Plant Science, 67 (1990) 237-244 237 Elsevier Scientific Publishers Ireland Ltd.

SUBCELLULAR LOCATION OF AVOCADO SUNBLOTCH VIROID IN AVOCADO LEAVES

J.F. MARCOS and R. FLORES

Unidad de Biologla Molecular y Celular de Plantas. Instituto de A gzoquim~ca y Tecnologta de Alimentos (CSIC). Calle Jaime Roi9 11, 46010 Valencia (Spain~

(Received June 26th, 1989) (Revision received October 20th, 1989) (Accepted October 31st, 1989)

The subcellular accumulation site of avocado sunblotch viroid (ASBV) in avocado leaves has been investigated. By differen- tial centrifugation techniques, ASBV was predominantly found in a form non-sedimentable at 100 000 × g for 2 h. A minor proportion of the viroid was also detected in association with larger cellular constituents. This association appeared to be mostly artefactual, since a very similar distribution pattern of ASBV was observed in control experiments with non-infected leaves processed with grinding medium containing purified viroid. Moreover, the ASBV contents of crude nuclear and chloro- plastic preparations were considerably reduced after their purification, and small amounts of viroid still bound to the purified organella were also observed in controls with non-infected tissue and externally added ASBV. In all these experiments the distribution of the viroid paralleled that of the cytoplasmic 4S RNAs. Therefore, ASBV appears to have the same subcellular location as coconut cadang-cadang viroid, being the two only exceptions to the so far existing generalization pointing to the nucleus as the major accumulation site for viroids.

Key words: avocado sunblotch viroid; viroid cellular location; subcellular fractionation; Persea americana

Introduction

Avocado sunblotch viroid (ASBV), composed of 247 nucleotide residues [1], is in the lower size limit of the viroids, a group of circular sin- gle-stranded RNAs with a highly base-paired structure [2]. Viroids can either cause specific diseases in higher plants, as is the case of ASBV, or replicate without inducing a host symptomatology [3].

Up to date the primary structure of 1 0 - 1 2 viroids is known [2]. Analysis of sequence data has revealed that ASBV is an unrelated viroid, which does not share a model of five structural domains proposed for the other members of the group [4]. This structural uniqueness of ASBV is accompanied by some functional pecularities, as is the in vitro self-cleavage of transcripts of both polarities [5], which so far has been only found in ASBV.

The knowledge of the subcellular accumula-

tion site of viroids is an important piece of information for the understanding of viroid- host cell interactions. In this respect the nucleus has been considered as the major accu- mulation organelle for viroids in the following system: potato spindle tuber viroid (PSTV) in tomato [6], citrus exocortis viroid (CEV) in Gynura aurantiaca [7-9], tomato planta macho viroid (TPMV) in tomato [10], and hop stunt viroid (HSV) in hop, cucumber and tomato [11]. In some of these cases, associations of viroids with membranous components were also detected [8,10,11], and a more refined analysis of the PSTV-tomato system has demonstrated that this viroid accumulates mainly in the nucleoli [12]. However, the information about the subcellular accumulation site of ASBV in its natural host is very scarce, with only an early report suggesting the association with chloro- plasts and/or endoplasmic reticulum [13]. In the present work we have re-examined this point

0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

238

by a combination of methods of fractionation and purification of subcellular components, with a highly selective and sensitive procedure for viroid detection. Our results indicate that ASBV accumulates mainly in the cytoplasm.

Materials and methods

Viroid and host plant sources Material from one Spanish avocado tree

(Persea americana Miller, cv. Fuerte) display- ing the symptoms of the sunblotch disease [14], was propagated in the greenhouse by bud grafting on healthy seedlings. The sequence of this ASBV isolate has been determined [15] and found to be highly homologous to that of an Australian isolate previously reported [1]. Young asymptomatic leaves (about 15 cm long) from ASBV-infected plants and from healthy controls, were used in all cases. In some experi- ments, apex leaves of Gynura aurantiaca DC plants showing the typical symptoms induced by a severe isolate of CEV, were also used.

SubceUular fractionation The fractionation procedure was based on

methods previously reported [8,10], with some modifications. All steps were carried out at 4 °C. Foliar tissue (5 g) freshly harvested, was gently ground for 2 - 3 rain with a mortar and pestle in 20 ml of a medium containing: 0.4 M sucrose, 20 mM KC1, 2 mM MgC12, 0.1o/0 (w/v) bovine serum albumin, 20 mM T r i s - H C I (pH 7.5) and 10 mM mercaptoethanol. The extract was passed through Miracloth and nylon (50 ~m), and the filtrate was centrifuged sequen- tially at 250 x g, 3000 x g and 10 000 x g for 10 min in each case in a Kontron AS4. 13 swing- ing-bucket rotor. The 10 000 x g supernatant was centrifuged at 80 000 x g for 30 min and then at 100 000 x g for 2 h in a Beckman 70 Ti fixed-angle rotor. The five pellets (A, B, C, D and E, respectively) and the final supernatant (S), were processed to determine their nucleic acid composition.

Preparation and purification of nuclei and chloroplasts

To purify the nuclei, the first pellet (fraction A) of the subcellular fractionation procedure described in the previous paragraph, was care-

fully resuspended in 20 ml of the grinding medium containing 0.40/0 (v/v) of Triton X-100, and then kept on ice for 10 min with occasional and gentle shaking. After centrifugation at 250 x g for 10 rain, the detergent t reatment was repeated. The final pellet of purified nuclei (N) and the first 250 x g supernatant (S1), as well as the two other ones resulting from the Triton X-100 washes ($2 and $3), were analyzed for their nucleic acid patterns.

Chloroplasts were isolated following the principles outlined previously [16,17]. Foliar tissue (5 g) was homogenized as stated above with 20 ml of a medium containing: 0.35 M suc- rose, 3 mM EDTA, 0. 1% (w/v) bovine serum albumin, 50 mM Tris--HCI (pH 7.2) and 10 mM mercaptoethanol. After passing through Mira- cloth and nylon (50 ~m), the filtrate was centri- fuged at 250 x g for 10 min to discard large cell debris and starch. The chloroplasts remaining in the supernatant were collected at 3000 x g for 10 rain. Aliquots of this crude chloroplasts sediment (CC) and of the corresponding 3000 × g supernatant (CS), were separated for nucleic acid analysis. Crude chloroplasts were further purified by centrifugation at 10 000 x g for 20 min in a discontinuous gradient of 10, 40 and 700/0 (v/v) Percoll in the grinding medium [18]. Purified and broken chloroplasts were collected from bands between the 70--40% and the 4 0 - 10% Percoll layers respectively, mixed with 10 vol. of the grinding medium, and recovered by centrifugation at 3000 x g for 10 min. The final pellets of purified (PC) and broken (BC) chloro- plasts were subjected to nucleic acid analysis. All the centrifugation steps were carried out at 4°C in a Kontron AS4. 13 swinging-bucket rotor.

The morphological quality of the different subcellular fractions was monitored by a Reich- ert Polyvar light microscope. Nuclei preparations were stained with 0.05°/0 Tolui- dine blue. Phase contrast microscopy was used in the case of chloroplast fractions, because unbroken plastids are easily distinguishable by their refractive and sharp contours [16,17].

Extraction and analysis of nucleic acids Pellets and supernatants, or aliquots

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thereof, were stored at - 3 0 ° C immediately after they were obtained. Before extraction, pellets were brought with grinding medium to the same volume as the corresponding superna- tants. Nucleic acids were extracted with buffer- saturated phenol [19], and recovered by precipitation with ethanol. The precipitates were resuspended in a small volume of distilled water and adjusted to STE (50 mM Tris-HC1, pH 7.2, 100 mM NaC1 and 1 mM EDTA) and 350/0 ethanol. After application to columns of 0.5 g of non-ionic cellulose (CF-11, Whatman) [20] pre-equilibrated with STE containing 35% ethanol, the cellulose was washed with 10 ml of the same solution and the nucleic acids were eluted with 4 ml of STE and recovered by ethanol precipitation.

Nucleic acids were analyzed by a method proposed previously [21] with some modifica- tions [22], consisting of two consecutive electrc~ phoresis in 5o/0 polyacrylamide gels, under non- denaturing and denaturing conditions respec- tively. Both gels were stained with ethidium bromide, and additionally with silver [23] in the case of the second denaturing one.

Resul ts

Distribution of ASBV in fractions obtained by differential centrifugation

In order to identify the predominant subcel- lular location of ASBV, avocado leaves were gently ground to minimize the destruction and aggregation of the organella, and the homogen- ate was fractioned by differential centrifuga- tion. Observations by light microscopy indicated that nuclei and chloroplasts were present mainly in the 250 x g and 3000 x g pellets, respectively (data not shown}. The nucleic acids components of the subcellular fractions were extracted, partially purified by cellulose chromatography, and analyzed by two cycles of electrophoresis under non-denaturing and denaturing conditions. The cellulose chro- matography step was included to reduce the background of the first non-denaturing gel, making in this way the host cellular RNAs more visible. The analysis by double electrophoresis allowed the detection of the

band of the circular ASBV forms in a zone of the denaturing gel free of other RNAs; se- quencing work reported previously [15] has demonstrated that this band is in fact com- posed of viroid molecules.

Figure la shows that ASBV was found pre- dominantly in the 100 000 x g supernatant In the same figure, the distribution of cellular RNAs, mainly ribosomal 5S and cytoplasmic 4S RNAs, can be seen, and also that ASBV levels paralleled those of 4S RNAs in the six fractions. To investigate the origin of the ASBV present in the pellets, we applied the same subcellular fractionation procedure to healthy avocado leaves including purified ASBV as an additional component of the grinding medium. The distri- bution of ASBV into the different fractions (Fig. lb), was very similar to that of the infected tissue, with a maximum in the final supernatant and decreasing amounts of viroid from the first to the fifth pellets. In order to have another control of the fractionation method, we applied it to Gynura aurantiaca leaves infected with CEV. The results obtained indicated that CEV was found in the first four fractions (with higher levels in the 250 x g and 80 000 x g pel- lets), whereas the viroid content presented a minimum in the final supernatant (data not shown). This agrees with observations reported previously in the CEV-Gynura aurantiaca sys- tem [8], indicating that the nuclei and the membranous system are the major accumula- tion sites of this viroid.

From these experiments, we concluded that the bulk of ASBV is in vivo in a form non-sedi- mentable at 100 000 × g for 2 h. The location of part of the viroid content in fractions sediment- ing at lower centrifugal fields was probably due to the non-specific association of the viroid with these fractions. To obtain additional experi- mental evidence in support of this assumption, we studied whether this association was main- tained when nuclei and chloroplasts were fur- ther purified.

ASBV consent of crude and purified nuclear and chloroplastic preparations

The initial pellet sedimenting at 250 x g for 10 min, which contained most of the nuclei, was

240

a b

A S B V J "

5 S J "

4 S ~"

DP"

ST A B C D E S A B C D E S

C - A S B V ~ . . -

L - A S B V ~ " rap-

Fig. L Polyacrylamide gel electrophoresis of the nucleic acids from the subcellular fractions of ASBV-infected leaves (a), and from non-infected leaves with purified ASBV added to the grinding medium (b). Lane ST, standard purified ASBV; lanes A - E, fractions corresponding to 250 x g for 10 rain, 3000 x g for 10 rain, 10 000 x g for 10 rain, 80 000 x g for 30 rain and 100 000 x g for 2 h pellets, respectively; lane S, 100 000 x g supernatant. The non-denaturing gels (upper panels) were stained with ethidium bromide, and segments of 0.75 cm containing the ASBV band were cut and applied on top of denatur- ing gels (lower panels) that were stained with silver. The arrows on the left indicate in upper panels the positions of ASBV, 5S and 4S RNAs, and in lower panels those of the circular and linear forms of ASBV.

subjected to a mild treatment with Triton X- 100 to remove the chloroplasts and other contaminating membranous components. Fig- ure 2a shows the purified nuclei, apparently undamaged, present in the final pellet. The analysis of nucleic acids revealed that the Tri- ton X-100 washes (specially the first one) released most of the ASBV initially associated with the unpurified pellet rich in nuclei (Fig. 3a). Moreover, the Triton X-100 treatment also released the cytoplasmic 4S RNAs in a similar

way as the viroid RNA. When the experiment was repeated with non-infected avocado leaves, adding purified ASBV to the homogenization medium, the viroid distribution among the frac- tions was reproduced (Fig. 3b). The similar results of both experiments support the hypothesis that the low viroid levels found in purified nuclei preparations were mainly due to the non-specific binding of the viroid RNA to these fractions.

Chloroplasts were purified by a specific pro-

241

cedure, with a centrffugation through a gra- dient of Percoll as a final step, designed to keep the integrity of this organelle. Phase contrast microscopy of the two bands recovered from the gradient revealed that the upper one con- tained broken chloroplasts and membranous components, whereas the lower one had a high number of refractive chloroplasts apparently intact (Fig. 2b). The analysis of nucleic acids showed (Fig. 4a) that the initial 3000 x g super- natant (CS), contained most of the viroid RNA when compared with the crude chloroplasts pellet (CC). The cytoplasmic 4S RNAs had also the same distribution as ASBV between these two fractions. When this crude chloroplast fraction was additionally purified through the Percoll gradient, small amounts of ASBV were observed in association with the broken and

purified chloroplasts (Fig. 4a). However, this association appeared to be for the most part spurious, since it was also found in parallel experiments with non-infected avocado leaves adding purified ASBV to the homogenization medium (Fig. 4b).

Discussion

At present it is well established that the nucleus is the main accumulation site for the viroids of the PSTV group [6-12]. However, the limiting existing evidence suggests that this is not the case of ASBV, which appears to be associated with chloroplasts and/or endo- plasmic reticulum [13]. We have investigated here whether this association reflects a situa- tion existing in vivo, or is the consequence o~ an

Fig. 2. Light micrographs of fractions containing purified organella from ASBV-infected leaf tissue. Toluidine blue stained nuclei under bright field (a), and chloroplasts under phase contrast (b). Bar = 10 ~an.

a b

243

ASBV-

C - ASBVm

L-ASBV-

CC CS PC BC ST CC CS PC BC ST

D

Fig. 4. Polyacrylamide gel electrophoresis of the nucleic acids from fractions obtained in the purification of chloroplasts from ASBV-infected leaves (a), and from non-infected leaves with purified ASBV added to the grinding medium lb). Lane CC, crude chloroplasts; lane CS, 3000 x g supernatant; lane PC, purified chloroplasts; lane BC, broken chloroplasts; lane ST, stand- ard purified ASBV. Other details as in the legend of Fig. 1.

observed in control experiments with healthy cadang viroid (CCCV), which in an early report leaves and purified ASBV added as indicated was found to be non-sedimentable by high cen- above. An additional evidence in support of the trifugal fields sufficient to pellet any cell com- cytoplasmic location of ASBV was the strong ponent of the size of ribosome or larger [24]. correlation observed between the concentra- CCCV shares with ASBV a very small size tions of viroid and cytoplasmic 4s RNAs in all (246 - 247 nucleotide residues), although their the fractions of the different experiments. sequences are very different [2].

We conclude therefore, that the major accu- Finally, we would like to point out that our mulation site of ASBV is the cytoplasm, a prop- data do not discard the possibility that a minor erty distinguishing ASBV from the other fraction(s) of ASBV could exist in association viroids whose subcellular location has been with organella, particularly with nuclei, and studied. The only previous exception in this have functional implications, e.g. ASBV could respect appears to be the coconut cadang- be synthesized in the nucleus, as it has been

a b

243

ASBV~,"

5 S ~

4 S ~ "

CC CS PC BC ST CC CS PC BC ST

C "ASBV ~"

L- ASBV

Fig. 4. Polyacrylamide gel electrophoresis of the nucleic acids from fractions obtained in the purification of chloroplasts from ASBV-infected leaves (a), and from non-infected leaves with purified ASBV added to the grinding medium (b). Lane CC, crude chloroplasts; lane CS, 3000 × g supernatant; lane PC, purified chloroplasts; lane BC, broken chloroplasts; lane ST, stand- ard purified ASBV. Other details as in the legend of Fig. 1.

observed in control experiments with healthy leaves and purified ASBV added as indicated above. An additional evidence in support of the cytoplasmic location of ASBV was the strong correlation observed between the concentra- tions of viroid and cytoplasmic 4S RNAs in all the fractions of the different experiments.

We conclude therefore, that the major accu- mulation site of ASBV is the cytoplasm, a prop- erty distinguishing ASBV from the other viroids whose subcellular location has been studied. The only previous exception in this respect appears to be the coconut cadang-

cadang viroid (CCCV), which in an early report was found to be non-sedimentable by high cen- trifugal fields sufficient to pellet any cell com- ponent of the size of ribosome or larger [24]. CCCV shares with ASBV a very small size (246-247 nucleotide residues), although their sequences are very different [2].

Finally, we would like to point out that our data do not discard the possibility that a minor fraction(s) of ASBV could exist in association with organella, particularly with nuclei, and have functional implications, e.g. ASBV could be synthesized in the nucleus, as it has been

244

shown for other viroids [25], and then translocated to the cytoplasm. This minor frac- tion might be also significant from a pathologi- cal perspective, since high ASBV levels in the cytoplasm are compatible with a symptomless condition of the tissue, suggesting that perhaps ASBV could trigger its pathogenic effects in a cellular location different from the accumula- tion site.

Acknowledgements

This work was partially supported by a grant from the Direccibn General de Investiga- cibn Cientifica y T~cnica de Espafla (PB87- 0346). We want to thank Dr. J.M. Farr~ for pro- viding the avocado material, and V. Monchol| and M. Climent for technical assistance. J.F. Marcos is the recipient of a pre-doctoral fellowship from the Ministerio de Educacibn y Ciencia de Espafla.

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