multiple viral determinants affect seed transmission of pea seed borne mosaic virus in pisum sativum

7/31/2019 Multiple Viral Determinants Affect Seed Transmission of Pea Seed Borne Mosaic Virus in Pisum Sativum

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J ~ u ~ n a l ~ ` ~ f ~ n e ~ r a l ~ V i r ~ l ~ g y . !~ 9 9 % ~ . 7 . ~ 3 ~ ! ~ 4 . 9 ~ 3 ~ 4 : ~ F ~ !n ~ e d ~ n ? e ~ a ! ~ B r ! ! a ~ . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . ..

M u l t ip l e v i r a l d e t e r m i n a n t s a f fe c t s e e d t ra n s m i s s io n o f p e aseedborne mosa ic v i rus in Pis u m s a t iv u mI. E . J o h a n s e n , 1 W . G . D o u g h e r t y , 2 K . E . K e l l e r , 3 D . W a n g 4 a n d R . O . H a m p t o n 31Biotechnology Group, The Dan ish Ins ti tu te o f P lan t and S o i l Sc ience, Lyngby, D K-2 80 0 Denmarkz Depar tment o f M icrob io logy and Center fo r Gene Research and B io techno logy, Oregon S ta te Un ivers ity , Corva l li s 97 33 1- 38 04 , USA3 Depar tment o f Botany and P lan t Patho logy, Oregon S ta te Un ivers ity , Corva ll is 97 33 1-2 90 2, USA4 Depar tment o f V i rus Research , John Innes Cent re , Norwich NR4 7UH, UK

T w o p e a s e e d b o r n e m o s a i c p o t y v i r u s ( P S b M V )i s o l a te s , P - 1 D P D 1 ( P - l ) , w h i c h is h i g h l y s e e d -t r a n s m i t t e d , a n d P - 4 N Y ( P - 4 ) , w h i c h is r a r e l y s e e d -t r a n s m i t t e d , a n d c h i m e r a s b e t w e e n P - 1 a n d P - 4w e r e a n a l y s e d t o m a p t h e v ir a l g e n e t ic d e t e r m i n a n t so f s e e d t r a n s m i s s i o n . I n f e c t i v it y o f c h i m e r i c v i ru s e sw a s e v a l u a t e d b y i n o c u l a t i n g P i s u m s a t i v u m w i t hR N A t r a n s c r i b e d in v i tro f r o m r e c o m b i n a n t f u l l -l e n g t h c D N A c l o n es . T h e c h i m e r i c vi ru s e s t h a t w e r eu s e d d e m o n s t r a t e d t h a t a g e n o m i c s e g m e n te n c o d i n g t h e 4 9 k D a p r o t e a s e a n d p u t a t i v e R N Ap o l y m e r a s e w a s r e s p o n s i b le f o r s y m p t o m i n d u c t io n .A t t e m p t s t o d e t e r m i n e t r a n s m i s s io n o f t h e c h i m e r ic

v i r u s e s i n P. sa t i vum c u l t i v a r s k n o w n t o t r a n s m i t P -1 a t h i g h f r e q u e n c ie s s h o w e d t h a t s e e d t r a n s m i s s io ni s a q u a n t i t a t i v e c h a r a c t e r i n f l u e n c e d b y m u l t i p l ev i ra l d e t e r m i n a n t s . S e e d t r a n s m i s s i o n f r e q u e n c y d i dn o t c o r r e l a t e w i t h a c c u m u l a t i o n o f v i ru s i n v e g e -t a t i v e t is s u e . T h e 5 ' 2 " 5 k b o f t h e 1 0 k b P S b M Vg e n o m e h a d a m a j o r i n f l u e n c e o n t h e s e e d t r a n s -m i s s i o n f r e q u e n c y a n d w a s a n a l y s e d f u r t h e r . T h i ss h o w e d t h a t , w h i l e t h e h e l p e r - c o m p o n e n t p r o t e a s ew a s a m a j o r d e t e r m i n a n t o f s e e d t r an s m i s s i o n , t h ep o t y v i ra l P l - p r o t e a s e e x e r t e d n o m e a s u r a b l ei n f l u e n c e .

I n t r o d u c t i o nTransmiss ion t h rough seed ha s been de sc r ibed fo r 108plant vi ruses in one or more of the i r hosts . For a l l of these ,except tobacco mosaic vi rus, successful seed t ransmissiondepends on t he v i rus en t e r ing and su rv iv ing i n t he embryo(Mink, 1993) . Seed t ransm ission is prec luded wh en the vi rus i sunable to infec t the gametes pr ior to fer t i l iza t ion, unable toen t e r t he embryo dur ing deve lopment , o r when the v i rus i si nac t iva t ed in t he em bryo dur ing seed ma tu ra t i on and s to rage(Maule & Wang , 1996) . In v i rus -ho s t com bina t ions wi thpotent ia l for seed t ransmission, the f requency of seed t rans-mi ss ion depends on bo th hos t and v i rus geno type and mayrange f rom 0% to a lmost 100% (Mink, 1993) .The mechan i sms o f r e s i st ance t o seed t r ansmiss ion a re no tresolved a nd inher i tance of resis tance has been inv est iga ted inon ly a few cases. In H o r d e u m v u lg a r e cul t ivar M odjo , resis tancewas r epor t ed t o be cond i t i oned by a s i ng l e r ece ss ive gene(Carroll e t al . , 1979), whereas resistance to seed t rans mission ofpea seedborne mosa i c po tyv i rus (PSbMV) in P i s u m s a t i v u m is

Autho r for correspondence: I. E. Johansen.Fax +4 5 45 9 3 22 13. e-mail e.johansen@ dips-lyngby.dk

inher i ted as a quant i ta t ive charac ter cont rol led by the ac t ion ofmul t iple maternal genes (W ang & M aule , 1994) . In a s inglehost cul t ivar , d i f ferent vi rus i sola tes a re seed- t ransmit ted todiffere nt degree s. The se differences m ay reflect differences inv i rus r ep li c a ti on and m ovem ent wh ich de t e rmine t he f r equencyat which the vi rus successful ly enters the gametes or theembryo (Carroll , 1981).

Pseudorecombina t i on s tud i e s wi th i so l a t ed v i r a l RNAsfrom st ra ins di f fer ing in seed t ransmissibi l i ty show ed tha t theseed t r ansmiss ion phen o type w as l inked t o RN A 1 o f r a spbe r ryr ingspo t and t oma to b lack, i ng nepov i ruse s (Hanada &Har r i son , I977) and t o RNA I o f cucumber mosa i c cucumo-vi rus (Ham pton & Francki , 1992) . Ana lyses of bar ley st r ipemosaic hord eivi rus (BSM V) chimeras fac i li ta ted mapping ofthe ma jo r de t e rminan t s o f seed t r ansmiss ion i n ba r l ey (H.v u l g a r e ) to the 5 ' un t ransla ted leader of RNAT, a 369 b p repeatin the 7a gene, and the 7b gene (Edwards, 1995).

The seed t r ansmiss ion f requency o f PSbM V i s in f luencedby the gen o typ e o f t he v i rus i so la t e (Kohnen e t al . , 1995) . Thecomple te nucleot ide sequences of two isola tes of PSbMVwhich di f fer wid ely in seed t ransm ission f requency in severa l P.s a t i v u m cult ivars h ave b een de term ined (Johansen e t al . , 1991,1996) . However , a comparison of the pr imary sequences

~ 1 4 c ~0001 - 4205 1996 S GM


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ii:iii:ii:i :.ig iiiii!ii ii! ii iii, ii.iiiiiiiiiiiiii',ir e v e a l e d t o o m a n y d i f f e r e n c e s t o c o n c l u d e wh i c h p a r t o f t h eg e n o m e d e t e r m i n e d s e e d t r a n s m i s s io n ( J o ha n s e n et al., I 9 9 6 ) .T h e r e f o r e a s t u d y w a s u n d e r t a k e n t o a n a l y s e t h e 1 0 k b R N Ag e n o m e o f P S b M V b y c r e a t i n g c h i m e r a s b e t w e e n t h e t w ov i r u s is o l a t e s a n d a n a l y s i n g t h e i r t r a n s m i s s i o n t h r o u g h s e e d i nP. sat ivum c u l ti v a rs k n o w n t o t r a n s m i t P S b M V a t h i g hf requencies .

M e t h o d s V i r u s i s o l a t e s . PSbMV isolate P-1 DPD1 was recovered from a peaseed sample analysed at the Dan ish Plant Directorate (Lyngby, Denmark).The PSbMV P-4 NY isolate was recovered from USDA Pisum P.I.accession 471128 and kindly provided by R. Provvide nti (New YorkState Agriculture Experiment Station, Geneva, NY, USA). For con-venien ce P-1 DP D1 and P-4 NY w ill be referred to hereafter as isolatesP-I and P-4; however, it should be noted that seed transmissibility s notnecessarily linked to the pathotyp e of PSbMV. Clo ning and sequencingof P-1 and P-4 were reported b y Johansen el al. (1991, 1996). cDNA modificationsand construction of full-length clones.A c istron map o f PShMV is show n in Fig. 1 (a). Locations o f natural andengineered restriction sites in the cDNA of isolates P-1 and P-4 areidentified by the first nucleotide (nt) of the recognition sequence. Mapsand names o f isolates P -I and P-4 intermediary clones are show n in Fig.I (b).

cDN A o f the 5' termini of isolates P-1 and P-4 covering nt 1-Sph[ 965,an d 1-SphI 924, respectively, were amplified by R T-P CR (Ko hnen et at.,

1992) with primers that added a DraI site at each 5' end an d an XbaI siteat each 3' end. The DraI site allowed precise excision of the viral 5'termini, and the XbaI site facilitated subsequent assembly of full-lengthclones. An XbaI linker was inserted 3' to the cDN A cov ering the viralpoly(A) tails, thus creating a unique XbaI site for linea rization of the full-leng th plasmids prior to in vitro transcription. The cDN A sequences w eremodified by site-directed in vitro mutagenesis (Sculptor, Amersham). AHindIII site was introduced at nt 1276 of P-l, a nd the P-4 sequence wasmodified in three po sitions, introdu cing a HindIII site at nt I234, a BamHIsite at nt 225 9 and a PstI site at nt 5812. All nucleotide substitutions weretranslatio nally silent.

Full-length clones for in vitro transcriptio n were assembled in thevecto r pTTE19( + ) (Petty, 1988), which was modified to remo ve the SphIand HindIII sites from the polylinker. The virus sequences were cloneddirectly behind the T7 promoter b y dige sting modified pT7E19( + ) withSad, treating with T4 DNA polymerase and then digesting with XbaIbefore inserting the 5' terminus of either P-1 or P-4 as a DraI-XbaIfragment. These plasmids were digested with SphI and XbaI, and clonedcDNAs (covering Sph1965 to BamH12300 of P-1 and SphI 924 to BamHI2259 of P-4) were inserted as SphI-XbaI fragmen ts creating plasmids pP aand pPb, respectively. Clon ed cDN As coverin g HindIII 8647 to thepoly(A) tail of isolate P-1 and HindIII 8585 t o th e poly(A) tail of isolateP-4 were inserted as BamHI-XbaI fragments 3' to both BamHI 2300 ofpPa and BamHI 2259 of pPb resulting in pPc, pPd, pPe a nd pPf.

pPg (containing cDN A covering BamH12300 to HindlII 8647 of P-l)and pPh (containing cDNA covering BamHI 2259 to HindlII 8585 of P-4) were digested with BamHI and PstI, and the BamHI-PstI fragment ofpPg was inserted into pPh, creating pPi.

Full-length plasmids pP-1, pP-111 4 and pP-411 1 were assembled by

(a)

/ / e'P1Pr HC-Pro Pa I I c, I ivPgl II 6kl 6k~

%

I IRdRp I CP ~poly(A)I

%(b)Dral Sphl BamHI Pstl4, $ 4, 4,

Hindlll XbalpP apPb

r - - - - - ~ pPcpPd

i i: ] p P eI ] I pP!

PPgpPhpPi

Fig. I . (o) Cistron map of PSbMVshowing the noncoding regions (solidline), the o pen reading frame encodingthe potyvirus polyprotein (open box) andputative pro teoly tic cleavage sites(vertical lines). Relevant restriction sitesin the cDNA are shown above (P-I) andbelow (P-4) the cistron map. Restrictionsites marked with an asterisk wereengineered into the sequences. PI Pro,PI protease; HC-Pro, helper-componentprotease; P3, P3 protein; 6ki, 6 kDaprotein I ; CI, cylindrical inclusion protein;6k2, 6 kDa protein 2; 49k-Pro, 49 kDaprotease; VPg, genom e linked virusprotein; RdRp, putative RNA dependentRNA polymeras e; CP, coat protein. (b)Maps and names of intermediary clonesused in the assembly of the full-lengthclones pP-1, pP-1114, pP-411 I, pP-114 4 and pP-4144. The P-I isolatesequences (light grey) and P-4 isolatesequences (dark grey) contained in theclones are shown as boxes, positionedaccording to the sequ ence in the ge nomethey represent. Areas represented by asolid line are not present in the clones.Vector sequences are not shown.


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(a) (b)Symp toms Virus acc. Inf./Tot. % ST % Germ inat ion

P-1 Severe 0.66 c 116/464 25 b 94 b,cP-4 " Mil d 0.37 d 1/505 0.2 d 91 c,dvP-1 ~ 4~ ~


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i i i ii ii i ii i i i i i ! i i ! i i i i i i i i iInfected plants were grown to maturity and seeds were harvested as theseeds matured. Seeds from infected plants were sow n and seedlinginfection was determined by DAS-ELISA 3 weeks aft er emergence.Confidence intervals (95 %) w ere calculated for the prob ability of theobserved seed transmission requency,assum ing a binom ial distributionof the data.

Re s u l ts a nd D i s c us s ionI n fec t iv ity o f R N A t ranscr ip ts f rom na t ive andr e c o m b i n a n t f u l l- l e n g t h c D N A c l o n e s

Inocula t ion of P . s a t i v u m w i t h i n v i t r o - s y n t h e s i z e d trans-cripts of pP-1, pP-1114, pP -4111, pP-1144 or pP-4144 (Fig.2a) , and pP-I(P-4 5 'UTR), pP-I(P-4 Plpro) or pP-I(P-4HC pro N) (Fig. 3 a) a l l resul ted in infec t ion of 50 to 100 % of theinoculated plants. In subsequent mechanical transfers, usinginfec ted plant mater ia l as inoculum, the infe c t ivi ty of vP-1 andthe chimeric vi ruses was comparable to na t ive i sola tes P-I andP-4. In contrast , transcripts of pP-4 and reciprocal chimeras totho se sh ow n in Fig. 2 (a) we re no t infectious. All these plasmidscon ta ined cDN A cove r ing n t s 2259-5812 o f P-4, sugges t i ngtha t t h is cDN A segmen t was de fec ti ve . How eve r , subs t i t u t ionof th i s r eg ion wi th cD NA f rom a new cD NA syn thes i s r e ac ti ondid not resul t in the genera t ion of biologica l ly ac t ivetranscripts.

S y m p t o m i nd u c ti on a n d a c c u m u l a t io n o f t r a n s c r i p t -de r ived v i rusesSym ptom s induced by vP-1, vF-1114 , vP-4111, vP- I (P-4

5 'UTR), vP-I(P-4 P lpro ) an d vP-I(P-4 HC pro ) were simi larto tho se of na t ive P-1 vi rus ( t ransient ve in c lear ing, d ow nw ardrol l ing of leafle ts and shorte ned internodes) . Sym ptom s of vP-1144 and vP-4144 were mi ld , causing only a s l ight growthreduct ion simi lar to plants inocula ted wi th na t ive P-4 vi rus(Fig. 2 b).

These obse rva t i ons sugges t t ha t t he r eg ion o f t he PSbMVg e n o m e b e t w e e n P s t I 5 8 7 4 / 5 8 1 2 a n d H i n d I I I 8 6 4 7 / 8 5 8 5 h a sa major inf luence on symptom sever i ty in P . s a l i v u m . Thisreg ion con t a ins t he po tyv i ra149 k Da p ro t ea se and t he pu t a t i veR N A d e p e n d e n t R N A p o l y m e r a s e , b o t h p r o p o s e d t o b einvo lved i n r ep l i c a ti on o f t he po tyv i ru s gen om e (Riechmann e tal . , 1992).

To de termine whether the di f ferences in symptom in-duct ion corre la ted w i th di f ferences in vi rus accumula t ion, there la t ive accumula t ion of vi rus in systemical ly infec ted leaveswas de term ined 3 w eeks af te r inocula t ion (Fig. 2 b).

vP-4111 accumula t ed t o a h igh e r concen t ra t i on t han P- l ,vP-1 and vP-1144, which, in turn, accumula ted to higherconcentra t ions than P-4, vP-1114 and vP-4144. These da tadem onst ra te tha t P - l , P-4 and the chimeras accum ula ted todi fferent leve ls in v egeta t ive t i ssues, b ut th a t the accumu la t ionof v irus was no t co r re la t ed wi th sy mp tom induc t ion . The re fo re,t he sym ptom s induced we re no t m e re ly a re su l t o f compe t i t i veinh ib it i on o f hos t g row th , bu t r a the r migh t have been due t o an

in te r ference wi th hos t g ro wth r egu l a to r me tabo l ism and g eneregula t ion (Fraser e t a l . , 1986; Wa ng & Maule , 1995) .S e e d t ra n sm i s si on o f v P - 1 1 1 4 , v P - 1 1 4 4 , v P - 4 1 4 4a n d v P - 4 1 1 1 in P. so t i vum ~ 5 4 9 '

The seed t ransmission f requencies of vP-1114, vP-1144,vP-4144 and vP-4111 w e re de t e rmined i n t h ree expe r imen ts .The abso lu t e seed t r ansmiss ion f r equency va r i ed be tweenexperiments, which was probably a resul t of seasonal changesaffec t ing greenhouse condi t ions. However , in a l l three experi-men t s , vP-1 w as t r ansm i t ted a t t he h ighes t f r equency , fo l l owedby P - l , vP-1114, vP-1144, vP-411 1 and vP-4144. P-4 wast ransm it ted in on ly one seed. Resul ts of the comb ined da ta a reshow n in Fig. 2 (b). The seed t ransm ission f requency o f vP-1was higher than P-1 in a l l three exper iments. The di f ference ,which was signi ficant (P < 0"05) , could be a resul t of themaintenance of P-1 by repeated mechanica l t ransfers wi thoutselection for seed transmissibil ity. In contrast , vP-1 w as deriv edf rom a cD NA of v ir al RN A i so l a ted f rom P-1 shor t l y a f t e r t hevi rus i sola te was f irs t recovered f rom an infec ted seed sample .The seed transmission frequencies of the chimeric viruseswere i n t e rmed ia t e be tween P-1 /vP-1 and P-4 . Of t he ch imera s,vP-4144 was t rans mit ted a t the lowest f requen cy (sl ight ly , bu tno t s ignif icantly , h igher tha n P-4) . The increasing cont r ibu t ionof sequences f rom P-1 found in vP-4144, vP-1144 and vP-1114corre la ted wi th increasing seed t ransmission, dem onst ra t ingtha t seed t r ansmiss ion o f PSbMV i s in f luenced by de t e rminan t scon ta ined in each o f t he exchanged geno me f ragment s . The 5 'r eg ion (n t 1 -2259) o f P-4 app ea red t o hav e t he s t ronges tinf luence on the seed t ransmission f requency, reducing theseed t r ansmiss ion f r equency o f vP-4111 to 4"6%, comparedwi th 37% fo r vP- I .The seed t r ansmission fr equency was no t co r re l a ted t o t hevi rus conc ent ra t ion in infec ted ve geta t iv e t i ssues (Fig. 2 b). Thisi s i n ag reemen t wi th r e su l t s ob t a ined by Wang e t a l . (1993)wh o found no obv ious r e l a ti onsh ip be tween v i rus con t en t andthe eff iciency of seed t ransm ission of PSbM V in di f ferent peacult ivars. A lso, Liga t & Randles (1993) observ ed th a t repea tedt ransfe rs t h rou gh seed i n t he cu l t i va r 'Dund a l e ' r e su l t ed i n ag radua l r educ t ion o f PSbMV accumula t ion i n vege t a t i ve t i s sueto a leve l where i t was n ot d e tec table by ELISA, whi le the seedt ransmiss ion f r equency exceeded 90 %.

The pe rcen t age ge rmina t ion o f seeds f rom in fec ted p l an t sranged f rom 88% (pP-1114) t o 98% (pP-4I I1 ) . Howeve r ,there was no apparent corre la t ion of seed viabi l i ty wi thsymptom seve r i t y , v i rus a ccumula t i on o r seed t r ansmiss ionfrequency.S e e d t r a n s m i s s i o n o f v P - 1 ( P - 4 5 ' U T R ) , v P - 1 ( P - 4P 1 p r o ) a n d v P - I ( P - 4 H C p r o N ) i n P. so t i vum ' V e d e t t e '

As descr ibed above , the region of i sola te P-4 f romnt 1-225 9, w hich covers the 5 ' -unt ran sla ted leader (UTR), theP1 p ro t ea se (P lp ro ) and t he N- t e rmina l two- th i rds o f t hehelper-co mp onen t proteas e (HCp ro ~) appeared to have a


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m a j o r e f f e c t o n s e e d t r a n s m i s s i o n . T h r e e r e c o m b i n a n t , f u l l -l e n g t h c l o n e s w e r e t h e r e f o r e c r e a t e d i n w h i c h t h e 5 ' U T R , t h eP l p r o a n d H C p r o N o f P - 1 w e r e e x c h a n g e d w i t h t h e c o r -r e s p o n d i n g r e g i o n o f P - 4 . S e e d t r a n s m i s s i o n o f t h e r e s u l t i n gc h i m e r i c v i r u s e s w a s a s s a y e d i n ' V e d e t t e ' , a n d t h e c o m b i n e dd a t a f r o m e x p e r i m e n t s f o u r a n d f i v e a r e s h o w n i n F ig . 3 ( b) .T h e s e e d t r an s m i s s io n f r e q u e n ci e s o f v P - I ( P - 4 5 ' U T R ) a n dv P - I ( P - 4 H C p r o N ) w e r e r e d u c e d t o 5 0 % a n d 2 0 % o f v P -1 ,r e s p e c t i v e l y , w h i l e v P - I ( P - 4 P l p r o ) w a s s e e d t r a n s m i t t e d a tt h e s a m e f r e q u e n c y a s v P - 1 .

T h e 5 ' U T R s o f P - 1 a n d P - 4 a r e 1 4 3 a n d 9 9 n t i n l e n g t h ,r e s p e c t i v e l y , a n d t h e r e a r e 3 2 a m i n o a c i d d i f f e r e n c e s i n t h eH C p r o N re g i o n b e t w e e n t h e t w o v i ru s es . T h e 5 ' U T R is l ik e lyt o a f f ec t b o t h t r a n s l a t i o n a n d r e p l i c a t io n o f t h e p o t y v i r u sg e n o m e ( R i e c h m a n n e t a l . , 1 9 92 ) . T h e r e g i o n o f H C p r oc o n t a i n e d i n H C p r o N a f fe c t s a p h i d t r a n s m i s s io n , r e p l i c a ti o n( A t r e y a e t a l . , 1 9 9 2 ) a n d lo n g - d i s t a n c e m o v e m e n t ( C r o n i n e t a l . ,1995) .

W a n g & M a u l e ( 1 9 9 4 ) d e m o n s t r a t e d t h a t t h e s u s p e n s o r c a na c t as a c h a n n el f o r t ra n s m i s si o n o f P S b M V t o t h e e m b r y op r o p e r . A p r e r e q u i s it e f o r e m b r y o i n f e c t io n i s, t h e r e f o r e , t h a tv i r u s re a c h e s t h e m i c r o p y l a r r e g i o n o f th e t e s t a b e f o r ed i s i n t e g r a t i o n o f t h e s u s p e n s o r . T h i s s u g g e s t s t h a t t h e H C p r o ~r e g i o n m a y a f fe c t r e p li c a ti o n a n d / o r l o n g - d i s ta n c e m o v e m e n ti n r e p r o d u c t i v e t i s s u e s a n d t h u s i n f l u e n c e t h e n u m b e r o fe m b r y o s w h i c h b e c o m e i n f e c t e d a n d h e n c e , t h e f i n a l s e e dt r a n s m i s s i o n f r e q u e n c y .

T h e o b s e r v a t i o n t h a t s e e d t r a n s m i s s i o n i s a f f e c t e d b ym u l t i p le d e t e r m i n a n t s i n t h e v ir a l g e n o m e a g r e e s w e l l w i t h t h er e su l ts o f W a n g & M a u l e ( 1 99 4 ) w h o d e m o n s t r a t e d t h a tr e s i s t a n c e t o s e e d t r a n s m i s s i o n i n P . s a t i v u m i n v o l v e s s e v e r a ln u c l e a r g e n e s . T h i s s u g g e s t s t h a t s e v e r a l v i r u s - h o s t f a c t o ri n t e r a c t io n s d e t e r m i n e t h e s p r e a d a n d a c c u m u l a t i o n o f P S b M Vi n t h e m a t e r n a l t e s t a t i s s u e . W h e r e a s P S b M V c a n e n t e r t h ee m b r y o a f t e r f e r t i l i z a t i o n ( W a n g & M a u l e , 1 9 9 2 ) , s e e dt r an s m i s s io n o f B S M V d e p e n d s m a i n l y o n t h e a b i li ty o f t h ev i r u s t o r e a c h t h e r e p r o d u c t i v e t i s s u e s p r i o r t o f e r t i l i z a t i o n(Edw ards , 1995) . D es p i t e t h i s d i f f e r ence , r ep l i ca t i on an dm o v e m e n t a l s o p l a y c e n t r a l r o l e s i n s e e d t r a n s m i s s i o n o fB S M V , t h e m a j o r d e te r m i n a n t s b e i n g t h e R N A ~ 5 ' U T R , a 3 6 9r e p e a t i n t h e 7 a g e n e , a n d t h e 7 b g e n e . T h e r e f o r e , s e e dt r a n sm i s s i o n o f b o t h P S b M V a n d B S M V a p p e a r s t o b ed e t e r m i n e d b y t h e a b i l i t y t o r e p l i c a t e a n d m o v e i n t h er e p r o d u c t i v e t i s s u e s a n d t o r e a c h t h e s u s p e n s o r o r m e g a -g a m e t o p h y t e , r e s p e c t i v e l y , b e f o r e a c e r t a i n c r i t i c a l p o i n t i nd e v e l o p m e n t .

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i n pea o f t he s eed t r ans mis s ion o f pea s eed- bor ne mos a ic v i rus . Journal o fGeneral Virology 7 3 , I 6 1 5 - I 6 2 0 .Wan g , D. & M aule , A . J. ( 1994 ) . A mod el f o r s eed tr ans mis s ion o f a p l an tv i r us : gene t i c and s t ruc tu r al ana lys is o f pea em br yo invas ion by pea s eed-bor ne mos a ic v i r us . Plant Cei l 6 , 777- 787 .Wang , D . & M aule , A . J . ( 19 95) . I nh ib i ti on o f hos t gen e expr es s ionassociated with plant virus repl icat ion. Science 2 6 7 , 2 2 9 - 2 3 1 .

W a n g , D . , W o o d s , R . D . , C o c k b a i n , A . J . , M a u l e , A . J . & B i d d l e , A . J.(1993). T he s us cep t ib i l i t y o f pea cu l t i var s to pea s eed- bor ne mos a icv i r us i n f ec t ion and s eed t r ans mis s ion in t he UK. Pl an t Pa t ho logy 42 ,4 2 - 4 7 .

R e c e i v e d 1 3 J u n e 1 9 9 6 ; A c c e p t e d 2 2 A u g u s t 1 9 9 6

multiple viral determinants affect seed transmission of pea seed borne mosaic virus in pisum sativum

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