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Micron 43 (2012) 839–850

Contents lists available at SciVerse ScienceDirect

Micron

j our na l ho me p age: www.elsev ier .com/ locate /micron

ytopathological Potato virus Y structures during Solanaceous plants infection

atarzyna Otulak ∗, Grazyna Garbaczewskaepartment of Botany, Faculty of Agriculture and Biology, Warsaw University of Life Sciences–SGGW (WULS – SGGW), Nowoursynowska 159, 02-776 Warsaw, Poland

r t i c l e i n f o

rticle history:eceived 18 October 2011eceived in revised form 17 February 2012ccepted 20 February 2012

eywords:morphous inclusionytoplasmic inclusionuclear inclusionotyvirusltrastructure

a b s t r a c t

The ultrastructural analysis of tobacco, potato and pepper tissues during infection with necrotic strainsand the ordinary Potato virus Y strain of revealed the presence of virus inclusions not only in the epidermisand mesophyll but also in the vascular tissues. For the first time cytoplasmic inclusions were documentedin companion cells and phloem parenchyma as well as in xylem tracheary elements. The ultrastructuralfeatures studied in this work consisted of mostly laminated inclusions (in the traverse and longitudi-nal section), which were frequently connected with enlarged cisternae of endoplasmic reticulum (ER)located in the direct vicinity of the cell wall attached to virus particles opposite to plasmodesmata. Itwas noticed that ER participates in synthesis and condensation of the PVY inclusions. During compatibleinteraction of tobacco and potato plants with PVY, amorphous and nuclear inclusions were observed.Such forms were not found in pepper tissues and potato revealing the hypersensitivity reaction to the

ytopathologyylem

infection with PVY necrotic strains. It was stated that the forms of cytoplasmic inclusions cannot serve asa cytological criterion to distinguish the potato virus Y strains and do not depend on host resistance level.Only in compatible interaction in Solanaceous plants tissues cytoplasmic inclusions were observed fromthe moment the morphological symptoms appeared. In the reaction of hypersensitivity, the inclusionswere found on the 24th day following the infection with the PVY necrotic strains, whereas the symptomswere observed 3 days after the PVY infection.

. Introduction

The main ultrastructural diagnostic criterion of the attendancef Potato virus Y in the host plant tissues is the presence of viralytoplasmic inclusions. Literature data define these structures as

result of the expression of proteins coded by a virus genome.wing to the large diversity of these deposits, their classificationas been attempted. A few models reflecting their 3D structuresere suggested. A conical type as a form of inclusion for the Tobacco

tch virus (TEV) was assigned by Andrews and Shalla (1974). Thenalyses of the Wheat streak mosaic virus (WSMV) undertaken byernaugh et al., 1980 proved that, considering a geometrical analy-

is, the three-dimensional (spatial) structure of the inclusion looksike an hourglass. However, the model that still seems to be valids a cylindrical inclusion, describing the 3D complex produced byotyvirus in the cytoplasm, first suggested by Edwardson (1966)nd supported by Martelli and Russo (1977) and Lesemann (1988).he above-mentioned research implied that cytoplasmic inclusions

ere made up of protein monomers as plates arranged in relation

o each other at 5 nm distances. A number of 5–15 protein struc-ures are connected to the central core of the inclusion, forming

∗ Corresponding author.E-mail address: katarzyna otulak@sggw.pl (K. Otulak).

968-4328/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.oi:10.1016/j.micron.2012.02.015

© 2012 Elsevier Ltd. All rights reserved.

sheets bent around the axis of the inclusion. The transversal cross-section of this type of inclusion presents a rosette shape, so it hasbeen named “pinwheel”, while the longitudinal section that lookslike a bundle is named, “bundles” (Martelli and Russo, 1977). Alsoan intermediate form between helical and plate inclusions was iso-lated, and it has been named “short curved laminated aggregates”(Edwardson et al., 1984).

As the large morphological diversity of the inclusion structures,an attempt to classify viruses into four subgroups with referenceto cytoplasmic inclusion forms was undertaken. The 1st subgroupincludes viruses generating tubular inclusions. The 2nd subgroupconsists of viruses capable of producing lamellar inclusions. The 3rdsubgroup was formed by merging the 1st and the 2nd subgroups,since these viruses induce inclusions characteristic of these twosubgroups. The 4th subgroup inclusions named “scrolls” and shortstacked band aggregates (Edwardson et al., 1984).

Apart from cytoplasmic inclusions (CI), we can also distinguishother types of inclusions, such as: nuclear inclusions (NI) and amor-phous inclusions (AI), which are not produced by all potyvirus.Most viruses that belong to the 2nd subgroup induce the produc-tion of these NIs that are located in nucleoplasm, or in some cases

also in the nucleolus. NIs were found in cytoplasm as electron-dense granular bodies. Immunolocalisation in tissues infected withTEV revealed that NI protein components first NI accumulate non-inclusively first in nucleolus as non-inclusive and subsequently

840 K. Otulak, G. Garbaczewska / Micron 43 (2012) 839–850

Fig. 1. Ultrastructure of tobacco cv. Samsun mesophyll after PVYNTN or PVYN Wi infection. (a) Fragment of palisade parenchyma (Pme) of the mesophyll of tobacco cv. Samsunafter infection with PVYNTN. In cytoplasm a present the following inclusions: laminated (LI), rosette-pinwheel type (Pw) and semi-rosette “scrolls” (Sc), and amorphous (AI)as well. Bar = 0.5 �m. (b) Spongy parenchyma (Sme) after PVYN Wi infection. Amorphous inclusions in cytoplasm (AI). Particles of PVYN Wi (VP) along tonoplast in cytoplasm(VP*). Bar = 0.2 �m. (c) Inclusions LI connected with the complex of nucleus pore (arrow) and with ER in a cell of spongy parenchyma of the tobacco infected with PVYNTN.B e, GA

–

p(attaop

ar = 0.2 �m. Abbreviations: Ch – chloroplast, CW – cell wall, Int – intercellular spac vacuole, VP – viral particles.

olymerise to form inclusive bodies accumulating in nucleoplasmBaunoch et al., 1988). NIs were also classified. For example, Martellind Russo (1977) isolated nucleoderivatives that were formed dueo the protein accumulation in nucleus, whereas the synthesis of

his type of proteins is observed in the cytoplasm. Therefore, it wasssumed that they are transported to the nucleus and where theybtain a crystalline form. Immunolocalisation of NI protein com-onents showed that NIs were formed in an area of cell nucleus

– Golgi apparatus, M – mitochondrion, N – nucleus, Sc – scrolls, Pr – peroxisome, V

by the participation of protein (Riedel et al., 1998). However, thesecomponents may also occur in the cytoplasm of the infected cell asaggregates (Martin et al., 1992).

The last type of NIs is represented by perinuclear inclusions

that accumulate between membranes of nuclear envelope at somestages of the infection (Martelli and Russo, 1977). The next typeof structures formed in cells infected by potyvirus is amorphousinclusions, formed as a result of the accumulation of non-crystalline

K. Otulak, G. Garbaczewska / Micron 43 (2012) 839–850 841

Fig. 2. Ultrastructure of tobacco cv. Samsun phloem infected with PVYNTN or PVYN Wi. (a) Pinwheels (Pw) and amorphous (AI) inclusions in a cytoplasm of phloem parenchyma(PP) with PVYN Wi (VP) particles. Bar = 0.2 �m. (b) Laminated inclusions (LI) opposite plasmodesmata (PD) in companion cells (CC). Visible particles of PVYNTN (VP) andamorphous inclusions (AI). Bar = 0.2 �m. (c) Laminated inclusions (LI) in contact with plasmalemma (PL) and cell wall (CW) in companion cell (CC). In cytoplasm rosetteinclusions PVYNTN–pinwheel type (Pw) are present. Bar = 0.1 �m. Abbreviations: CC – companion cell„ CW – cell wall, Ch – chloroplast, M – mitochondrion, N – nucleus, PD –p

c(Hgfi(t

om

lasmodesmata, SE – sieve element, Sc – scrolls, V – vacuole.

omponents; as demonstrated for: PVY, Panicum mosaic virusPMV), Tobacco vein mottling virus (TVMV, Baunoch et al., 1990;ellmann et al., 1988). AI may be of different morphology, e.g.ranular, like in cells infected by Papaya ringspot virus (PRSV) orbrilar–granular like for infection with Pepper veinal mottle virusPVMV). Potyviruses may also form amorphous inclusions inside

he nucleus (Edwardson and Christie, 1983; Hari, 1995).

Ultrastructural analyses during PVY infection have concentratednly on indicating the presence of the cytoplasmic inclusions inesophyll cells. These observations have important diagnostic

value as markers of potyvirus infection. Our investigations focuson in situ ultrastructural analysis of PVY inclusion forms and itsarrangement in different Solanaceous plants tissues. We examineddifferent hosts tissue types – especially the vascular ones. More-over, our observations concentrated on occurrence of CIs in potatocultivars showing different level of resistance to a necrotic strain

of PVY. Our study presents an ultrastructural analysis for the formand arrangement of cytoplasmic inclusions during infections of dif-ferent hosts from the Solanaceae family (potato, tobacco, pepper)with potato virus Y (strains PVY0 or PVYN Wi, PVYNTN).

842 K. Otulak, G. Garbaczewska / Micron 43 (2012) 839–850

Fig. 3. Ultrastructure of tobacco cv. Samsun mesophyll infected with PVYNTN or PVYNWi. (a) Laminated inclusions (LI) in contact with plasmalemma (PL) opposite plas-modesmata (PD). Plasmalemma removed from the cell wall (CW) in the area of plasmodesmata. In cytoplasm, amorphous inclusions (Al) and a bunch of vesicles with ane fromp 0.2 �mV

2

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(

lectron-dense content (arrow). Bar = 0.2 �m. (b) Protoplast of mesophyll removedarticles of PVYNTN (VP) in contact with laminated inclusions (LI) in protoplast. Bar =

– vacuole.

. Materials and methods

.1. Plant material and PVY strains

Solanum tuberosum cv. Igor, cv. Rywal, Nicotiana tabacum cv.amsun, Capsicum annum cv. Monsun plants were grown under phy-otron conditions (growth chamber), at temperature 18 ◦C, with6 h light of intensity 400 �mol m−2 s−1 PAR (photosyntheticallyctive radiation). Plants with four levels of leaves were infectedith ordinary or two necrotic strains of PVY: PVY0, PVYN Wi or

VYNTN (Chrzanowska, 1991, 1994). For 3 years, three times perear, ten plants were inoculated mechanically by using carborun-um. Plants were inoculated with 20 drops of PVY suspension (onerop 25 �l), obtained from leaf blades of tobacco cv. Samsun 15 daysollowing the infection in 0.1 M phosphate buffer (pH 7.4). Controllants material was inoculated with phosphate buffer.

Tobacco cv. Samsun plants, used as the material for infection,nd infected plants were tested using DAS-ELISA procedure inHAR Młochów (according to Clark and Adams, 1977). Leaf bladesnd petioles were obtained 15 and 30 days after PVY0, PVYN Wir PVYNTN infection for compatible interaction, and 24 days afterVYN Wi or PVYNTN infection for (incompatible) hypersensitiveesponse. An example of a secondary infection was light sproutsbtained from infected tubers of potato cultivar Igor. Tissue sam-les were collected from five plants of a given variety of leaveshat were directly infected and non-infected. Additionally, extraissue samples were obtained from necroses and area located closeo necroses.

.2. Ultrastructural analysis in transmission electron microscopy

Material was fixed in 2% (w/v) paraformaldehyde and 2%v/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2–7.4)

the cell wall (CW) in plasmodesmata region (PD). In the area of apoplast, visible. Abbreviations: ER – endoplasmic reticulum, M – mitochondrion, Pr – peroxisome,

(Karnovsky, 1965) for 2 h at room temperature. Then, the sampleswere contrasted and fixed in 2% (w/v) OsO4 in cacodylate bufferfor 2 h at 4 ◦C. The material was rinsed with sodium cacodylate andthen dehydrated in a series of increasingly strong water solutionsof ethanol. The material was gradually saturated with resin Epon812 (Fluka) and polymerised for 24 h at 60 ◦C.

Observations were conducted in transmission electron micro-scope Morgagni 268D (FEI- Eindoven, The Netherlands). Photo-graphic documentation was prepared with the use of digital camera“Morada” (SIS) and computer program iTEM (SIS).

3. Results

Ultrastructural analysis for the arrangement and form of cyto-plasmic inclusions during infection with potato virus Y was done.Tissues infected with necrotic (PVYN Wi, PVYNTN) or ordinarystrains PVY0 of different plant hosts (tobacco, potato and pepper)were examined.

In tissues of tobacco cv. Samsun, irrespective of the virus strain,all types of cytoplasmic inclusions were observed in the meso-phyll (Fig. 1a and b) and vascular tissues (Figs. 2a and 4a and b). Incells of palisade parenchyma and wall cytoplasm, inclusions wereobserved such as: laminated (LI), rosette (Pw) of pinwheels typeand semi-rosette (Sc) of scrolls type (Fig. 1a). Large areas in cyto-plasm of palisade and spongy parenchyma cells were occupied byamorphous inclusions (Fig. 1a and b). LI inclusions were in con-nection with ER cisternae, some of them were clearly connectedwith a nuclear pore complex (Fig. 1c). In the phloem area, theoccurrence of laminated and rosette inclusions were found, and in

phloem parenchyma cells, there were also amorphous inclusionsobserved (Fig. 2a). In companion cells, infected with PVYNTN, lam-inated inclusions were observed attached to plasmalemma and tothe cell wall (Fig. 2b and c). Inclusions may also be located on the

K. Otulak, G. Garbaczewska / Micron 43 (2012) 839–850 843

Fig. 4. Ultrastructure of tobacco cv. Samsun mesophyll infected with PVYNTN or PVYN Wi. (a) Laminated (LI) and semi-rosette (Sc) inclusions connected with ER in a cello ted ax m parB roxiso

bmwttIu(dippwwa

f xylem parenchyma (XP) after infection with PVYN Wi. Bar =0.2 �m. (b) Laminaylem parenchyma (XP) cell. Bar = 0.1 �m. (c) Nuclear inclusion (NI, arrow) in xylear = 0.1 �m. Abbreviations: CW – cell wall, M – mitochondrion, N – nucleus, Pr – pe

oth sides of the cell wall (Fig. 2b). The occurrence of LI in the plas-odesmata area (Figs. 2b, 3a and b) was frequently observed. Itas accompanied by enlargement of the wall apoplast space by

he removal of the protoplast of mesophyll cells. In this space, par-icles of PVY were observed attached to the inclusions (Fig. 3b).n xylem, laminated inclusions associated with endoplasmic retic-lum in xylem parenchyma cells were most frequently observedFig. 4a). Laminated and rosette inclusions were documented insideifferent tracheary elements of xylem (Fig. 4b). In tissues of tobacco,

rrespective of PVY strain, the inclusions LI and Pw prevailed. Amor-hous inclusions were mostly located in mesophyll and phloem

arenchyma; although, such localisation of amorphous inclusionsas rare in comparison to other tissue types. Nuclear inclusionsere also observed rarely in other tissue types except mesophyll

nd phloem parenchyma (Fig. 4c).

nd rosette inclusion (CI) of PVYNTN inside the xylem tracheal element (X) and inenchyma (XP) cell after PVYNTN infection. Asterixes indicate the perinuclear areas.me, X – xylem tracheary element.

In tissues of pepper cv. Monsun infected with the ordinarystrain PVY0 or necrotic strains, mainly laminated inclusions wereobserved (Fig. 5a and b) and rosette inclusions with stacked band,which are the characteristics of this host plant (Fig. 5a, c and d). Thisform of inclusion was not observed in tissues of either tobacco orpotato, irrespective of PVY strain. Inclusions LI occurred togetherwith ER cisternae (Fig. 5a), but also in direct contact with cell wallopposite the plasmodesmata (Fig. 5b). The inclusions in tissues ofCapsicum annum cv. Monsun were mainly found in mesophyll andphloem parenchyma, but without the amorphous inclusion types.

In leaf tissues of potato cv. Igor, infected with necrotic strains,

laminated and scrolls inclusions were observed (Figs. 6c and 7b).Numerous LI and Pw inclusions were noticed in connection withdistended ER cisternae; inside the cisternae, a fibrilar materialwas visible (Fig. 7b). Inclusions of amorphous type were revealed

844 K. Otulak, G. Garbaczewska / Micron 43 (2012) 839–850

Fig. 5. Ultrastructure of pepper cv. Monsun tissues infected with PVY0, PVYNTN or PVYN Wi. (a) Laminated (LI) and rosette inclusions with stacked band (LAs), some of themconnected with ER in cell of sponge parenchyma (Sme) after infection with PVY0. Bar = 500 nm. (b) Laminated inclusions (LI) located opposite plasmodesmata (PD) in cellof palisade parenchyma (Pme) infected with PVYN Wi. Bar = 500 nm. (c) Rosette inclusions with stacked band (LAs) PVY0 in a cell of palisade parenchyma (Pme), some oft nclusic cell w

ppsoem

ttwnoN

hem connected with ER. Visible bundle of vesicles, in the vicinity of condensing iompanion cell (CC) of phloem. Bar = 500 nm. Abbreviations: Ch – chloroplast, CW –

revailingly in cells of xylem parenchyma (Fig. 6b and c). In xylemarenchyma (Fig. 6b and c) and in mesophyll (Fig. 7b), inclu-ions fulfilled a considerable part of protoplast of cells. LI was alsobserved in collenchyma cells (Fig. 6a) and inside a tracheary xylemlement (Fig. 6d). Occasionally nuclear inclusions were observed inesophyll cells (Fig. 7a).The analysis of tissues in light sprouts of potato cv. Igor showed

he presence of viral traces mainly in tissues of primary cor-ex parenchyma (Fig. 8a and b) and xylem parenchyma, which

as a result of secondary infection with strains of PVY0 andecrotic strains (Fig. 8c). Semi-rosette scrolls inclusions werebserved, especially atin an early stage of condensation (Fig. 8b).ear inclusion and ER cisternae ER, vesicles were found in the

ons (arrows). Bar = 1 �m. (d) Rosette inclusions (LAs) and particles PVYNTN (VP) inall, M – mitochondrion, N – nucleus, Pr – peroxisome, SE – sieve element.

cytoplasm (Fig. 8a). In xylem parenchyma, LI inclusions con-nected with ER cisternae ER and rosette inclusions prevailed(Fig. 8c). No amorphous or nuclear inclusions were observed inthe analysed tissues of light sprouts during the secondary infec-tion.

Also the tissues of potato cultivar Rywal infected with necroticstrains of PVY were tested by ultrastructural analysis. These plantsreveal hypersensitivity reaction (HR) to the infection with PVY, theyhave gene Ny-1 with reference to PVYN Wi and PVYNTN (Szajko

et al., 2008). Potato cv. Rywal demonstrates morphological symp-toms 3 days after the infection with PVYN Wi or PVYNTN, and theparticles of virus were found in tissues 10 h after the infection(Otulak and Garbaczewska, 2010). Apart from this, PVY cytoplasmic

K. Otulak, G. Garbaczewska / Micron 43 (2012) 839–850 845

Fig. 6. Ultrastructure of potato cv. Igor collenchyma and xylem infected with necrotic strains PVY. (a) Laminar inclusions (LI) in the area of angular collenchyma (Co)a N N Wi in

a = 0.5 �w m, GAe

iwnfdnotos

fter interaction with PVY Wi. Bar = 0.2 �m. (b) Amorphous inclusions (AI) PVYmorphous (AI) inclusions in xylem parenchyma (XP) cell after PVYNTN infection. Barith PVYNTN. Bar =1 �m. Abbreviations: CW – cell wall, ER – endoplasmic reticulu

lement.

nclusions were first observed only 24 days after the infectionith necrotic strains of PVY. Laminated inclusions directly con-ected with ER cisternae or with plasmalemma were dominant

orms (Fig. 9a and c). Scrolls inclusions at the early stage of con-ensation in direct connection with ER were also observed nearbyumerous vesicles in the cytoplasm (Fig. 9b, d and d). LI and Pw

ccurred mainly in tissues of xylem parenchyma and mesophyll. Inhe analysed material, no amorphous and nuclear inclusions werebserved during the hypersensitivity of potato on the PVY necrotictrains.

xylem parenchyma (XP) cell. Bar = 0.2 �m. (c) Concentration of laminated (LI) andm. (d) Laminated inclusions (LI) inside xylem tracheary element (X) after infection

– Golgi apparatus, M – mitochondrion, PL – plasmalemma, X – xylem tracheary

4. Discussion

The presence of cytoplasmic inclusions, usually in epidermisand mesophyll cell, has become an important diagnostic tool toidentify the infection caused by potyviruses. Our findings indicatethat CIs as well as virus particles were located in phloem, espe-

cially in parenchyma and phloem companion cell. The presence ofCIs in sieve element has been rarely demonstrated, for examplein mixed infection PLRV-PVY (Garbaczewska and Kerlan, 2001). Inour findings, inclusions were seen opposite to or in the proximity of

846 K. Otulak, G. Garbaczewska / M

Fig. 7. Ultrastructure of potato cv. Igor mesophyll after PVYN Wi infection. (a)Nuclear inclusion (NI, arrows) in mesophyll cell. Bar = 0.5 �m. (b) Rosette (Pw) andliN

pepcaipCotTim2hmtialmtpaAe

aminated (LI) inclusions connected with ER distended cisternae. Fibrile materialnside cistern. Bar = 0.1 �m. Abbreviations: GA – Golgi apparatus, M – mitochondrion,

– nucleus.

lasmodesmata between sieve element and companion cell. Inter-stingly, we also noticed PVY inclusions directly attached to virusarticles opposite plasmodesmata as an effect of an active translo-ation mechanism. During compatible interaction, viral particlesnd inclusions of necrotic strains PVY were observed in compan-on cells, phloem parenchyma, as well as the particles of PVY insidelasmodesmata between a sieve element and a companion cell.ytoplasmic inclusions of the virus (LI, Pw) were often locatedpposite to or in the vicinity of the plasmodesmata. The data men-ioned above imply systemic transport of PVY by phloem route.he above-mentioned analysis is also true for the incompatiblenteraction, but the difference consists in the fact that cytoplas-

ic inclusions of PVY appear in vascular tissues and parenchyma4 days after the infection. Our observations during potato-PVYypersensitive response have identified viral particles in xylem ele-ents (Otulak and Garbaczewska, 2010). Our results, for the first

ime, have documented PVY cytoplasmic inclusions in xylem. Allnclusion types were found in xylem parenchyma in compatibles well as incompatible interaction. Potato virus Y inclusions wereocalised in mature and differentiating xylem tracheary elements

uch more often than in sieve element – typical for virus sys-emic translocation. There are only a few data demonstrating which

roteins are included into cytoplasmic inclusions, and knowledgebout function of such proteins is poor. The basic component ofI is protein HC-Pro for the viruses: PVY, PMV, TVMV (Baunocht al., 1990; Hellmann et al., 1988), and such inclusions may be

icron 43 (2012) 839–850

located in cell nucleus. Typical nuclear inclusions which are syn-thesised with the use of NIa and NIb proteins, are coded by thevirus (Riedel et al., 1998). Our investigations demonstrated nuclearinclusions as an effect of compatible interaction between host andnecrotic strains of PVY in the examined tissues potato and tobacco.Some independent teams of researchers found that CI protein is abasic protein immunotagged in cytoplasmic inclusions Potyviridae(Murphy et al., 1991; Rojas et al., 1997). The comparison betweensequences of PVYN and PVYNTN necrotic strains, which proved highhomology, made a reference point for the comparison performedby Thole and others (Thole et al., 1993). Further CI protein, stud-ies conducted on potyvirus TEV and TVMV by Murphy et al. (1991)and Langenberg and Zhang, 1997 demonstrated that another com-ponent that forms inclusions is protein P3. Moreover, it has beensuggested that, in the vicinity of cytoplasmic inclusions built by CIprotein, the coat protein (CP) also submits to tagging (Ammar et al.,1994).

In this paper, the nomenclature of cytoplasmic inclusion typesfor PVY necrotic strains was applied according to the nomenclaturefound in literature (e.g. Edwardson, 1966; Lesemann, 1988). Basedon marking potyvirus proteins mentioned as inclusion structures, ithas been reported that they are different cross-sections of the sameforms, which are laminated inclusions. Longitudinal cross section(LI) of the CI inclusion of PVY results in morphology defined as LI;however, when the cross section is transverse, then its shape willassume the form of rosette inclusion (i.e. pinwheel). Therefore, itwould be probably more appropriate to use only a symbol CIs forvirus inclusions.

The main division of PVY includes 3 groups of strains: ordinaryPVY0, necrotic PVYN isolate PVYN Wi described as more infec-tious for potato and achieving higher concentration, but producingsymptoms of gentle mosaic (Chrzanowska, 1994). PVYNTN wasidentified in Europe as producing necrotic symptoms on potatotubers in the form of necrotic ringspot disease PTNRD (Kus, 1990;LeRomancer and Kerlan, 1991). Despite symptomatic diversitybetween the strains, the ultrastructural analysis revealed that cyto-plasmic inclusions and their forms cannot be a cytological criterionfor distinguishing strains.

If potyvirus infects a host–plant and is able to multiply itself,it deposits different proteins in tissues of such a plant. Since over-production of these proteins is so high, which can be observed inultrastructural analysis, these proteins have to perform a significantfunction for pathogen. Unfortunately, the knowledge on this issue isstill incomplete. Hari (1995) suggested that polymerisation of sur-plus potyvirus non-structural proteins in the form of cytoplasmicinclusions permits the process of merging particles of virus (link-ing the genome with protein of capsid), and also rendering virusspread easy in the plant. Eagles et al. (1994), Klein et al. (1994)and Lain et al. (1990) claimed that CI protein, being a component ofcytoplasmic inclusions, is not only as active as helicase during repli-cation but it also assists during inter-cellular transport. Wei et al.(2010) show that newly identified protein P3N-PIPO as a PD-locatedprotein and directs the CI protein to PD, facilitating the deposi-tion of the cone-shaped structures of CI at PD by interacting withCI protein. Wen and Hajimorad (2010) reported that mutation ofthe putative SMV (Soybean mosaic virus) PIPO impedes cell-to-cellmovement, providing genetic evidence that P3N-PIPO is a potyvi-ral movement protein. Taking these new findings into account,Wei et al. (2010) propose a model for the formation of movementcomplexes that facilitate intercellular transport of potyviruses [forthe TEV and TuMV (Turnip mosaic virus) examples]. The virion–CImovement complex is intracellularly transported to the modified

PD where CI forms conical structures anchored by the PD-locatedP3N-PIPO. The virion is then fed through the CI structures and PD toenter the adjacent cell. Analysing ultrastructural changes, an effectof both compatible and incompatible interaction of Solanaceae

K. Otulak, G. Garbaczewska / Micron 43 (2012) 839–850 847

Fig. 8. Ultrastructure of potato cv. Igor light sprouts tissues infected with PVY0, PVYNTN or PVYN Wi. (a) Semi-rosette inclusions (Sc) in different condensation stage andlaminated ones (LI) connected with ER in the area of primary cortex parenchyma (PCP) after infection with PVY0. Bar = 0.1 �m. (b) Semi-rosette inclusions (Sc) in differentcondensation stage near cell wall (CW) in primary cortex parenchyma (PCP) after infection with PVYN Wi. Bar = 0.5 �m. (c) Laminated (LI), rosette (Pw) and semi-rosette( s. Bar

t

ppptetpoc1vc

Sc) inclusions connected with ER xylem parenchyma (XP). Visible PVYNTN particleracheary element.

lants with PVY, frequent connections of laminar inclusions withlasmolemma near a cell wall were observed, especially oppositelasmodesmata. Occasionally, also rosette inclusions of pinwheelsype (Pw) underwent such connections. Inclusions near plasmod-smata were sometimes associated with ER cisternae at the sameime. They are usually also attached to virus particles in proto-last as well as in apoplast space between cell wall and cytoplasmpposite to the plasmodesmata. The above correlations were also

onfirmed by studies done on virus TVMV (Rodriguez-Cerezo et al.,997) and in tissues infected with PSbMV (Pea seed-borne mosaicirus) (Roberts et al., 1998). The presence of CI protein (as inclusion)onnected with plasmalemma near plasmodesmata (occasionally

= 0.1 �m. Abbreviations: CW – cell wall, M – mitochondrion, P – plastid, X – xylem

together with capsid) was observed, providing the continuation ofa canal linking the neighbouring cells through a core of the inclu-sion together with a cell membrane. The above data justify thehypothesis that cytoplasmic inclusions take part in positioning avirus complex (through inclusion into plasmodesmata), enablingtranslocation. According to Carrington et al. (1998) such a repeti-tive system proves the interaction between CP and CI in the transferof virus from cell to cell. Perhaps the inclusion in such a relation-

ship functions for a short time. The inclusion CI with CP localisesnear the cell wall only during the replication and after that it disso-ciates from the cell wall/membrane, taking place in cytoplasm (assuggested by Roberts et al., 1998).

848 K. Otulak, G. Garbaczewska / Micron 43 (2012) 839–850

Fig. 9. Ultrastructure of potato cv. Rywal tissues during HR 24 days after PVYNTN or PVYN Wi infection. (a) Laminated (LI) inclusions connected with plasmalemma (PL) inmesophyll cell after infection with PVYN Wi. Bar = 1 �m. (b) Scrolls inclusions (Sc) in early stage of condensation connected with ER in xylem parenchyma (XP) infected withPVYN Wi. Bundle of vesicles (from ER) indicated with arrows. Bar = 2 �m. (c) Laminated inclusions (LI) connected with ER in mesophyll cell infected with PVYNTN. In thev ons (S NTN

( ndoplt

Psdmtota

icinity, a bundle of vesicles indicated with an arrow. Bar = 1 �m. (d) Scrolls inclusifrom GA) indicated with arrows. Bar = 0.2 �m. Abbreviations: CW – cell wall, ER – eracheary element.

The analysis of compatible interaction of tobacco/PVYN andVYNTN done by Felczak and Garbaczewska (2004) stated thatmall vesicle structures filled with condensed material occur withifferent cytoplasmic inclusions of PVY in a symplast of tobaccoesophyll, and the authors conclude after Lesemann (1988) that

he structures may be significant for the replication of virus. Thebservations concerning compatible and incompatible interac-ions (HR) suggest that vesicles filled with condensed materialre located in protoplasts in the areas where PVY cytoplasmic

c) in different condensation stage in mesophyll cell infected with PVY . Vesiclesasmic reticulum, GA – Golgi apparatus, PL – plasmalemma, V – vacuole, X – xylem

inclusions (of different types) were deposited. Some of them havetheir membrane thorn, and in cytoplasm, just beside it, thereare inclusions with different stages of advanced condensation.Moreover, during the HR reaction, vesicles of this type were firstobserved when the inclusions were found, i.e. 24 days after the

infection with necrotic strains of PVY. Another stage is unusuallyfrequent connection of viral cytoplasmic inclusions with ER cis-ternae – mostly of laminated type (never amorphous type). Thissituation concerns both the observations during HR interactions

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K. Otulak, G. Garbaczewsk

nd the compatible interactions (in tissues of tobacco, potato,ncluding light sprouts). The above-mentioned data suggest thatesicle structures are probably involved in synthesis and/or sort-ng of PVY virus cytoplasmic proteins. The specialisation of ERomains in plant cell may not only be regulated by the growthtage, environment conditions (Sherameti et al., 2008; Yamadat al., 2009), but also by stress conditions of the plant, whichs undoubtedly the defence against the pathogen activity. Sub-ellular structures formed from the plant ER may perform variousunctions. They are surrounded with a single membrane from ERisternae and are able to conduct synthesis and compartmentationf some specific proteins. These sub-cellular structures may includeR bodies accumulating some amounts of proteins that do not func-ion inside a given compartment (Staehelin, 1997). The structuresarlier referred to as replication vesicles are a form of ER, in whichroteins of PVY cytoplasmic inclusions are synthesised in the ERisternae. It was observed that growth of ER bodies was induced bytress conditions. The appearance of this vesicular structures maye a strategy of the cell to exist or in defence response under stressonditions and named iER (induced ER bodies) (Hara-Nishimurat al., 2005; Ibl and Stoger, 2011).

In our investigations, we examined inclusions localisation inotato cultivars with different resistance level to PVY necrotictrains. In incompatible interaction (reaction HR), the formation ofytoplasmic inclusions (laminated, pinweels and scrolls) PVY wasrst observed in tissues of potato cv. Rywal 24 days after the infec-ion. It may be an effect of the plant–host defence reaction. Hinrichst al. (1998) and Hinrichs-Berger et al. (1999) argue that duringhe interaction with immune plants, the reaction is carried out soapidly that there is no time to form viral cytoplasmic inclusionsnd to form complete particles. Our earlier analyses indicated thatt is probable that viral inclusions are not indispensible to replicatehe PVY genome and is not necessary for intercellular transport ofhis pathogen (Otulak and Garbaczewska, 2010). It is confirmed bynalyses made by Carrington et al. (1998) concerning the protein CIf virus TEV. The analyses showed that mutational changes at the-end of this protein induce disturbance in intercellular transport,ot affecting replication. However, in case of hypersensitivity reac-ion, there is no complete certainty if the restrictions of transportre really a result of mutations, or perhaps a defence response of thelant. The form and arrangement of the cytoplasmic inclusions are

ndependent of the level of resistance to PVY infection. Moreover,he HR interaction indicates that the symptoms appearance and theresence of the cytoplasmic inclusion do not correlate, but the timef inclusion presence depends on type of virus–host interaction.

onflict of interest statement

The authors declare that they have no conflict of interest.

cknowledgements

We would like to thank Prof. Mirosława Chrzanowska (IHAR,łochów) for providing the plant material and inz. Ewa Znojek for

xpert technical assistance.

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