Ultrastructural studies of plasmodesmatal and vasc

Authors: Grażyna Garbaczewska Katarzyna Otulak Marcin Chouda Mirosława Chrzanowska

Publish Date: 2012/02/23

Volume: 34, Issue: 3, Pages: 1229-1238

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Abstract

The studies focus on an ultrastructural analysis of the phenomenon of intercellular and systemic vascular transport of tobacco rattle virus TRV in tissues of the infected plants TRV is a dangerous pathogen of cultivated and ornamental plants due to its wide range of plant hosts and continuous transmission by vectors—ectoparasitic nematodes Two weeks after infection with the PSG strain of TRV tobacco plants of the Samsun variety and potato plants of the Glada variety responded with spot surface necroses on inoculated leaf blades Four weeks after the infection a typical systemic response was observed on tobacco and potato leaves necroses on stems and lesions referred to as corky ringspot Ultrastructural analysis revealed the presence of two types of TRV virions capsidated and noncapsidated forms in tobacco and potato tissues In the protoplast area viral particles either occurred in a dispersed form or they formed organised inclusions of virions We demonstrated for the first time the presence of noncapsidatedtype TRV in the vicinity of and inside plasmodesmata Capsidated particles of TRV were observed in intercellular spaces of the tissues of aboveground and underground organs Expanded apoplast area was noted at the cell wall with numerous dispersed noncapsidatedtype TRV particles These phenomena suggest active intercellular transport Our ultrastructure studies showed for the first time that xylem can be a possible route of TRV systemic transport We demonstrated that both capsidated and noncapsidated virions of varied length participate in longdistance transport TRV virions were more often documented in xylem tracheary elements and parenchyma than in phloem Noncapsidated TRV particles were observed inside tracheary elements in a dispersed form and in regular arrangements in potato and tobacco xylem The presence of TRV virions inside the bordered pits was demonstrated in aboveground organs and in the root of the tested plants We documented that both forms of TRV virions can be transported systemically via tracheary elements of xylemTobacco rattle virus TRV is a widespread pathogen in the plant world Over 400 plant species from monocotyledon and dicotyledon classes can serve as hosts for the virus The most common hosts are cultivated plants such as potato sugar beet tobacco and tomato Allen 1963 Huth and Lesemann 1984 TRV belongs to the Tobravirus genus It has a bipartite genome and it is made of two single strands of positive polarity +ssRNA The genetic material undergoes separate encapsidation into simple rodshaped helical capsids with identical diameters of 225 nm The virions of TRV vary in terms of length longer particles L range from 180 to 197 nm and shorter particles S from 55 to 114 nm Both types of virions have 5 RNA and 95 of protein The virions of these two kinds fulfil different functions in the processes of viral infection and multiplication in a plant The longer particle containing RNA1 induces infection whereas the shorter one containing RNA2 is responsible for creating a coat protein CP for both strands Bergh et al 1985 If the plant is infected only by L particles only RNA of these particles multiplies Frost et al 1967 Such TRV form was referred to as nonmultiplying NM It is sensitive to ribonucleases and it is easily degradable in plant sap Complete virions known as capsidated forms appear in the plant when both types of TRV virions and/or their RNA are present Harrison and Robinson 1978 MacFarlane 1999 2010RNA1 has four open reading frames ORF which encode the proteins that participate in virus replication and transmission by vectorsnematodes and it is believed that they also take part in celltocell movement of the virus Hamilton and Baulcombe 1989 RNA2 encodes coat protein and plays a part in the transmission by nematodes protein 16 K Ploeg et al 1993 MacFarlane and Brown 1995 The TRV strains have been classified and those isolated from potatoes that are relatively well described include PRN Scotland strains from Oregon Lister and Bracker 1969 and PSG—a strain isolated in the Netherlands and described for the first time by Cornelissen et al 1986Current studies on TRV focus on strains and their recombinants that are transmitted mostly by nematodes of the Trichodorus and Paratrichodorus species In our studies we used the method of mechanical inoculation—one of the basic possibilities of infection in field greenhouse or laboratory conditions The objective of these studies was to present the phenomenon of intercellular and vascular translocation of the PSG strain of TRV in potato and tobacco tissues at the ultrastructural levelPlants of Solanum tuberosum cv Glada and Nicotiana tabacum cv Samsun were grown in a growth chamber at a temperature of 18°C and a 16h light cycle with the intensity of 400 μmol m−2 s−1 PAR Plants at four leaf stage were infected with the PSG strain of TRV Plants were mechanically inoculated with TRV suspension using carborundum in a 01 M phosphate buffer pH 74 The virus isolate was received from Institute of Phytopathological Research Wageningen The Netherlands Control plant material was inoculated only with phosphate buffer The plants of tobacco cv Samsun used as a material for infection and the infected plants of potato were tested using the DASELISA procedure at IHAR Młochów according to Clark and Adams 1977 immunoglobulins received from D Z Maat Wageningen The Netherlands The plant organs were collected depending on symptoms 2 3 and 4 weeks after TRV infection The investigations were repeated three timesThe material was fixed in 2 w/v paraformaldehyde and 2 v/v glutaraldehyde in a 005 M cacodylate buffer pH 72–74 Karnovsky 1965 2 h at room temperature Next the samples were contrasted and fixed in 2 w/v OsO4 in cacodylate buffer for 2 h at 4ºC The material was rinsed with sodium cacodylate and then dehydrated in a series of increasingly strong water solutions of ethanol The material was gradually saturated with resin Epon 812 Fluka and polymerized for 24 h at 60ºC The ultrathin sections on copper grids were stained with uranyl acetate and lead citrate

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