t r o n m i c r o s c o p y o f t h e b e a n a n d c o w p e a s t r a i n s o f s o u t h e r nb e a n m o s a i c v i r u s w i t h i n l e a f ce ll s. Journal of Ultrastructure Research 32, 1 6 7 .

(Received 31 March 1971)

inclusion bodies in cells infected with radish mosaic virus

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