Effects of chloroplast targeted potato virus Y coat protein on transgenic plants

Physiological and Molecular Plant Pathology (1997) 50, 67–83

Effects of chloroplast targeted potato virus Y coat

protein on transgenic plants

M. N and P. H. B*

Department of Plant, Soil and Entomological Sciences, Di�ision of Plant Pathology, Uni�ersity of Idaho, Moscow,ID 83844-2339, U.S.A.

(Accepted for publication August 1996)

Studies of several plant viruses have shown that virus coat protein (CP) may play a role insymptom development. In order to determine how and where potato virus Y (PVY)-CP maycontribute to symptom development, two sets of plants were generated, one expressing PVY-CP,and one transformed with a chimeric construct consisting of PVY-CP fused to the transit peptide(TP) of pea ribulose bisphosphate carboxylase small subunit (rbcs) to target PVY-CP to thechloroplast stroma. Numerous transgenic plants developed chlorotic or white mosaic symptomsafter transformation with the TP-CP construct, but not after transformation with PVY-CP alone.Both western blot analysis of stroma and thylakoid protein fractions and immunocytochemistryrevealed that the imported PVY-CP was preferentially associated with thylakoid membranes inTP-CP+ plants. Light and electron microscopy showed that PVY-CP disrupted chloroplastdevelopment and cell structure in TP-CP+ transformants. Virus challenge of non-transformedCP+ and a range of TP-CP+ tobacco plants indicated that high level PVY-CP expressing TP-CP+

plants were more susceptible to PVY. These observations suggest that PVY-CP action inchloroplasts may contribute to symptom development in PYV-infected plants.

# 1997 Academic Press Limited

INTRODUCTION

Potyviruses cause a wide range of symptoms in their hosts, dependent on the virus–

host combination as well as environmental conditions. The precise manner by which

plant viruses cause disease is unknown. Since a common symptom is chlorosis, as is

typical when mosaic or mottle symptoms are observed, it is reasonable to assume that

virus infection has some bearing on chloroplast function. Several mechanisms

underlying virus pathogenicity have been proposed, including the possibility that ac-

cumulation of one or more viral products in the chloroplast leads to symptom

development [for reviews see 5, 46]. The interaction of viruses or specific viral gene

products with host chloroplasts has been observed in a variety of virus–host

combinations [5, 28, 46].

Disruption of chloroplast development or function in virus-infected plants could be

*To whom correspondence should be addressed.Abbreviations used in text : CP, coat protein; LM, light microscopy; PVY, potato virus Y; TEM,

transmission electron microscopy; TMV, tobacco mosaic virus ; TP, transit peptide ; TVMV, tobacco veinmottling virus.

0885–5765}97}02006717 $25.00}0}pp960071 # 1997 Academic Press Limited

68 M. Naderi and P. H. Berger

due to a variety of factors. Among these may be the result of an interaction(s) of virus

coat protein and host chloroplasts. Here, we have focused on the coat protein of potato

virus Y (PVY), and have done so for two reasons : (1) we previously observed that not

only genomic RNA but also two PVY gene products, coat protein (CP) and the helper

component-proteinase protein, are found inside chloroplasts of infected plant cells

[15]. Moreover, PVY-CP is not distributed uniformly in chloroplasts but is almost

exclusively associated with isolated thylakoid membranes [15]. We have also observed

that viral RNA isolated from chloroplasts of infected plants is infectious (Gunasinghe

and Berger, unpublished data). Further, there can be a specific reduction or inhibition

of photosystem II activity in potyvirus-infected plants [29, 30] or a reduction in

chloroplast numbers and size [44]. (2) While it is likely, and has been demonstrated in

several cases, that virus gene products other than coat protein correlate or are somehow

involved in alterations of chloroplast function [4,11, 18, 19, 27, 32, 33, 41], there is

considerable evidence indicating that CPs of a number of viruses are involved in

chlorosis induction [7, 16, 31, 34, 40].

In order to investigate the effects of PVY-CP in different compartments of the cell,

we have generated several transgenic tobacco lines. One series produces and

accumulates CP in the cytoplasm, while another series accumulates CP in the

chloroplast stroma compartment by use of a stroma-specific transit peptide (TP). We

report here the effects of chloroplast targeted CP on host cells.

MATERIALS AND METHODS

Vector construction

An 800 bp SalI}EcoR I fragment encoding the PVY-CP gene was cloned into plasmid

pJIT117 [14] to create a transcriptional}translational fusion of PVY! coat protein and

the transit peptide (TP¯ 80 amino acids) of pea ribulose bisphosphate carboxylase

small subunit (rbcs). The TP-CP chimeric gene is flanked at the 5«-end with a CaMV

35S promoter (with duplicated enhancers) and at the 3«-end with a CaMV transcription

terminator}poly(A) addition signal. This expression cassette, as a Xho I}Sac I

fragment, was cloned into pKYLX [39] binary vector, flanked by another transcription

terminator}poly(A) addition signal (rbcs). The resultant plasmid was designated

pMNLX1.0. The PVY-CP gene without the transit peptide sequence and with an in-

frame, translationally optimal initiation codon was cloned into another vector,

designated pKYLX68.1.

Plant transformation

Nicotiana tabacum cv. Xanthi, a systemic host for PVY!, was used throughout this study.

Binary vectors were mobilized into Agrobacterium tumefaciens LBA4404 using the method

of An [1]. Nicotinia tabacum plants were transformed with the engineered Agrobacterium

using the leaf disk method and transformants selected on modified Linsmaier–Skoog

media [39] using kanamycin concentrations of 25, 50 and 300 mg l−". Plants

transformed with PVY-CP without the transit peptide sequences were selected on a

medium containing 300 mg l−" kanamycin. Shoots were transferred to shooting media

without antibiotics and, if roots developed, transferred to soil and grown in growth

PVY coat protein and chloroplasts 69

chambers at 25 °C with a 16 h photoperiod. R"seeds were germinated in the presence

of 50 mg l−" kanamycin under the above conditions on modified Linsmaier–Skoog

media [39].

Protein isolation and western blot analysis

Total leaf proteins were extracted as described previously [15] and stored at ®20 °Cuntil use. For western blot analysis, total soluble proteins from each sample were

separated by 12% SDS PAGE [23], electrophoretically transferred to nitrocellulose,

and probed with anti-PVY sera as described previously [15].

Chloroplast isolation and separation of stroma and thylakoid protein fractions

Intact chloroplasts were isolated as described by Fish and Jagendorf [12] as modified

by Reinero and Beachy [34]. Chloroplasts were resuspended in resuspension buffer,

thermolysin (200 µg ml−") and CaCl#(0±5 m) added, and incubated on ice for 1 h [3].

Chloroplasts were washed twice in five volumes of resuspension buffer. Thylakoid and

stromal protein fractions were obtained as described [37]. Proteins from both fractions

were precipitated with nine volumes of cold acetone, equalized on the basis of OD#)!

,

and stored at ®20 °C for later use in SDS PAGE and western blotting.

RNA isolation and RNA gel blot analysis

Total RNA was isolated as described by Sambrook et al. [38]. RNA accumulation was

determined by northern blot hybridization using 10 µg of total RNA per lane. The blot

was exposed to Kodak XAR-5 film for 12 h at ®80 °C.

Light and electron microscopy

Developmentally similar tobacco leaf tissue was fixed and infiltrated with LR White

resin and polymerized in gelatin capsules at 60 °C for 24 h, as described elsewhere by

Tranbarger et al. [43], except that 2% osmium tetroxide postfixation was used for

tissues for transmission electron microscopy (TEM) but not for immunolocalization.

Plant tissue was selected for collection at approximately the same time of day (approx.

10:00 am). Sections were prepared on an LKB 8800 Ultratome III (Gaithersburg,

MD, U.S.A.) ultramicrotome for light microscopy (LM) or TEM. For light microscopy,

sections 2 µm thick were affixed to gelatin-coated glass microscope slides and stained

with methylene blue. Stained sections were photographed under bright-field

illumination using a JENAVAL (aus JENA, Germany) transmitted-light research

microscope. For TEM, thin sections were picked up onto formvar-coated nickel grids,

and stained with uranyl acetate and potassium permanganate. For TEM immuno-

cytochemistry, anti-PVY sera was used as described by Grimes et al. [13]. Electron

micrographs were taken with a Zeiss EM10A electron microscope.

Virus challenge tests

R"progeny seed was surface sterilized as described above and plated on tissue culture

media containing 50 µg ml−" kanamycin. Plantlets that germinated and survived to the

three leaf stage, and thus were homo- or heterozygous for the gene of interest, were


transplanted to soil and mechanically inoculated with PVY. Inoculum was prepared

by grinding PVY-infected tobacco in phosphate buffer (0±05 , pH 7±0) at 1:5 (w}v),

aliquoting this suspension and freezing aliquots at ®80 °C until use, when it was mixed

with carborundum. Ten plants per line were inoculated, and samples for ELISA tests

were collected just prior to inoculation and at 5 day intervals thereafter. Virus titre was

determined by relative amounts of helper component produced using anti-PVY HC

(kindly provided by D. Thornbury, University of Kentucky).

RESULTS

Symptom de�elopment in TP-CP+ plants

Leaf discs of N. tabacum cv. Xanthi were infected with A. tumefaciens harbouring

pMNLX1.0 (Fig. 1) and transformants selected on different concentrations of

kanamycin up to 300 mg l−". When only PVY-CP (pKYLX68.1) lacking the rbcs

transit peptide (CP+) was transformed into tobacco, nearly all CP+ transformants were

green and phenotypically normal, regardless of which kanamycin concentration was

used (Table 1). Two of these lines were chosen for comparative purposes, based on

western analysis (see below). The few white shoots that did develop initially became

green and developed normally after transfer onto non-selective media (data not shown)

and appeared to be non-transformed, antibiotic sensitive plants which had escaped

selection during transformation. Antibiotic resistant TP-CP+ plantlets or calli could

only be obtained using kanamycin concentrations of 50 mg l−" or less. Many TP-CP+

transformants were chlorotic or pale green (Table 1). Chlorotic plants stopped growing

after two to five leaves developed. Some of these chlorotic TP-CP+ plantlets were

transferred onto non-selective media where they remained chlorotic (Table 1),

indicating that this phenotype was not the characteristic response of non-transformed

plants to toxic levels of kanamycin.

Some primary TP-CP+ pale green transgenic plants started showing white mosaic

(white and green areas) symptoms 4 weeks after transfer to soil. These symptoms were

generally pronounced on the adaxial surface, but the abaxial leaf surface had no

apparent symptoms (data not shown). When white spot symptoms progressed beyond

the upper cell layers, leaves became deformed (data not shown). Figure 2 shows a

kanamycin resistant R"progeny TP-19 from plants with these symptoms. These plants

showed symptoms on both adaxial and abaxial leaf surfaces, and on both selective and

non-selective media, indicating that white mosaic symptoms were not due to side-

effects of kanamycin treatment. These disease-like symptoms, therefore, were not due

to culture conditions but rather to the presence of the TP-CP allele. In addition, the

more rapid and pronounced development of symptoms in the R"progeny compared to

the primary transformants may indicate that the phenotype penetrance increases with

gene dosage. Moreover, white mosaic symptoms are more pronounced along the leaf

vein system (Fig. 2). This could be explained by higher activity of 35S promoter along

the vein system in transgenic plants [2]. Many TP-CP+ transgenic plantlets that

survived initial selection at 50 mg l−" kanamycin were severely chlorotic and unable to

develop further. No shoot regeneration was observed when kanamycin at 300 mg l−"

was used. Since the number of integrated genes tends to increase as selection stringency


F. 1. Organization of the binary vector pMNLX1.0 which was used to target PVY-CP intothe stroma compartment of tobacco chloroplasts. RB, right T-DNA border ; LB, left T-DNAborder ; P35 S#, cauliflower mosaic virus 35 S promoter with duplicated enhancers ; TP, transitpeptide of ribulose bisphosphate carboxylase small subunit from pea; CP, PVY coat protein;TCaMV, cauliflower mosaic virus 35 S transcriptional terminator ; Trbcs, ribulose bisphosphatecarboxylase transcriptional terminator.

T 1Effects of transformation of tobacco with PVY coat protein with (TP-CP) or without (CP) the rbcs transitpeptide (TP). Numbers of transformants based on number of calli after selection on 50 mg l−" kanamycin

for TP-CP or 300 mg l−" for CP

TP-CP

chlorotic

Experiment No. transformants No. %

1 41 12 292 74 66 89Total 115 78 68

CP

1 40 2 52 50 4 8Total 90 6 7


increases [45], it is possible that higher concentrations of CP in chloroplasts were

detrimental or perhaps lethal to the plant cell. CP+ plants could be obtained at high

kanamycin concentrations and these did not develop any symptoms and remained

normal.

F. 2. White mosaic symptom development on TP-CP+ transformant which were selected on50 mg l−" kanamycin. R1 seedlings from the white mosaic TP-CP+ transformant TP-19.

Expression of TP-CP and PVY-CP transcripts

We were unable to maintain the most severely affected TP-CP+ plants. However,

northern blot analysis (Fig. 3) showed that chlorotic (Fig. 3, lane 10), pale green TP-

CP+ (Fig. 3, lanes 1–4 and 8), and green CP+ plants (Fig. 3, lanes 5 and 6) expressed

TP-CP and PVY-CP transcripts corresponding to their expected sizes (PVY-CPE1±6 kb and TP-CPE 1±8 kb, respectively). In order to show that the RNA was being

translated in the plants, protein extracts were analysed using anti-PVY serum (Fig. 4).

Several polypeptides were recognized by the antiserum that had mobilities

corresponding to unprocessed TP-CP fusion protein (PVY-CPE 30 kDaTPE6 kDa), mature PVY-CP, and smaller peptides (Fig. 4, lane 6). The smaller peptides

were probably break-down products of PVY-CP. Since rbcs TP-mediated protein

import and processing into chloroplasts is normally very efficient and rapid (Fig. 4,

lanes 9–14) such that unprocessed protein is often undetected in transgenic plants

[6, 8, 14], we believe the unprocessed product, which was apparent only in samples

from chlorotic TP-CP+ plants, might be an indication of the poor physiological state

of these plantlets and their chloroplasts.

Western blot analysis demonstrated that PVY-CP was synthesized in CP+ transgenic

plants (Fig. 4, lanes 3–5). While none of the CP+ plants analysed accumulated as much


1 11102 3 4 5 6 7 8 9

TP–CP+ TP–CP+CP+

1.8

1.6

kb

F. 3. Presence of PVY-CP and TP-CP transcripts in transgenic plants. Total RNA wasisolated from independent transgenic tobacco plants, and 10 µg RNA}lane was analysed bynorthern blot analysis using a $#P-labeled CP cDNA as probe. Lanes 1–4 are from pale green TP-CP+ plants TP-19, -45, -26, and -55 respectively; lane 8 is from pale green TP-CP+ transformantTP-79; lanes 7 and 9, green kanamycin resistant plants which were not expressing TP-CPtranscript ; lane 10, chlorotic TP-CP+ transformant TP-18; lanes 5 and 6 are from CP+

transformant CP-20 and -16; lane 11, healthy untransformed plant. The numbers on the rightare positions of molecular markers. kb, kilobase.

1 102 3 4 5 6 7 8 9

CP+ TPCP+

36

30

kDa11 12 13 14

Chlorotic

F. 4. Processing of TP-CP fusion protein into CP in TP-CP+ plants. Total soluble proteinswere extracted from independent transformants, and extracts containing 12 µg protein}lane(lanes 2–14) subjected to SDS PAGE and western blot analysis, using anti-PVY IgG as probe.Lane 1, 1±0 µg purified PVY! ; lane 2, untransformed non-infected plant ; lanes 3–5, are fromCP+ transgenic plants CP-16, -42, and -20, respectively; lane 6, chlorotic TP-CP+ transformantTP-18; lanes 7–14, pale green TP-CP+ plants. Lanes 9–14 are from transformant TP-45, -55,-26, -37, -68, and -19, respectively. The numbers on the right show positions of molecular weightstandards in kDa.

protein as the highest expressing TP-CP+ plants, ranges in expression of this transgenic

series overlapped the range of the TP-CP+ series. As shown in Fig. 4, lanes 3, 4 and 5,

CP+ plants had similar amounts of CP as TP-CP+ plants analysed in lanes 8, 9, 12 and

13.


F. 5. For legend see opposite.


Effects on chloroplast structure and de�elopment

Cell morphology did not change in CP+ plants (data not shown), but light microscopy

studies indicated that chloroplast-directed PVY-CP affected cell and chloroplast

development (Fig. 5). Figure 5 represents typical cellular changes which were observed

in different TP-CP+ plants with mosaic symptoms. Cells were enlarged, cell shape

altered, and the number and size of chloroplasts decreased in chlorotic TP-CP+ plants

(Fig. 5B). TP-CP+ plant TP-19 was selected to show the differences between adjacent

green and white areas in plants showing symptoms. Cell enlargement, cell shape

alteration, and a decrease in number and size of chloroplasts were more pronounced

in white areas of TP-CP+ transformant TP-19 with white mosaic symptoms than in

nearby green areas (Fig. 5C). More chloroplasts could be seen, for example, as the field

of view moved away from white (centre) areas towards green (left) areas (Fig. 5C).

Control tobacco plants had 16±2 chloroplasts per palisade parenchyma cell, while TP-

CP+ plant TP-19, as an example, had 5±2 chloroplasts per palisade parenchyma cell.

In equivalent areas of tissue sections, TP-CP+ plants had 38% fewer cells.

Since we observed chlorosis (Fig. 2), and light microscopy indicated that cell size and

chloroplast number had decreased in TP-CP+ plants, we used TEM to investigate the

ultrastructure of TP-CP+ plants. Fig. 6 shows a TEM analysis of TP-CP+ transformants

TP-18 (Fig. 6C) and TP-19 (Fig. 6B). Thylakoid membrane stacking was abnormal

and the number of starch granules per chloroplast reduced in the transformants (Fig.

6B, C) in comparison with untransformed plants (Fig. 6A). Expression of TP-CP

resulted in the formation of filamentous or rod shaped structures in some chloroplasts

(Fig. 8A and C). The chloroplasts in Fig. 6B were typical for those observed in the

border area between green (left) and white (right) sections in white mosaic TP-CP+

transformant TP-19. Thylakoid membrane stacking appeared abnormal in chloroplasts

from green areas of TP-CP+ transformant TP-19 with white mosaic symptoms (data

not shown). The ultrastructure of chloroplasts from PVY-infected plants was also

changed (Fig. 7A and B). In contrast to TP-CP+ plants, there were no apparent

ultrastructural alterations in chloroplast structure of CP+ plants (data not shown).

Localization of PVY-CP in abnormal chloroplasts from TP-CP+ transformants

The presence and subcellular localization of CP in chloroplasts of TP-CP+ plants was

determined by immunocytochemistry and western blot analysis using anti-PVY IgG

(Fig. 8B–D). TEM immunocytochemistry of chlorotic transformant TP-18, and white

mosaic transformant TP-19 indicated that PVY-CP was localized in abnormal

chloroplasts (Fig. 8B and C). PVY-CP was localized mainly in filamentous structures

(Fig. 8C). No labelling was observed in normal chloroplasts from green areas of white

mosaic TP-CP+ plants (data not shown), indicating that chloroplast abnormalities

were correlated with the presence of CP inside these organelles. No labelling was

observed when sections from chlorotic tissue were treated with preimmune serum (Fig.

F. 5. Abnormal cell and chloroplast development associated with chloroplast-directed PVY-CP. (A) Leaf cross section from an untransformed non-infected tobacco. (B) Leaf cross-sectionfrom chlorotic TP-CP+ transformant TP-18. (C) Leaf cross-section from white mosaic area of aTP-CP+ transformant TP-19. Bar¯ 40 µm. UE, upper epidermis ; P, palisade parenchyma; S,spongy mesophyll. ; C, chloroplast ; N, nucleus ; LE, lower epidermis ; T, trichome.


F. 6. Transmission electron micrograph of the chloroplasts from TP-CP+ plants showingabnormal chloroplast development. (A) Mature chloroplast from an untransformed, non-infected, tobacco cell. Grana thylakoids (G) are present as well as starch grains (S) and the stromais uniformly dense. (B) A chloroplast from white mosaic TP-CP+ transformant TP-19. (C) Achloroplast from chlorotic TP-CP+ transformant TP-18. Bar¯ 0±5 µm. S, starch granule ; G,grana; L, lipid bodies ; B, electron translucent bodies ; F, filamentous structure ; T, thylakoidmembrane; C, chloroplast.

8A) or when leaf sections from untransformed healthy tobacco were treated with anti-

PVY IgG (data not shown). Total numbers of gold particles were counted in cells from

five different sections that had been treated immunocytochemically. About 94% of all


F. 7. Transmission electron micrograph of chloroplasts showing effects of PVY! infection ontobacco chloroplasts. (A, B) Chloroplasts from a PVY!-infected tobacco with severe symptoms(Fig. 2D). Bar¯ 0±5 µm. C, chloroplast ; M, mitochondria; I, cytoplasmic inclusion body; L,lipid bodies.

gold particles were observed in association with abnormal chloroplasts of TP-CP+

plants, of which about 72% were associated with filamentous structures. No gold

particles were observed in sections of TP-CP+ plants that had been treated with pre-

immune rabbit IgG.

Association of PVY-CP with thylakoid membranes in TP-CP+ transformants

Immunolocalization tests indicated that CP in TP-CP+ transformant TP-19

preferentially associated with filamentous structures. Note that the rbcs transit peptide

only targets proteins to the chloroplast stroma. To further investigate the CP-thylakoid

association, stroma and thylakoid membrane protein fractions from isolated intact

chloroplasts were analysed by western blotting. This analysis indicated that most of the

PVY-CP from TP-CP+ transformant TP-19 was present in the thylakoid fraction (Fig.

8D, lanes 1 and 3), and little signal was observed from the stromal fraction (Fig. 8D,

lanes 2 and 4). No signal was detected in a sample of total proteins from isolated intact

chloroplasts of CP+ plants (Fig. 8D, lane 5).


1 2 3 4 5

30kDa

F. 8. Immunolocalization of PVY-CP in sections of TP-CP+ plants, showing specificlocalization of CP to the thylakoid membranes. (A) Preimmune serum control. Chloroplasts fromthe white region of TP-CP+ transformant TP-19. (B) Immunolocalization of CP in sections froma chlorotic TP-CP+ transformant TP-18. (C) Immunolocalization of CP in a section from achloroplast from a white region of TP-CP+ transformant TP-19 showing immunogold labelling offilamentous structures. (D) Western blot analysis of plastid proteins from transformant TP-19.Lanes 1 and 3 are thylakoid fraction proteins, lanes 2 and 4 are stroma fraction proteins and lane5 total chloroplast proteins from CP+ tobacco plant CP-20. Bar¯ 0±5 µm. C, chloroplast ; F,filamentous structures ; M, mitochondria. Numbers on the left side show migration positions ofmolecular weight standards in kDa.

Effect of �irus challenge on TP-CP+ plants

Ten plants each of 10 transgenic lines were challenged with PVY at the four leaf stage,

as was non-transformed Xanthi tobacco. ELISA assays were done, with samples taken

over time, using antiserum produced against HC-Pro, since some TP-CP+ lines

produced large quantities of CP and thus made a standard ELISA unusable. The

CP+ lines were used as transgenic control plants and, as we had determined previously

(data not shown) were kan+ but showed little or no resistance to PVY. A range of TP-

CP+ plants were chosen for testing, on the basis of CP expression levels. TP-CP+ lines


with the highest level of CP expression had the greatest susceptibility to PVY (Fig. 9).

TP-CP+ transformants TP-19, -26, and -55 (high expressive lines) show visible

symptoms by 4 days post-inoculation. However, untransformed plants and CP+

transformant CP-16 and -20 show visible symptoms by 10 days post-inoculation. The

medium (TP-45, -68, and -79) and low expressive (TP-37) TP-CP+ transformants

show visible symptoms by 8 days post-inoculation. The increased susceptibility of high

expressing TP-CP+ lines was most evident at 5 and 10 days post-inoculation, and HC-

Pro levels thereafter decreased to the level of susceptible control plants.

0.4

0

Days post-inoculation

Abs

orba

nce

405

0.3

0.2

0.1

Con

trol

CP

-16

CP

-20

TP

-37

TP

-79

TP

-68

TP

-45

TP

-55

TP

-19

TP

-26

Con

trol

CP

-16

CP

-20

TP

-37

TP

-79

TP

-68

TP

-45

TP

-55

TP

-19

TP

-26

Con

trol

CP

-16

CP

-20

TP

-37

TP

-79

TP

-68

TP

-45

TP

-55

TP

-19

TP

-26

Con

trol

CP

-16

CP

-20

TP

-37

TP

-79

TP

-68

TP

-45

TP

-55

TP

-19

TP

-26

Con

trol

CP

-16

CP

-20

TP

-37

TP

-79

TP

-68

TP

-45

TP

-55

TP

-19

TP

-26

Con

trol

0 5 10 20 30

F. 9. Virus titre, as measured by HC-Pro ELISA, in CP+ and TP-CP+ progeny plants at 5,10, 20 and 30 days post-inoculation. Y axis is absorbance at 405 nm, bars are mean A

%!&, and error

bars represent³1 SD from the mean. Solid bars are control (untransformed) plants, hatched barsresults from two CP+ lines (CP-16 and -20), and white bars results from seven TP-CP+ lines. LineTP-37 expresses low level CP, lines TP-79, -68 and -45 express moderate levels of CP and linesTP-55, -19, and -26 express high levels of CP.

DISCUSSION

Transgenic expression of PVY coat protein, as a fusion with the rbcs transit peptide,

results in accumulation of PVY CP in the chloroplast compartment. Western blot

analyses (Fig. 4) indicate that CP in TP-CP+ plants is processed and accumulated in

chloroplasts of transgenic plants, including those which develop chlorotic or white

mosaic symptoms. In some plants this accumulation equals or exceeds total CP

accumulation in PVY-infected tobacco. Significantly, transgenic lines expressing high

levels of TP-CP can be adversely affected, and this effect appears to be solely due to

CP. At times this effect can be so pronounced that transformant growth is arrested.

There are other reports of foreign gene expression in transgenic tobacco using the same

transit peptide with no apparent detrimental effects [9, 10, 21, 22, 25, 42, 47], further

supporting our contention that effects we have observed are exclusively due to CP

accumulating in the chloroplast. It would be most interesting to target other potyvirus


proteins to the chloroplasts and determine if similar effects are observed, particularly

HC-Pro, as we know this gene product is present in infected chloroplasts [15].

Detrimental effects due to TP-CP expression can be observed at the macroscopic and

microscopic levels. While the symptoms of PVY are not faithfully reproduced in

abnormal cases, the symptoms seen are reminiscent of virus infection. Light microscopy

(Fig. 5) and electron microscopy (Fig. 6) reveals that chloroplast targeted PVY-CP

disrupts chloroplast development and structure in cells. It also reduces chloroplast

numbers per cell, and these cells appear less differentiated. Mesophyll cells in yellow

areas of infected leaves with mosaic symptoms are commonly less differentiated with

fewer chloroplasts and fewer or no intracellular spaces [28].

Western analysis of fractionated chloroplast proteins from TP-CP plants indicates

that chloroplastic CP is associated with the thylakoid membrane fraction. This

observation is consistent with our previous observation that chloroplast-associated CP

from infected plants is almost exclusively associated with the thylakoid membrane

system, a situation very similar to what has been reported for tobacco mosaic virus

(TMV) [34], and consistent with our previous observations on PVY-infected tobacco

[15]. Our observation of abnormal thylakoids and lack of chlorophyll (chlorosis—Fig.

2) suggests that their function is also probably impaired, which is consistent with

previous reports on TMV [17, 35] and a potyvirus that photosystem II is adversely

affected after virus infection [29, 30]. Since CP also associates with the thylakoids in the

absence of any other virus-encoded proteins, there must be an inherent property of CP

that directs this protein to the thylakoids, possibly in association with chloroplast

protein(s).

The presence of CP in chloroplasts (Fig. 8D) and the appearance of filamentous

structures, as observed by TEM (Fig. 8A and C), suggests that these may be aggregates

of CP, and thus with sufficient quantities of CP these molecules self-assemble or

aggregate spontaneously inside chloroplasts. Additionally, the approximate diameter

of these filamentous structures, as determined by comparison with the 10 nm gold

particles, is within the range of the diameter of PVY virions [20, 28]. Considering the

high level of CP expression in some TP-CP+ transformants and the fact that CP is

concentrated within the chloroplasts, the appearance of these potyvirus-like rods is not

surprising. Whether these particles have encapsidated any chloroplast RNA is

unknown, but assembly of potyvirus CP molecules into virus-like particles in the

absence of the viral RNA has been reported by Jagadish et al. [20].

TP-CP+ lines with the highest expression levels of TP-CP are more susceptible to

PVY. This observation indirectly supports the hypothesis that CP is toxic to the

chloroplast and CP that is already present in the organelle, after import of TP-CP, is

additive in terms of disease.

Our data indicate that PVY-CP action in host chloroplasts can affect this organelle

and even induce symptoms suggestive of virus infection, but it is unlikely that this is the

sole explanation for the symptoms we see in potyvirus-infected plants. For example,

Lindbeck et al. [24] showed that there is a correlation between the production of

mutated TMV coat protein and chloroplast degradation. Very little of this mutated

CP was detected in the chloroplasts of infected cells by immunolocalization, and the CP

was mainly localized to discrete-staining coat protein bodies in the cytoplasm. They

suggested that the mutated CP exerted its influence on chloroplasts from the cytoplasm


instead of from inside of the chloroplasts. Recently, Masuta et al. [26] reported that

chlorosis induced by CMV Y satellite RNA (Y-satRNA) is attributed to a massive

reduction in chlorophyll content. The chlorosis induction by Y-satRNA was apparently

due to specific inhibition of chlorophyll accumulation rather than to chloroplast

degradation since chloroplast ultrastructure was not adversely affected. In the case of

tobacco vein mottling potyvirus (TVMV), the 3«-untranslated region of the genome

can have a direct effect on symptom severity [36].

This work could also have implications for the use of coat protein-mediated

resistance for crop protection. In lieu of a known mechanism for potyvirus CP import

into chloroplasts, if CP somehow enters chloroplasts of transgenic plants expressing

viral CP in the cytoplasm, one could speculate that the transgenic plants could lose

their acquired CP-mediated resistance. If enough CP enters host cell chloroplasts, there

could also be deleterious effects on photosynthesis. Of course, the quantity of CP

expressed in most transgenic plants is considerably less than we have seen with some

TP-CP+ plants, but lower levels of CP in chloroplasts could presumably have subtle

effects on plant vigour. Many groups generate transgenic plants which express viral CP

but do not show CP-mediated-resistance. Perhaps this could be due, in part, to import

of CP into chloroplasts of transgenic plants which is then manifested by susceptibility

of transformants or lack of resistance.

There are many aspects of PVY-host chloroplast interactions which remain

unresolved. Our work shows that the presence of PVY-CP inside chloroplasts induces

disease-like symptoms. Our data suggest that CP contains a thylakoid targeting

mechanism, but do not indicate how CP is imported into chloroplasts in infected

plants. Nor do we have any indication as to whether or not virus RNA translation

occurs in organello but we do know that PVY RNA is present in chloroplasts of infected

plants [15]. Further experimentation should address these questions as to how specific

chloroplast biochemical functions are affected by CP action inside the organelle. As

mentioned above, we have also observed the HC-pro protein in chloroplasts [15], but

have no information as to whether this gene product (or perhaps others from the PVY

genome) affects chloroplast function. The present study shows, nonetheless, that PVY-

CP is toxic to chloroplasts, at least under the conditions of our experiments, and may

be a factor in symptom development in infected plants.

We wish to thank A. Caplan for helpful suggestions, F. Bailey for assistance with

electron microscopy, P. J. Shiel for help with tissue culture media, S. Chowrira and

V. Francheschi for assistance with electron microscopy immunolocalization, and A.

Caplan, S. Wyatt, and M. Wiese for critical reading of the manuscript. This is Idaho

Agricultural Experiment Station publication No. 94728.

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Effects of chloroplast targeted potato virus Y coat protein on transgenic plants