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Spontaneous infection of Chelidonium majus and Ligustrum sp. with cucumber mosaic Cucumovirus (CMV) encapsidating satellite RNA

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Spontaneous infection of Chelidonium majus and Ligustrum sp. with cucumber mosaic Cucumovirus (CMV) encapsidating satellite RNA

Abstract

Background and purpose: Cucumber mosaic Cucumovirus (CMV) infects many plant species. Nuturally occurring CMV is occasionally associated with a satellite RNA (satRNA) which is capable of modulating CMV-induced symptoms, making them more destructive to host plants. A lot of research has been done lately to investigate which plant species can be natural hosts or reservoirs for CMV-satRNA and if, how and when can those pathogens spread from their natural hosts to agronomically important species. Material and methods: CMV encapsidating satRNA was isolated from Chelidonium majus and Ligustrum sp. and studied serologically, by means of double-stranded RNA (dsRNA) analysis and regarding reaction of mechanically inoculated test plants including agronomically important species. Results: The type of symptoms on mechanically inoculated experimental hosts implied. CMV infection, which was confirmed by serological and dsRNA analysis. Agronomically important species like pepper, Swiss chard and spinach expressed severe mosaic after inoculation. Cucumber expressed systemic ring-shaped chlorosis. SatRNA was consistently present in dsRNA isolates from natural and experimental hosts except in Chenopodium quinoa in which satRNA replication was suppressed. Conclusions: Our results present the first report on Chelidonium majus and Ligustrum sp. as natural hosts of CMV encapsidating satRNA. Even symptomless Chelidonium plants were found to be natural hosts of those pathogens. Since virus with the associated satRNA caused significant symptoms on some vegetable species, potential spreading of those pathogens from their natural, widely distributed hosts could cause serious epidemics.
PERIODICI JM I]IOLOGORUM
VOL. l0l No3,229 232,1999 "?#Rrffirffi El
Onqinal scientific papel
Spontcrneous infection of Cxtelidonium morius qnd Ligustrumsp. with
cucumbr moscric Cucumovirus (CIW/) encqpsidcrting scrtellite nNA
ANÐELKo HnZnru¡Rx',
MrRN¡, óunrovró PERIC.# *,
MI,.A,DEN IaùUAÖIó'z,
ÐORÐE MAMUL"#
I Karl-Iranzens Urriversity,
lrslitrrtc oI Mcdic¡tl Biochetnislry,
Harractrgasse 2lllll,
B0l0 Graz,
Auslria
2 University of Zagreb,
I'hculty of Science
Dcpt. of llioÌoc,y,
Marulióev lrçy 20/ll
10000 Zagrcb,
Croalia
Correspondence:
Mirna Óurkovió Pcnca
University of Zagrcb,
Facrrlty ol Scicrrcc
Dept. of tliology,
MarLrlióev Irq20lll
Ì0000 Zacrrcb,
Croatia
Kev words: Cucutnber mosatc
CtrcLtmovirus (CM\0, satellite RNA,
Cltelidonium majus, Liguslrum sP.
.Abstract
Background and purpose: Cucumber mosaic Cucumovirus (CMl/) in
fects many plattl species. Naturally occurring CMV is occasionaily associ-
ated with a satellite RNA (satRNA) wltich is capable modulating CMV
induced synptoms, maktng them more destructive to host plants. A lot
research åas /¡ee¡l done lately to investigate which plant spectes can be
naturaÌ iosls or reservoirs for CMVsatRNA and i[, ]tow and when can
those pathogens spread t'rom their natural¡losts to agrononically impor-
fanl specres.
Material and methods: CMV encapsidaüng salÄlt/,4 was isolated lrom
Chelìdonium majus and Ligustrum sp, andsl¿¡diedserologically by means
of doub]e-strancted RNA (dsÃ/V,A.) analysis and regarding reaction of me-
chanically inoculated lesl planls ittcluding agronomically important spe-
cles.
.Resu,lts; The type of sytnptoms on nechanically irtoculated experimental
hosts impliect CMV nfectiott, whiclt was cont'irmed by serological and
dsRN,{ analysrs. Agronornically important species }ike pepper, Swiss chard
and spinach expressed severe nosaic after inoculation. Cucumber ex'
pressedsysl enic ring-shaped chlorosis, Sal.R.Ay',4 r,rzas consistently present
tn c/sRl/Ärso1a tes from natural and experimental hosts exceptin Chenopo-
clium quinoa rl whiclt satRNA replication was suppressed.
Conclusions: Our results present the first report on Chelidonium majus and
Ligustrum sp. as natural hosts of CMV encapsidating satRNA' Even
symptonless Clielidoniumpla nls were fouttd to be natural hosts tlnse patho-
gers. Sritce w nt s wi th lhe assocla led sa lRN.{ ca use d s i g ni fi c a nt sym pton s o n
sone vegetab.le specleg potential spreading of those pathogens from their
natural, wdely distt'ibuledåosfs co¿¡/d cause serìous epidemÌcs'
INTRODUCTION
ucumber mosaic Cucttmovints infects many agronomically importanl
plant species throughout the lemperate regions of the worìd (B). fu all
other nlembers of the genus Cucumovirus, CMV has triparlite genome of
positive stngle stranclecì RN,As, designated as RNÄ I, 2 and 3. Viral particles
also encapsiclate IìNÄ.4, which rs a subgenomic species encoding the coat
proterrr (l l, 12) Naturally occurring CMV is occasionally assoclated with a
satellite RNA (satRNA), SatRN,A. is a linear smaÌl molecule, 330-405 nucle-
otides long. It rs completely dependent on the helper vrrus (CMV) for its
replicatron, encapsidation and transmission and is therefore regarded as a
molecular parasite (6). One of the most interesting CMV satRNÄ. properties
is the capability of modulating CMV-induced symptoms. Some of the most
clestructive CMV epidemics reported lately in tomato crops were associat-
ecl with the presence of CMV satRNA wilh necrogenic phenotype (5, 9, I3),
It was aìso reportecl that many weed species can serve as natltral reservoirs
llcccuccl Sol)lorrìlx)r 2, I993.
229
A. llrärrlak t'l irì
of CMV and the assoctatecl satRNA, fron which e¡ticlem-
ic can spread agatn to agronomicaìly itrportant crops
(2, 5). Chelidoniunt najus was already founcl to be a
nattrral host of CMV l/), l.¡ut Ligttslt-unt sp. was llot pre'
vrously reportecl as a CMV host. Therefore thts paper
presents the first relrort on Ligttsûzrrl sp. as lratural host
of CMV ancl the first cletection of the vlrus-associatecì
satRNA in bolìr spectes, as well as the experimental re-
sults consrdenng urfluence of the nrus etrcapsidating
satRN,A on some vegetable species,
MATERIÄIS ^ãND METHODS
Moteriols
Symptornless ancì sym¡ttorn ltearing, wild Cl¡ellclo-
nittnt najus and cullrvatecl Ligttstrun sp. plants were
collected in Botanical qarden of the Faculty of Science,
Unrversity of Zagreb.
CMV containing satRNA was tnechanically transmit.
ted from Chelidoniutn rlajus to healthy experimental
lrost plants: Beta vulgaris, Capsicun annuum, Cttcutttis
salir,'¿¡s, Cheno¡toditttn quinoa, Gonphrena globosa,
Nicotiana megalosi¡tlton, N. glttttnosa, N. tabacun, Pe-
tunìa lrybricla. Spr)lacla oleracea, Zirtnia elegans. AII
tliose plants were ¡tlautecl in sterile soil ancl kept at 20"C
in greenhouse conditions. TV'renty plants of each men-
tioned host specres were used for the t¡ansmission of
the vlrr.rs with satRNr\.
Methods
Portiol purificotion of CMV ond serology
Method introduced by Habili and Flancki (3) was
used for partial purification of CMV Double- and single
radial irnmunodiffirsion tests were performed Llsing a
polyclonal antiserum to CMV a caruation isolate kindly
sr-rpplied by Dr E. Luisoni (lstituto di Fitovrrologia Ap-
plicata del C.N.R Thrin, ItalY),
Nucleic qcid extroction ond double-strc¡nded
RNA onalysis
T\ivo grams of fresli or frozelt leaf tissue were ltomoç.¡'
enizccl irr STE-exl¡action ltr,tffer (0. I M NaCl 50 rnM'llts,
I nlM ED'll\, pl{ 6 B) ancl centrifuged at40009. Total utt-
cleic acicls were phenoVcliloroforu exlracted and appìied
to CF-ll Sllhatntan) cellulose column pre'treated u'rth
STE buffer containing l6% ethanol. Irurificatiotl nrethocl
introduced ÌryVa-lverde et al. (14) and modifieclby Kajaöió
and l,orkovió (10) was usecì for purifyng dslìNÄ.. Electro-
1:lioretic analysis of dslìNA fron all testcd platrt hosts was
pcrformed tn 1.2% açJarose gels ancl 'l'llE Ìrtrffer (90 tnM
'IYrs, 90 nrM boric acicl, I niM lll)TA, pH I 3) for I h at l0
V/crn. DslìNÄ. of CMV assocrated with satlìNÄ, iclenti-
frecl prevrously by ucans of ntolecul¿rr liybnclìzations (13),
served as an electrophoretic stanclarcl. Nucleic acid
l:ands were vlsualized alter ethidtum-bromide starning
(50 nclrrl) on UV transillurrinator (300 ntn).
230
Clrcliclot¡iu¡t¡ rlaTus atttl Liguslrutn sp, its ttittttral hosts of CMV wilh satllNÄ
RESULTS
Symptomotology ond serology
CMVwas rsolated from symptom bearing (express-
ing light systemic chlorosis and leaf deformation) and
symptom-free plants of Cå. majtts, T\.n¡o out of l0 appar-
ently healthy Chelidoniun plants tested in our experi'
ments were infected with CMV encapsidatinq satRNÄ'
in its particles. Ligustrun specimens containing CMV
together with its satRNÄ expressed ring-shaped chlo-
rotic symptoms.
Serologicaì tests in which a polyclonal anliserum to
CMV was usecì, ¡troved wthout any doubt that those
Chelidoniunt ancl Lþttslrtrm plants were infected wtth
CMV (data not shown).
Tìre infection was readily transmilted to the range of
host plants, which expressed symptoms characteristic
of CMV (Table l) Vtrus transmission from symptom
bearing and synptomless Cllelldo¡ium plants had ex-
actly the sarne effect on host ¡rlant symptomatology,
DsRNA
DsRNÄs obtained in ottr experiments were identifiecl
by compartng tìre patterns to those of CMV-CrFG ancl
The associated CrFG-satRN,4 (/ 3) that were used as elec-
trophoretic standa¡ds. In adclition to 4 viral dsRNÄ
bands, the fifth sateìlite-like band was consistently
present rn tsolates from naturaìly infected Ch, majus
pìants with and without symptoms and Lìgusttum sp.
plants with symptoms. Mechanically inoculated hosts
G. globosa, N. ntegalosiphon, N. glututosa, N. tabacun,
P hybrida, Z. elegans,ancl agronomically important spe-
cies C annuLun, B. wlgaris and S. o/eracea also re-
vealed the satne electrophoretic pattern (Figure l).
SatRNÄ band, as all other CMV-dsRNA bands, was re-
vealed already after the first tnoculation of host plants.
TABLE I
Synptonts infection witlt CMV encapsrdaÛhg salRNA
on different host plants.
I Iost species 'l!pc of infection Synrptorns
Cherto¡>odnun r¡inoa
Pclunia hybrkla
Corn¡threna gld;osa
necrottc lcsrons
nccrolic lesions
necrolic lcsions rvilh ¡ccl
rìng borclers
vcinclearing anrl lnosaic
syslcnlc lcaf mottìing ancl
(listorti(nr
loaf üìottling ancl cìistortion
l nosaìr;
s;r:vcrc Illosaic
sevcrc mosaic
severe tnosaic
rinq.shapecì r:lrlorosis
toiracco lNrr.'olrhrn litbaant) systclnic
N. nrcgalosphon
N. glulntosa
Znuna ck:g,tns
Srvrss clrrr¡ri (llal.t vttl¡,uis) syslcrrl(:
s¡linlrl t (S¡rn,rr l'r ol r: t. t, :c:t) syslot nic
o¡oltnìror (Cucutt¡is s,l/ivus) syslcrnic
local
local
syslenrìr;
systct rìt(;
systetrtir;
Period biol, Vol 101, No 3, 7999
A. HrZenjak et al,
The exception among tested hosts was C¡. gumoa as
the species which did not support the satellite RNA rep-
lication. Electrophoretic pattern of dsRN^A efracted from
Ch. quinoa revealed onÌy 4 bands, 3 of lhem represent-
ing replicative forms of genomicviral RN,As correspond-
ing to MWs 1.0, 0.9 and 0.7x106 and fourth representing
subgenomic RN.A, with MW 0.3 x106. The fifth band of
satRNA corresponding to MW 0, lxl 00 was not revealed
(Figure 2)..Although satRNA replication was suppressed
in Ch. quìnoa, satellite survived at least lB days in that
plant tissue. This was confirmed by the experrment in
which M glutinosa and N. megalosiphon were mechan-
icaìly inoculated with sap from leaves of the quoted Ci.
quinoa pìants. Symptoms that appeared on those ly'ico-
lr'ana plants were the same as those mentioned before
('lhble i) and dsRNA analysis revealed again an inten-
sive satRNA band. The attempt to use Ch. quinoa for
biologicaì separation of the virus from its satRN.A, was
unsuccessful, because leaves of Ch. quinoa that exhib-
lted local lesions died lB days after inoculation with CMV
and at that point satRNÄ was still present in the plant
tissue, Biological assay and dsRNA analysis showed that
seedlings raised from 40 seeds of infected Cåelidonium
plants were vrrus-free.
RNAI -+
RNAz ->
RNA3 +
RNA4
soIRNA-l
f lGUlìE | . EleclropÌtorettc analysis, ott L20/o aEI¿?t ose
gel, of CMV (l, 2, 3, 4) ancl satellite (saf) c1sllN,4s rso-
lated [rotn: Iìeta vulgJaris (b) ancl Nicottana gltttinosa
(c) which were inoculated wttlt sap preparecl frotn
Cheliclonirrm majus. C MV C t' F G' t C
r FG - s a t R NA c out -
plex serued as a positive contt'ol (a).
Period biol, Vol 101, No 3, 7999
Chelidoniu¡n rnaTus and Liguslnnt sp. as natural hosts of CMV with satRNA
DISCUSSION
Until now Chelidonium maTus and Ligustrum sp.
\¡Íere not listed among the species already reported to
maintain CMV-satRNA, (2, 5). CNN has not even been
previously recorded inügustrum sp,ln our experiments,
CMV plus satRN.A,was detected in symptomless as welÌ
as in symptom exhibiting CI1. majus plants, showtng that
even a symptom-free plant can be a carrier of CMV en-
capsidating satRNÄ, and could therefore be a potential
danger to agronomically important species. However,
we did not find any dsRN,{s in seedÌings raised from
the seeds of infected Chelidonium. So, seed-transmis-
sion of the pathogenwas not confirmed, possiblydue to
a small number of the examined seeds, Pepper, spin-
ach, Swiss chard and cucumber as weìì as alì the other
experimentaì plants expressed severe symptoms after
inoculation wth this virus.
The electrophoretic pattern of dsRNÄ isolated from
Chelidonium was usually not so clear as the one isolat-
ed from Ligustrum, Nevertheless, using the modified
method for dsRNÄ purification (10) we succeeded in
detecting satRN.A, directly in naturally infected hosts and
in experimental hosts following the first inoculation. In
some prevrous reports satRNAwas usually detected only
after several passages through the experimental host
plants, which enabled better satRN,A, amplification (4).
RNAI -)
RN,Az -+
RN.A3 _>
RNA4 _)
sotRNA-+
FIGURE 2. ElectropÌtoreLic analysis, o¡t 1.20/o agarose
gal, of CMV- (l, 2, 3, 4) and satellite (sat) dsRl/ls rso
lated Íron; inoculatecl (a) and healtlty Chenopodium
quìrioa (c) and from inoculated (b) and healtÌty
Nicoliana negalosiphon plants (d). Both plant species
were inoculatecl wilh sap preparecl lron Chelidonium
maJus.
t?ì
l\. I'lrZcn¡ak ct al
Tlre fact that satRNA was present in Cltelido¡tttt¡tt
plants inclependently of sFnptom e)ostence, provecl that
rt was not responsible for the presence/absence of CMV
induced symptoms on those plants. We were not able
to evaluate the ability of this satRNÄ. to modulate CMV-
induced symptoms on other host species, since tl.re at-
tempt of biological elimination of the satRN¡\ from vrri'
ons was unsuccessfitl. It is known that satRNA re¡lìica-
tron is often su¡r¡:ressed in hosts from familles Cttcttr'
bitaceae and Cäenopodiaceae (7). Unfortunatelly, al-
though satRNAwíìs not revealecl in clsRNA isolates from
Ch. quinoa, it survlvedlìl vivo as ìong as its host, reveal-
ing again a band of hiç¡h intensity when inocltlated on
Nicotiana spccies. Whether some other plant spectes
coulcì serve for biological elitnination of this, l)l tzitzo clttite
stable satRN,\, rentains to be estaltlished,
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a1a Pcriod biol, Vol 101, No 3, 1999
... To my best knowledge, the only plant virus, which was shown to infect C. majus in its natural habitats, is cucumber mosaic virus (CMV), which produces only very mild mottling on infected plants [180] or does not produce any visible disease symptoms [179]. Symptomless C. majus plants were found to be natural hosts of CMV encapsidating satRNA [181]. Information about viral infection is wider for opium poppy (P. ...
... For C. majus, which belongs to Papaveraceae, the family of mostly latex-bearing species producing diverse natural compounds, such evolutionary benefits seems to be con-nected with strong antiviral and antimicrobial activity of latex ingredients. The shortage of studies on viruses or other pathogens infecting C. majus plants could be connected with their strong defense system presenting the lack of disease symptoms even after infection [179,181]. Such strong defense mechanism of C. majus is connected with the presence of predominant and constantly expressed pathogenesisrelated proteins contained in its latex, like MLP, PPO, POX (on the contrary to their inducible definition), acting synergistically with small-molecular compounds (mostly benzylisoquinoline alkaloids) [32]. ...
Article
Full-text available
The aim of this review is to cover most recent research on plant pathogenesis- and defense-related proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesis-related (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.
... Both proteins potentially possess ribonucleic and deoxyribonucleic activities, and hence could be able to digest plant viral RNA or DNA (Huh & Paek, 2013) or act differently using unknown mechanisms (Nawrot, 2017). Data known so far on the proteomic content of C. majus milky sap support the hypothesis that the plant possess strong defense system presenting the lack of disease symptoms even after infection, what could be also indirectly supported by the lack of studies on viruses or other pathogens infecting C. majus plants (Hrzenjak, Curkovic-Perica, Krajacic, & Mamula, 1999;Pospieszny, Borodynko, & Jonczyk, 2004). The only plant virus which was shown to infect C. majus in its natural habitats is the cucumber mosaic virus (CMV) (Br c ak, 1979). ...
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In this review we have collected information regarding latex proteins and their contribution to latex-borne defense among a variety of latex-bearing plants. We comprehensively describe the functions and properties of different pathogenesis- and defense-related proteins found in plant latex, including peroxidases, lipoxygenases, polyphenol oxidases, major latex proteins, β-1,3-glucanases, chitinases, osmotins, proteases, and others. The last section describes proteomic changes occurring during plant-virus interactions in latex-bearing papaya infected with Papaya meleira virus as an example.
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Article
Some physical and chemical properties of the V strain of tomato aspermy virus (TAV) and the Q strain of cucumber mosaic virus (CMV) have been compared. The size, morphology, sedimentation rate, RNA base ratio, and buoyant density of the two viruses are indistinguishable. Preparations of RNA from both viruses were each resolved into four distinct species by polyacrylamide-gel electrophoresis. TAV-RNA preparations contained species with molecular weights of 1.26 × 106,1.10 × 106, 0.90 × 106, and 0.43 × 106 daltons, and CMV-RNA, species of 1.26 × 106, 1.10 × 106, 0.77 × 106, and 0.34 × 106 daltons. Analysis of sodium dodecyl sulphate (SDS)-treated viral proteins by polyacrylamide-gel electrophoresis showed that both viruses have protein subunits of molecular weight 24,500 daltons. The amino acid compositions of proteins from the two viruses, although similar, were distinguishable, and the calculated molecular weights of protein subunits were 26,100 and 26,300 daltons for TAV and CMV, respectively. The two viruses were serologically distinct. On the data presented it is suggested that in preparations of both TAV and CMV three distinct particles are present in each with identical protein shells, but different RNA cores.
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