Genetic variability of potato spindle tuber viroid RNA replicon

The ge netic con ti nu ity of the po tato spin dle tu ber viroid (PSTVd) ge nome was ana - lysed af ter in fec tion of to mato plants with cloned cDNAs of pa ren tal strains. Dur ing the six weeks of the ex per i ment, sev eral new se quence vari ants ap peared. The se - quence vari ants de tected in the prog eny pop u la tion in duced se quence-specific dis ease symp toms. The PSTVd ge nome there fore fol lows the pat tern ex pected for typ i cal pseudo-strains prop a gat ing in plants as a pop u la tion of sim i lar se quences. As sessing fur ther the replicon con ti nu ity, a PSTVd cDNA mu tant with a de le tion in the cen tral con served re gion was con structed and proven to be non-infectious. Sur pris ingly, in a sub-population of po tato transformants ex press ing the same de leted PSTVd RNA an in fec tious viroid was de tected. This sug gests spe cific tran script con ver sion fol lowed

The po tato spin dle tu ber dis ease was the first of all viroid-induced dis eases to be re cognised and stud ied by plant pa thol o gists.During the ef forts to pu rify a pu ta tive in fec tious agent from po tato spin dle tu ber-affected plants the un usual prop er ties of the patho gen were re cog nised, the first viroid iso lated, and the viroid con cept de vel oped.The term "viroid" was pro posed (Diener, 1971) in or der to dif fer en ti ate these small, pro tein-free in fectious RNAs from con ven tional vi ruses with an encapsidated ge nome.To date, over 20 dif fer -ent vir oids have been de tected, the ma jor ity caus ing dis eases of eco nom i cally im por tant crop plants.
The symp toms of po tato spin dle tu ber viroid dis ease may vary con sid er ably de pend ing on the PSTVd strain, po tato cultivar and en vi ronmen tal con di tions.Typ i cal fo liar symp toms in clude stunt ing, up right ness, and small leaflets.Tu bers are smaller, fewer in num ber, elon gated, with nu mer ous shal low eyes, and have ab nor mal skin col our and tex ture (Pfannenstiel & Slack, 1980 As a stan dard, Rutgers to mato plants are used as test plants to prop a gate PSTVd and de ter mine the symp tom se ver ity.Ac cord ing to the symp toms in duced, the PSTVd strains are clas si fied as mild, in ter me di ate, se vere, and le thal. The first viroid se quence to be de ter mined was that of the PSTVd in ter me di ate strain (PSTVd-DI) (Gross et al., 1978).This single-stranded RNA con sists of 359 nu cleo tides, and is cir cu lar.A unique rod-like struc ture, with a se rial ar range ment of dou ble-helical sec tions and small in ter nal loops was proposed.Taking into ac count all re ported data, it was con cluded that viroid RNAs are not trans lated into viroid-specific polypeptides (Zaitlin & Hariharasubramanian, 1972;Conejero & Semancik, 1977;Gross et al., 1978;Conejero et al., 1979;Camacho Henri quez & Sänger, 1982a;1982b).Com par a tive se quence anal y sis of dif fer ent PSTVd fam ily mem bers has in di cated the pres ence of five struc tural do mains, des ignated: TL, P, C, V and TR (TL -left ter mi nal, P -patho ge nic ity, C -cen tral, V -vari able, TR -right ter mi nal), each re spon si ble for differ ent func tions (Keese & Symons, 1985;1987).The cen tral con served re gion (CCR) of the C do main may rep re sent an im por tant con trol re gion in viroid rep li ca tion, po tentially as sum ing al ter na tive sec ond ary structures in dif fer ent rep li ca tion steps (Keese & Symons, 1985;Diener, 1986;Sänger, 1987, Steger et al., 1992 al., 1994; 1997).Se quence het er o ge ne ity has been ob served in nat u ral viroid iso lates.Such ob ser va tions in di cate that PSTVd, like many other RNA patho gens, prop a gates in the host as a pop u la tion of sim i lar but non-identical sequences com pris ing quasi-species.The quasispe cies con cept de vel oped by Eigen and co-wor kers de scribes the com plex be hav iour of such pop u la tions act ing as a whole (Eigen & Winkler-Oswatitsch, 1990;Eigen, 1993).To un der stand better the be hav iour of quasi-species, as well as to de scribe it on math e mat i cal foun da tions, the se quence space con cept was in tro duced.In the space of given se quences, each pos si ble se quence vari ant is rep re sented as a sin gle point.The points are or dered to form dis crete, cel lu lar and multi-dimensional space vol umes re flect ing the in for ma tional rela tion ships be tween the se quences they rep resent.To each point in the se quence space a sequence fit ness value can be as signed, re sulting in a fit ness land scape con tain ing peaks and ridges of high fit ness sep a rated by saddles and val leys of lower fit ness.Due to nat ural se lec tion, higher po si tions of the fit ness land scape be come more pop u lated than others.New mu tants re sult ing from rep li ca tion of se quences with a high fit ness ap pear close to their pa ren tal se quences, thus also in the high fit ness re gions.This means that the cloud of points rep re sent ing the quasi-species as a whole is con densed by nat u ral se lec tion on the high fit ness peaks and ridges.Thus, rep li ca tion er ror rate and the shape of the fitness land scape are the two fac tors that fi nally de ter mine the be hav iour of the quasi-species in the se quence space.Such un der stand ing of the re la tion ships be tween in di vid ual mu tants leads to the con clu sion that the RNA ge nome may os cil late be tween sev eral vi a ble se quence ver sions of the replicon.If this is true, the specific PSTVd se quences de tected while sequenc ing a pop u la tion rep re sent in fact tran sitory en ti ties be ing mo men tarily pre ferred by con di tions of the ex per i ment, host phys i ol ogy, or se lec tive pres sures cre ated by the ob server seek ing spe cific phe no types.To prove this hypoth e sis mas sive clon ing and se quenc ing of the prog eny of de fined pa ren tal PSTVd strains were per formed.Test plants were infected with prep a ra tions of cDNAs car ry ing the ap pro pri ate PSTVd se quences.Ho mo gene ity of the pa ren tal cDNAs was con firmed by di rect DNA se quenc ing.The vari a tions observed in prog eny se quences could point to the quasi-species na ture of the PSTVd replicon.

Viroid iso lates and se quence vari ants nomen cla ture.
The PSTVd iso lates and sequence vari ants have been re ported pre viously (Góra et al., 1994;1997).Three iso lates of PSTVd: mild, in ter me di ate and se vere had been kept in the col lec tion by con sec u tive passages in Rutgers to mato plants grown in green houses.cDNA syn the sis and clon ing.Pu rified viroid RNAs ex tracted from PSTVd in fected to mato leaves were re verse tran scribed and the re sult ing cDNAs PCR am pli fied.Two specific prim ers cor re spond ing to the CCR were used (Góra et al., 1994).To gen er ate fulllength monomeric in fec tious clones the cDNAs were li gated into the SmaI site of the pUC9 vec tor.The cloned PSTVd cDNAs were se quenced us ing a flu o res cent primer sequenc ing kit and an au to mated A.L.F.sequencer (Pharmacia).
Infectivity as says.To mato seed lings (cv.Rutgers) were in oc u lated with plasmids (2 mg/plant) con tain ing a monomeric fulllength cDNA of the ap pro pri ate se quence variant ac cord ing to Cress et al. (1983) and Candresse et al. (1990).
Clon ing of PSTVd cDNA into a bi nary vec tor.The SmaI-BamHI re stric tion fragment of the S23 PSTVd cDNA was li gated into HincII cleaved pUC1813 vec tor (Kay & Mc -Pherson, 1987).This step was per formed to add sym met ric HindIII sites on both sides of the cDNA.The re sult ing HindIII frag ment was re-cloned into the HindIII site of the binary pKYLX71-35S 2 vec tor (Maiti et al., 1993) un der the con trol of the cau li flower mo saic virus (CaMV) 35S pro moter.The plant ex pression cas sette of the pKYLX71-35S 2 car ries an en hanced 35S pro moter of CaMV and the termi na tion sig nal of the small sub unit of the pea ribulose bisphosphate carboxylase gene (3¢ rbcS).The re sult ing con structs with PSTVd cDNA (356 bp) in (+) and (-) ori en ta tion were used to trans form Agrobacterium tumefaciens LBA 4404.Po tato trans for ma tion.Trans for ma tion of Solanum tuberosum (cv.Irga) was car ried out ac cord ing to the method of Mar tini et al. (1993) with some mod i fi ca tions (Góra-Sochacka et al., 2000).

Se quence het er o ge ne ity in phenotypically es tab lished PSTVd iso lates
PSTVd field strains iso lated af ter sev eral pas sages in test plants are gen er ally as sumed by phytopathologists to be pure strains.However, un der rig or ous anal y sis they may show signs of he red i tary in sta bil ity.In oc u la tion with PSTVd RNAs iso lated from a de fined pa -ren tal plant may re sult, in prog eny plants, in a col lec tion of phe no types scat tered around the typ i cal one.In stan dard phytopathological exper i ments on type vari ant prop a ga tion, such vari abil ity of phe no types is gen er ally overlooked, only sub-populations of plants with desired phe no types be ing se lected for fur ther patho gen prop a ga tion.This in duces a bias in the eval u a tion of the ge netic sta bil ity and sequence ho mo ge ne ity of PSTVd vari ants.It should be stressed that vir oids iso lated from field plants of ten rep re sent rather het er o geneous pop u la tions.The in ten si fi ca tion of symp toms dur ing con sec u tive pas sages, the scat ter ing of phe no types in in fected pop u lations and the het er o ge ne ity of nat u ral iso lates ques tion the stan dard view on ge netic con tinu ity/sta bil ity of a de fined PSTVd strain.In fact, the quasi-species con cept by it self asks for a re-analysis of the ge netic con ti nu ity of PSTVd strains.Ac cord ing to the sim plest hypoth e sis (Fig. 1), high ge netic sta bil ity of the PSTVd replicon would re sult in the ho mo gene ity of prog eny pop u la tions of PSTVd variants.As suming a low ge netic sta bil ity re sulting from a high er ror rate of RNA rep li ca tion, the prog eny RNA pop u la tions should be het ero ge neous, with a sig nif i cant por tion of sequence vari ants dis tant from the pa ren tal one.This could even lead to al tered phe notypes in the prog eny pop u la tion (Fig. 1).
To test the ge netic sta bil ity of PSTVd we took ad van tage of re ports show ing infectivity of cloned viroid cDNA (Candresse et al., 1990;Cress et al., 1983;Owens et al., 1986).The start ing RNA prep a ra tions were de fined by phytopathologists as type iso lates in duc ing either se vere, in ter me di ate or mild dis ease symp toms.Such prep a ra tions (Ta ble 1, left col umn) are used as stan dard PSTVd iso lates in bi o log i cal tests for viroid de tec tion (Diener, 1987).Pu rified PSTVd RNA from these isolates was re verse tran scribed and the re sulting cDNAs were en zy mat i cally am pli fied.The full-length PSTVd cDNAs ob tained were cloned in the pUC9 vec tor.In this man ner, a num ber of se quen tially ho mo ge neous, in fectious PSTVd cDNA clones were ob tained.These cDNA clones were se quenced and assayed for patho ge nic ity.The re sults are shown in Ta ble 1.The se vere and in ter me diate iso lates were a mix ture con tain ing a few se quence vari ants.In the mild iso late only one type of se quence was de tected.The patho genic ity of each in di vid ual se quence vari ant was as sayed.As shown in Fig. 2 and Ta ble 1 the detected se quence vari ants in duced dif fer ent dis ease symp toms -from mild to se vere.Surpris ingly, di ver gence in symp tom se ver ity was ob served even among the se quence variants pres ent in the same RNA iso late de fined by phytopathologists as the stan dard strain (Ta ble 1).

He red i tary phe no type fluc tu a tion in PSTVd
Cloned PSTVd se quence vari ants as a rule in duce well-defined dis ease symp toms in the ma jor ity of pri mary in fected plants.How ever, the pri mary in fec tion with PSTVd vari ant S27 was fol lowed by ev i dent dis ease phe no type insta bil ity.Only two of the ten in oc u lated plants de vel oped the ex pected se vere symp toms, whereas eight were symptomless.Looking fur ther into phe no type he red ity, the prog eny pop u la tion of S27 was iso lated from symptomless hosts and in oc u lated to a next se ries of plants.In fected plants were al lowed to grow for 6 weeks and the pro ce dure of progeny iso la tion, clon ing, se quenc ing and in fection of the next gen er a tion of plants was repeated 5 times.The prog eny PSTVd pop u lations were ana lysed at the se quence level af ter the first and the sixth plant pas sage.Taking ad van tage of the cDNA clon ing pro ce dure, which makes it pos si ble to ob tain in fec tious clones, a spe cific phe no type was as signed to each se quence vari ant de tected.The sequences of S27 prog eny genomes were aligned.Fig ure 3 shows the graph of the sequence vari ants or dered in such a way that the se quences, which are neigh bours in the graph, dif fer by a sin gle point mu ta tion (in dicated to the right in the graph).Rep re sen tatives of this se ries show sur pris ingly dis parate phe no types.It seems that the pa ren tal vari ant S27 (in duc ing se vere symp toms) can be eas ily con verted by a point mu ta tion to the S27-I-8 vari ant, which in duces mild symptoms.S27-I-8 it self can be con verted to a severe mu tant (S27-VI-106) by an other ad ditional point mu ta tion.Next, this last vari ant can be fur ther point mu tated to yield an other mild vari ant (S27-VI-19).Such rather un usual Vol. 48 Ge netic vari abil ity of PSTVd 471 Ta ble 1. Se quence vari ants de tected in PSTVd iso lates.
RNA phytopathological stan dards were ob tained from PSTVd strain col lec tion.RNAs rep re sent ing se vere, in ter medi ate and mild iso lates were re verse tran scribed and PCR-amplified (see Ma te rials and Methods).The re sult ing PSTVd cDNAs were then cloned in the pUC9 vec tor.A col lec tion of cDNA clones de rived from each stan dard iso late was se quenced.Each mo lec u lar vari ant was sep a rately tested for infectivity and phytopathological phe no type (see Ma te rial and Methods).PSTVd-I2 is iden ti cal to the pre vi ously de scribed PSTVd-DI (Gross et al., 1978).Fre quency of a given se quence vari ant is ex pressed as the num ber of cDNA clones with the de tected se quence per num ber of sequenced clones.
fluc tu a tions could be re spon si ble for the dispa rate dis ease phe no types fre quently observed in pop u la tions of the pri mary in fected host.

Re cov ery of in fec tive viroid mol e cules from a trun cated PSTVd tran script
The prog eny of dif fer ent PSTVd vari ants included a sub set of vi a ble genomes car ry ing short de le tions.The vi a bil ity of spe cific deletants was also re ported in the lit er a ture (Wassenegger et al., 1994).In such cases, dele tions pres ent in the pa ren tal ge no type were also de tected in the prog eny genomes.It there fore ap pears that short de le tions could be con sid ered as well-conserved in PSTVd.Expect ing con ser va tion of de le tions, we de cided to con struct a non-infective PSTVd deletant trun cated in the cen tral con served re gion be -lieved to be cru cial for PSTVd rep li ca tion.The cDNA of the S23 PSTVd se quence vari ant with a two-nucleotide de le tion (C 93 C 94 ) in the cen tral con served re gion was cloned into the bi nary pKYLX71-35S2 vec tor un der con trol of the CaMV 35S pro moter.Con structs with the cDNA in serted in (+) and (-) ori en ta tions were ob tained and used in Agrobacterium-medi ated trans for ma tion of Solanum tuberosum (cv.Irga) leaf discs.This was ex pected to result in transgene-driven ex pres sion of the trun cated, non-replicating and there fore non-infectious PSTVd ge nome.Such a truncated, non-viable PSTVd mol e cule could be of in ter est for stud ies on plant re sis tance to viroids.A to tal of 113 trans gen ic lines were re gen erated and grown in vi tro.PCR anal y sis of the plant DNA con firmed the pres ence of the intro duced con structs.Fifty se lected trans gen ic plants were tested to eval u ate their de gree of re sis tance to PSTVd in fec tion.Pre lim i nary re sults in di cate that none of them was substan tially re sis tant to PSTVd.How ever, bi olog i cal as says led to an un ex pected ob ser vation.A few trans gen ic plants car ry ing the PSTVd cDNA con struct in the (+) ori en ta tion (with ex pected (+) RNA ex pres sion) showed dis tinc tive mor pho log i cal changes -growth stunt ing and leaf mal for ma tion -sim i lar to the dis ease symp toms caused by PSTVd.No such symp toms were ob served in trans gen ic plants car ry ing the PSTVd cDNA in the (-) ori en ta tion.Se quence anal y sis re vealed that these transformants ac cu mu lated in fec tious full-length viroid mol e cules.

DIS CUS SION
Ab in itio prog eny anal y sis with the cloned pa ren tal stan dards led to rather un ex pected ob ser va tions on PSTVd vari ant phe no type hered ity.The in fec tive cDNA clones rep re sent by def i ni tion pop u la tions of iden ti cal sequence.Ho mo ge ne ity of the cDNA clones was ver i fied by di rect cDNA se quenc ing.Ac cord - ing to the stan dard un der stand ing (Fig. 1A) in fec tion with such clones should lead to proge nies iden ti cal in se quence and func tion to the pa ren tal one.This is ev i dently not the case as il lus trated with the ex per i ments done with the S27 se quence.Af ter a sin gle plant pas sage, the PSTVd prog eny was al ready het er o geneous, with the het er o ge ne ity in creas ing with each con sec u tive plant pas sage.There fore, the hy poth e sis pre sented in Fig. 1B was confirmed.As the clon ing pro ce dure makes it possi ble to ob tain in fec tious PSTVd cDNA clones, the dis ease phe no type in duced by each sequence vari ant could be tested.This al lowed us to pin point among the prog eny se quences those which dif fer only by a sin gle point mu tation or de le tion; sur pris ingly these mu tants some times dif fered heavily in dis ease phe notype (Fig. 3).This means that start ing from the mas ter S27 se quence, the phe no type fluctu ates in con cert with point mu ta tions, the first mu ta tion con vert ing the phe no type to mild, the sec ond re sult ing in a se vere phe notype, the third re-inducing mild symp toms.The mu ta tions in ques tion are lo cated in the PSTVd P do main and for mally can be compared to intragenic phe no type suppressors.One could ex pect at ten u a tion fol lowed by mild dis ease phe no type to be ben e fi cial to pathogen prop a ga tion.In evo lu tion ary time scales, the mild ver sion should be pre ferred over the se vere one, del e te ri ous to the host.In fact, the mild se quence can re-create the se vere sequence and os cil la tion of the phe no type (mild « se vere) is ob served in the S27 fam ily.With ap pro pri ate res er va tions, it seems that the dis ease phe no type in duced is of im por tance to plant grow ers while prob a bly be ing neu tral to the patho gen.Taking into ac count that mild in fec tion in a 6-week-old to mato plant gives around 6 ´ 10 13 PSTVd mol e cules, and assum ing that the se vere dis ease di min ishes the plant weight three fold, the se vere ge nome still rep li cates in num bers as sur ing its suc cess ful prop a ga tion.It seems that the mutational rate in PSTVd 'prob ing' the se quence space is high enough to cre ate, even dur ing the time of the ex per i ment, evo lu tion ary fluc tu a tions between par a sit ism and com men sal isms.Therefore, se lec tive pres sure to wards the elim i nation of the se vere phe no type is pe ri od i cally alle vi ated by the ap pear ance of PSTVd ver sions prop a gat ing with out dam age or with re duced dam age to the host.In gen eral, such a "fluc tuat ing" phe no type could be con ceived as a new mech a nism of chronic dis ease.In deed it is a warn ing to phytopathologists.The mild in fection -un de tect able in the field -can lead to lo cal se vere dis ease foci ap pear ing with out con tact with se verely dis eased plants dur ing the growth sea son.This also means that in fection with the mild vari ant can not be con sidered a bar rier for dis ease spread, since the mild vari ant by it self could be the source of severe mu tants.Af ter iden ti fi ca tion of the ef fects of base substi tu tions, we fo cused our at ten tion on the genetic con ti nu ity of spe cific de le tions in the PSTVd replicon.In prog eny pop u la tion of paren tal ge no types, a few one-and two-nu cle otide de le tions in the P or the TL do main were de tected (data not shown).Such de le tions were fol lowed by strong re duc tion in infectivity.As ex pected, de le tions were not detected in the re gion be lieved to be cru cial for viroid rep li ca tion.In deed, the re com bi nant plasmid car ry ing the PSTVd cDNA with a two base de le tion in the CCR (see Methods) was not in fec tious (re sults in prep a ra tion).This strongly sug gests that the ef fect of de le tions in this re gion would be le thal.There fore, we as sume that ex pres sion of a transgene car rying this de le tion in trans gen ic plants would lead to the ap pear ance of a trun cated PSTVd tran script un able to prop a gate in the host.How ever, con trary to ex pec ta tions, in a sub-population of trans gen ic plants in fec tious full-length PSTVd mol e cules were ob served.Di rect se quenc ing of transgene con firmed the pres ence of the in tro duced de le tion at the transgene level (to be pub lished).At the present stage we as sume that mas sive PSTVd-like tran script pro duc tion in the trans gen ic plants may lead to oc ca sional tran scrip tion of full-length PSTVd mol e cules due to misin corporations by RNA poly mer ase.Even if such events are rare, they would be de tected in the sys tem we used, due to the en su ing am pli fi cation of any PSTVd mu tant tran script hav ing re cov ered the ca pac ity to rep li cate.One can spec u late that in for ma tion lost at the DNA level un der rather spe cific con di tions could thus be re trieved at the RNA level.

Fig ure 1 .
Fig ure 1. Hy po thet i cal re sults of test plant in fec tion with ho mo ge neous PSTVd strain.D: sche ma tises a mu ta tion.

Figure 2 .
Figure 2. Symp toms in duced by in di vid ual PSTVd se quence vari ants in Rutgers to mato plants one month af ter in oc u la tion.

Fig ure 3 .
Fig ure3.Pro posed "fam ily tree" of mu ta tions in the S27 PSTVd fam ily.S27 -pa ren tal se quence (de tected in the se vere PSTVd iso late, see Ta ble 1); S27-I-8 -se quence vari ant detected among prog eny of S27 pa ren tal se quence af ter the first plant pas sage; S27-VI-106 and S27-VI-19 -sequence vari ants de tected af ter the sixth plant pas sage.All nu cle o tide changes re fer to dif fer ences with re spect to the S27 se quence.
; Baumstark & Riesner, 1997).Since the de ter mi na tion in 1978 of the first com plete nu cle o tide se quence of the PSTVd in ter me di ate strain (PSTVd-DI), the se quence of about forty dif fer ent PSTVd se quence variants has been de ter mined.Se quence anal y ses re vealed that they dif fer from PSTVd-DI by only a few nu cle o tide changes such as sub stitu tions, in ser tions, and de le tions.The RNA chain length var ies from 356 to 360 nu cleotides.The mu ta tions are mostly lo cated in the P and V do mains (Kuo, 1979; Gross et al.