Some Effects of Turnip Yellow Mosaic Virus Infection on Nucleic Acid Metabolism in Brassica Pekinensis Rupr.

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SOMR E!?FTTS 02" P YYLLOV! MOSAIC WXDS I ~ T I O N ON NUCLRIC A C I D P@X'ABOLISM

IN

BRASSICA -P?TKINT!XSXS R U P R o

J T . Randlee, M. A g . Sc

.

( ~ d e l a i d e ) Department of Cell Biologg

Unlvereity

of

Auakland.

Thesls _submLtted t o the University of Auoklmcl

for

the degree of Doctor of PMlosoghy

Deaember, l968.

I 1 . I I ~ ~ ! ~ ~ ~ ~ T Y @f , s C C ~ C A ~ . L . ? ~ -

I I,- " ^'

.,

^{*.t -2:'}

q r c x 0 G Y THESIS

4994 - bS

%N a w

4 '7

L -

7.3 ..>

P-.

\

;

cf?

i,, A. r:

Growth of _{W w t d} plants Smpl* procedure

(i) F i e a y ~ t a m i c a l l y Fnfsated leaf.

(ii) Led sharing mosaic.

(iii) Light rdcroaaopy.

Radiochenriaal r n e t h ~ d s - ~ ~ ~ and 3 5 ~ Isolatim

of

chloroplasts and nnalei

Asaay of riboeomes and virus on aueraee density gradients Extraction of total nucleic acids

flucleic acid f r a o t i o n a t l m an methylated albuadn aolunsls Purifiuatf of DRA

Preparation of DEA from organelles CsCl equilibrium density &iente

b c l e o t i d s composition of 3 2 ~ labelled

M A

PolyacrglmAde gel eleotrophoresis

Fatimations of t a t 8 1 leaf ribmucleaae activity

CAAPPER 111. CHI\RACTmJ:STICS OF mE FIRST SYSTEYICALLII U93PMm:

LEAF

22 7. Growth

of

tho

first

aysteadcally infeoted leaf

(i) Rate of expansion.

(ii) Fresh and dry weifbt.

(u) Leaf thiolmeas.

(iv) Cell nnmber and sice.

2.

Time

course of virus Inorease

3.

Changes in ohloroplaate

(i)

Chloroplaets s i t u .

(ii) Isolated chloroplast 8.

(i) Growth of the leaf.

(ii) ^{The first} systemically infected _leaf a s an axper5rnental system.

ml!8

m.

W W S

IR

.WCLXIC ACIDS, RIBOSO??!% ^hFII) POL=@3Q?%S

I??

T)f3;: FIRST SPSTmCALLY

xrmx?I?ED

XrEA7"

37

^{I '}

1. Rucleio aaide

37

(i) T o t a l

nucleic

acids.

38

.' (ii) llucleio aoids separated

an

MAX ooluma.

39'

(a)

Lanr

moleoular w e i g h t

RHA

(~RIPA).

39

^'

(b) DXA. A0

(c)

a&

moleuular weight

M A

(wA). 44 (d) Interrelatimahipa betwean mcleio mid c b a a e s . 43

2. Ribosmee 42

(i)

Propertiee of r i b a s o d fractions isolatsd from

sucruse denrsity gradientrs, 42

(ii) ^Time c m e e of changes

in

the ribosome content

of

healthy an& M e c t e d leaves, 43 (a) Riboames axtraoted in Ma-B&-SR buffer.

43

(b) ^Ribosome8 extracted in _the presence of

T r i t o n X-?OO.

( c ) RLboaomee extracted

in

the preaenoe of

panareati c ribonnolease.

4

(w) Ribosomes in excised healthy and infected leaf tieme.

A5

(a) Discs

floated for 4

hours.

6

(b) Leavea sxoised 3 days after the time of j~noculationm

(ir) Rate of incmporaticm of 3 2 ~ into ribos-s. 47

3. Time

^{aburse of cbmgea in} polyribasamea during e s r l y

vf rus replica$lcln 49

(i) The polyribosome content of -ding h e d t w snd

first systemically inTeotei! havea.

15.9

(ii) lncozporaticm of S 2 ~ into polyribosomea. 51

4. Dieuueaion 52

CRAJ?TFB V. A SPXRCR

FOR

!!TISO-SPECIFIC POLYPXBOSOP'?X D I R I l J G

EARLY

VIRUS S'YFTKESLS 62

1. Procedures f o r extracting an& preserving polyribosomee

(i)

h c t i o n a t i a n

on

sucrose density gradfenta.

(ii) ESctr~ction and ppreaervation of polyribasoma.

2. Factora affecting polyribosome levels and r i r a s ayntheais (I) Wfeot of cutting and floating leaf dfaes.

( l i ) Xffect

of

darkness.

(a) mciaed leaf discs.

(b) m o l e plants.

(tii)

Eff eat of actinoqyoin D

(m)

^an polpibosame p?.itms.

(ir) Effect of NvD on the incorporation of 333? in dieas.

3.

Virus induced uhanges in pol@.bosomes

in

AMD-treated

leaf d2scs.

69

(i)

3 5 ~

labelled leaf d i s c s . 70 (a) Distribution of TCA insoluble

3 5 ~

in @ & i d s . 70

(ii) 3 2 ~ labelled leaf discs. 71

(a) Distribution of TCA insoluble 3 2 ~ in gradients. 71 (b) Contamination of f r a c t i a e d t h h o l e v i r u s . 72

(iii) Isolation of nucleic actd.da from polyribosmes.

74

(a) Fk~ctlonation falloaing trmtmsnt rr~th DTA. 76 (b) Fractionation f o l l o r i n ~ Rnase treatment.

77

(d) Polyt~orylamide gel elsatrophoreaia. 78 4. Virus induoed ahanma in polyribosomes from whole leares

83

5. Discussion 84

CHAPTER

VI.

^FE?7 DDA 03' CKIlmE CABBAGE AND

ITS

PRO-FEJ?TIES

SYSTDTCALLY 1-ED 3Jbl.F 92

1 Some properties of the DNA iaolated from Chinese aabb-

93

\

(i) &traction of

DM. 93 '

(ii)

DNA components in CsCl equilibrium gradients. 95

(ti9

bsociatian of

D M

with c h l o r ~ l ~ s t ~ ~ 2. T h e course of 3 2 ~ incorporation i n t o the nucleic acids

of _dark green and yellow tieme of leaf showing mosaic 3. ^Amounts af 3 2 ~ incorporated F n t o the BnA of dark green t ? r

h e d t b tissue compared with infected t i e m e

(ti) Specific r a i o a c t i r i t y of naj w EUXI m i n o r components

of DIIA.

(a) ^{D a r k p e e n} and yellow t i a s a e in moasic leaf up %a 14 om long,

(b) Realtby leaf compared with roa-e first ,s,vstemfenlly i n f e o f d leaf.

4 ^Isolation of a r g m e l l e ~

(i) Some properties of the

T,

M and

A

fraotions frm

emlc p e e n or h e a l t v tinsue and yellow tisaue.

(a) Composition of

T,

)I and 1P f h c t i o ~ ~ s . (b) Protein pattern8 easoeiated d t h

T

and N

fractions.

( c ) Absenoe o f TYBV A o m cell organelles.

(ii)

Incorporation of 3 2 ~ i n t o the DlOA of

T,

M and

R

f r ~ c t f ons.

(a) Aealtby leaf.

(b) Dark e e a n ^and yellow tissue of mossier l e d .

(id DRA

oomponents in the

T,

M and 19 fraations of heabt%y and -f ectsd leaves.

1. T o t a l Mane m t i v i t r (i) Method of assay.

(11) A c t i v i t p d t m b g %he growth o f healthy nnd first 8ystemicall;y infeefed leaves,

(Fii] Activity in

TY!fl infected leaves sholrlng; moaaio.

2. Separation and assay of PXase ieozymes

(A) Development of a technique for as- isozpes.

(ii) ^Change6

in

Rhse iao?ymes in leaves systemically inTected wtth !ITrnT,

(w) ^{ChRnges in -Waae} iao.ryl~es in leaf eyetemically inlecterl n i t h txmiip n o s d c virus (%I:;).

(iv) ^{IOJaae isomas} in _!IT397 inoculated leaf.

3.

Associstian o f RlTase activitg with cell fraations ( i ) T o t a l RNase sotivity of chloraplasta md nuclei.

(ii) ^RRm9 iaozymes aseaciatted with cell f ~ i c t i ~ n s . 4. Acid 2 h a s ~ h a t ~ s s activttr

5. Disaussim

C3lGTER VXII. PROBLmS ASSOCIATED KPFR STUDSIB OF TrRUS-INDUCED C M E B

IN

TEE HOST PLANT

I am

indebted to Profesam R.E.F. Matthem f o r supervision, &dance and helpful discuesfon t h d u ~ h o a t the course of _{t h i a} work.

f

@ a t e M & v aclmorledge the encmragement and advice of other members of the Depact- ment of Cell Biology, and rrf.sh to thank in particular the following people far seaistance d t h aspects

of

this workr

Dr.

S o SalUvant

for

eleotron nicroecov, and in partioulat the ~ e ~ o a r a t i o n of chloraplaat DAA specimens) 1%. R. VaMyama f a r a a z r y b g out mch of the

thin

seotioning and photcmfmosco~;

Bk.

J.J. F i e l d s

for

photo@aphJta aemicae and assietanoe Fn the ohatocowinng of figures;

I ! x .

C. JF/hAttingham

for

glass-house semices; Dr. P.L.Bergquist for p r d . & b g _E. a o q ' 4 ~ - a m ~ # Pk. MeV. Barridge for p r a v i d w yesat 8FRAo ?hRnks are a l s o due t o mmbtrrs

of staff of the D.S.I.R., and in particular to Dr. R.L. BielasM and

Dr.

M.S. Reid for: inertmotion in the use of ver.f;iual flat-aheet pol$- ucrylemide gel electro-ihmeais,

Dr.

_P.R. for m i d i r i g tprnip moatdo vim8 i n d w n , and to b. G.B. Pettxmen f o r camylng out _the CsCl equ5Ubrian C e n a i t y gradient centrifuaation of early DNA preparatim8 in the k n m i c a l ultracentrifuge 81: Paherston Rorth.

I

am g r s t e h t l for the help of q y d f e in the preyxation o f Figwes, and thank Miss A.

Thompson for t y ? b g services,

I

acrhorrldge with t h e the finnnctal saaistance gained from a H w Zealad Post~ar?l,uate Saholarahip.

snn3qnarr

i. Some changes induced in Chinese cabbage nuclei0 aoid metabolism by infeotion with turnip yellow mosaic Pima ( T P ~ W ) have been examined

in

the first leaf t o develop systamia symptoms. Thia leaf was used in an attempt t o avoid w a u n d f n g effects, and its charactariatius have been defined t o allow infection-induced changes t o be related t o the stags

of l e a f development, the time course oP infeatl.cn and v i r u a inaresse, and the onset of microscopio Rnd maaroscopic _symptom and stunting effeots.

In

some expeflments, where effeets at a late stage of infeation rere studied, leaves showing massic p p t o m a were used.

2. In healthy leaveer which w e r e comparable with f i r a t ~ s t e d o s l l j

infected leaves, the extr~atcrbillty of 68s a& 83s rfbosomee deckhied during expxpanaion, appsrentv ,va a result of inoreaasd binding to

manbranas by eome ribonnolease sensitive meabniam.

In

infeated leaves, a sharp r i s e was o b s e r ~ e d in the amounts of ribosclnnes erhaeted j u s t before virus could be detected, and concentrations remained higher

than in healthy leaves ^during the period of _active v i r u s replieation.

Thle

infection-induced rise i a attributed t o t (i) the increaeed synthesis of ribosmea, and (ii) t o the increaeed axtrnotability of rib osomea

.

3.

^No virus-induced changes could be observed in polyribosumes up t o the time when virus was f i r s t detecrted. %en host polyribosornee were

suppressed by excising leaf tinsue and treating with actinamyoin

D,

infeat ion-apeoif i c 32~-labelled 50-608 and 9 60-21 2s oomponsnts were

isolated at 6-7 days after the time of inomlation, ^{and R} high

malacular ^weight

FTA

VBR isolated from then on 2$ poly8,crylamide e l s e An infeotirm-opeaif5.0 xdioaotive ??Qe aampamt ^was ale0 iaaletled.

from 3 2 ~ - l ~ b c l l e d intaat i d e a t e d leaf treated with aatinomgcin

D.

4.

In

healthy lesves, the eontent of t o t a l

rmcleio

aoids, md

low

moleuulax w e i g h t RWA,

DFA

and high maleau3.ar weight FXA per ctell. did not appear t o o m markedly f r o m the latter afa~pae of the aell ^I d i v i s i o n phase of l e d dweloplaent to the time when leaves r m e fnlljr expanded. ^Canteat per Leaf inoreased until cell d i v i s i o n aeescd then

remainsd, relatively cmatant. Conaenfration f e l l aa the leaf exr~yliaed.

I

Chmges induaed i?;v infection w e r e s m d ! l , bnt resalts were incancl~rsive beaauee of high v~~Aah1.lliw. I

5. ^{Chineso cabbage D3A has n m j o r oanyonen*}

( y

7.696)s and a minor oomponent

(p

= 1.703) nMah appenrs t o be n nnclear aat ellif e

DAA.

30

chloroplast DNA satellite has been obeer~ed in C s C l equilibrium dmai*

1

padietlta, &though electron microscopic e x a m t i o n 2 m ~ ahom that

DHA

is associated with ohloroplasts. The yellow areaa of leavee aver 91

crm

long shoring mosaio ls~nnptonur, and old f i r a t systemically infected leatea, inoorporated more 3 2 ~ i n t o DHA thm did idomppacable dark gresll or healthy tissue.

In

emller beeves s h m h g mosaio, the differenae was e m U . e r and more variable. Attemyte, t o deterndns the intraaellular _{s i t e} of higher 3 2 ~ h a r p r e t i o n i n t o

D M

have given inconclasLvtve results.

6.

An

electrophoretic method haa been dweloped f o r carrying aut rapid and semiquantitative essays of ribonuclease ieozyme aatipity in crude

were detected. 3.n Chiaeee c a b b w leavea, pad d 1 were fmt! at *M~hesf

~ ( ~ f t v i t y f.n the 705,000 F: e,v*op3a~mic oupemztent;, V m r l5ff;le ~ o t i v j l t ~ r

was

Neb

^{dur- the} emQr @%ages of leG m d h , htat declined to a

1

v % r 3 a d u undeteo),~b?.e level rrhm le@h exseedd.

?O

cg. RFTcasee

I

lsnd 11 ahowed no mc&ed. change5 %n ~ c t i v i w d t h tb.c.

7.

³³ leave8 q y ~ t e m f c ~ ~ tnfected d-th !?'YW, the PXaae Isozyme yattena m a id-entlaal t o +hnt of healthy 3-eaves

mil

^after v h ~ s I?ad be@- t o inme~ats rapidly. P ~ R , E ~ 111 then show&?- rerlevd- atrow sqt5dtg, ELad J.nsalses 1 and 11 alea a h m d a 9rr(R11! $~creanc 3m activity. ?% nm iaoz.ymas w e r e obeemwt- ^j31 qyatevdcal1,y infect &!! leayea.

8. S+mtemia infmtim *ath f ~ ~ o ~ 9 . l ~ ~ v F N n p (WV) c ~ s d c h m : ~ s in ieazyme p s t t e m e very ~kn12l.15~~ t o thoae obsemred in _leave8 a y ~ t e m l c a l l y infeotecl d t h !TYFT. Leaves w-ed by rubbin6 with CarbontnC!um d o n e shored increased FVaae

fIX

activity. Leaves mecheniaally inoculated with TTT retained high R b ~ e XI1 aotivity for R lonmr time than _rubbed control leaves, an& the level m\s compere.ble with t h s t

in

s y s t e n i c a l b fnfeotd lenves of the same size. Thua, the x t e e in. _P?Sase 111 activity appears to be a non-x-rpecific p5enomman a~eoaiateit with increassd. PFA synthesris.