Cucumber Mosaic Virus D Satellite RNA–Induced Programmed Cell Death in Tomato
Ping Xu and Marilyn J. Roossinck
1
Plant Biology Division, Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, Oklahoma 73402
D satellite RNA (satRNA) with its helper virus, namely, cucumber mosaic virus, causes systemic necrosis in tomato. Theinfected plant exhibits a distinct spatial and temporal cell death pattern. The distinct features of chromatin condensa-tion and nuclear DNA fragmentation indicate that programmed cell death is involved. In addition, satRNA localizationand terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling show that cell death is initiated from the in-fected phloem or cambium cells and spreads to other nearby infected cells. Timing of the onset of necrosis after inoc-ulation implicates the involvement of cell developmental processes in initiating tomato cell death. Analysis of theaccumulation of minus- and plus-strand satRNAs in the infected plants indicates a correlation between high amountsof minus-strand satRNA and tomato cell death.
INTRODUCTION
Cucumber mosaic virus (CMV) is an isometric plant virus witha tripartite plus-sense RNA genome (Palukaitis et al., 1992).Some strains of CMV harbor satellite RNAs (satRNAs)—small, linear molecules ranging from 332 to 405 nucleo-tides long. The satRNAs are dependent on CMV for theirreplication, encapsidation, and dispersion, but they are notnecessary for the life cycle of the virus. They can attenuateor exacerbate the symptoms induced by the helper virusesin specific plant hosts. For example, D-satRNA, B-satRNA,and WL1-satRNA induce necrosis, chlorosis, and attenua-tion, respectively, in infected tomato plants in the presenceof any CMV helper virus. In tobacco, however, D-satRNAand WL1-satRNA attenuate symptoms, whereas minor nu-cleotide sequence variants of the B-satRNA either attenu-ate symptoms or induce chlorosis in a helper virus–specific manner (reviewed in García-Arenal and Palukaitis,1999).
D-satRNA (335 nucleotides long) induces a lethal sys-temic necrosis in tomato that has been reported as epi-demic in France, Italy, and Spain (Kaper and Waterworth,1977; Jordá et al., 1992). The molecular and cellular mech-anisms of the disease, which can cause a catastrophic re-duction in tomato production, remain unknown, althoughthe sequences in D-satRNA responsible for the lethal ne-crosis have been determined (Sleat and Palukaitis, 1990;
Sleat et al., 1994). By using in vitro and in vivo analyses ofits secondary structure, researchers have correlated a helix
and a tetraloop region near the 3
9
end of the satRNA withthe necrotic syndrome (Bernal and García-Arenal, 1997;Rodríguez-Alvarado and Roossinck, 1997). For some spe-cific CMV and satRNA combinations, RNA 2 of CMV hasbeen shown to be involved in determining pathogenicity(Sleat et al., 1994). Recently, however, the minus-strandD-satRNA expressed from a potato virus X vector wasshown to induce similar necrosis in tomato, thereby exclud-ing the specific role of CMV in the pathogenicity of D-satRNA,except for its function as a helper virus in the host plants(Taliansky et al., 1998).
The conspicuous symptom of tomato plants infected byD-satRNA and CMV is cell death, the cause of the lethal sys-temic necrosis (Kaper and Waterworth, 1977). In recentyears, plant cell death has been studied extensively (Danglet al., 1996; Jones and Dangl, 1996; Greenberg, 1997;Pennell and Lamb, 1997). Programmed cell death (PCD) wasfirst defined on the basis of the predictable and distin-guished cell morphology in animal cells (Kerr et al., 1972).Some kinds of plant cell death share features that are similarto animal PCD or apoptosis, such as the cell death duringcell differentiation of tracheary elements, somatic embryo-genesis, and leaf senescence. Similarity also exists in thecell death during the interactions between plants and patho-gens or environmental factors (Pennell and Lamb, 1997;Danon and Gallois, 1998; Yen and Yang, 1998; Gao andShowalter, 1999; Navarre and Wolpert, 1999; Stein andHansen, 1999).
PCD is an ordered cell-suicide process. It includes the
1
To whom correspondence should be addressed. E-mail [email protected]; fax 580-221-7380.
1080 The Plant Cell
condensation, shrinkage, and fragmentation of the cyto-plasm and nucleus and also the fragmentation of nuclearDNA into
z
50-kb fragments or, in some cases, into oligonu-cleosome lengths (180 bp and multiples thereof) (Ellis et al.,1991). However, not all of these events occur in every PCDsituation. The hallmark of PCD is nuclear DNA fragmenta-tion. The DNA fragments can be visualized by using the ter-minal deoxynucleotidyltransferase-mediated dUTP nick endlabeling (TUNEL) technique in cell sections. A laddered pat-tern of total DNA also can be visualized after agarose gelelectrophoresis (Gavrieli et al., 1992). When the dying cellshows condensation of the chromatin in the nucleus, break-age of the cytoplasm and nucleus into small, sealed pack-ets, and processing of the nuclear DNA at nucleosome linkersites, this process is termed apoptosis.
Animal PCD or apoptosis has very conserved cell deathexecution machinery and multiple regulatory pathways. Todate, not all aspects of plant PCD are understood. Somemorphological features of cell death in plants are similar tothe features of animal PCD. Some biochemical events arealso similar, including calcium flux, membrane exposure ofphosphatidylserine, and activation of specific cysteine pro-teases or aspartate protease (Drake et al., 1996; May et al.,1996; Wang et al., 1996b; Chen and Foolad, 1997; Solomonet al., 1999).
The hypersensitive response (HR) is the most well-studiedcell death in plants. A result of interactions of plants and in-compatible pathogens, it causes the rapid collapse of the in-fected tissue that can lead to resistance. PCD is involved inthe HR, as shown by genetic, biochemical, and cell biologi-cal studies (Dangl et al., 1996; Greenberg, 1997; Pennell andLamb, 1997). Cell death is often a feature of disease symp-toms during the susceptible interaction between plants andnecrotrophic pathogens. Usually, the cells are killed by theaction of pathogen-derived toxins or else die at a late stageafter infection. However, cell death is not well understood inmost cases. In Alternaria stem canker disease of tomatocaused by the toxin of the fungus (
Alternaria alternata
f sp
ly-copersici
), cell death in the toxin-treated protoplasts andleaflets has the morphological characteristics of apoptosis(Wang et al., 1996a). In victoria blight of oats, cell death in-duced by victorin also has some apoptotic features (Navarreand Wolpert, 1999).
Here, we studied the cell death of the lethal systemic ne-crosis of tomato induced by D4-satRNA with its helper virus,CMV. Cell death occurs at a late stage of infection, makingthis an excellent system for studying the interaction be-tween host cells and viruses. The satRNA molecule has awell-studied sequence and structure, and this lethal sys-temic necrosis has a strong host-specificity component. Thebiological characteristics of cells during cell death were ana-lyzed, and the formation of systemic necrosis with a specifictemporal and spatial cell death pattern was assessed. Thecorrelation of vascular cell development and high amountsof minus-strand satRNA with the initiation of tomato celldeath are shown.
RESULTS
D4-satRNA–Induced Systemic Necrosis
Tomato plants (cv Rutgers) were inoculated on the first trueleaves of seedlings at the three-leaf stage with RNA from theFny strain of CMV (Fny-CMV) and D4-satRNA, Fny-CMVRNA alone, or viral RNA dilution buffer. After
z
10 days, typ-ical mild mosaic symptoms appeared on the systemically in-fected leaves of Fny-CMV–inoculated tomato plants (Figure1B). However, epinasty of the leaflets and systemic necrosisoccurred in the plants infected by Fny-CMV and D4-satRNA(Figures 1C and 1D). Necrosis first occurred on the secondnode below the meristem and spread upward and down-ward along one side of the stem and the midrib of the leaflet(Figure 1D). The mock-inoculated tomato plant showed nosymptoms (Figure 1A).
Light Microscopy of Fny-CMV and D4-satRNA–Infected Tissues and Cells
The stems above the inoculated leaves were cut, sectioned,and observed by light and fluorescence microscopy, shownin Figure 2. One side of the stem in the area of the secondnode below the meristem exhibited symptoms of necrosis(Figure 2C), and the necrotic cell walls emitted yellow fluo-rescence when exposed to blue light (Figures 2D and 2F to2H). The xylem cells ordinarily emit green autofluorescence(Figures 2D and 2F to 2H), and the healthy cells emit red au-tofluorescence (Figures 2B, 2D, and 2F). The first signs ofnecrosis in stems of infected tomato plants at 9 and 10 dayspostinoculation (DPI) were observed in the prevascular cellsclose to the meristem (data not shown) or in some phloemor cambium cells along one side of the stem (Figures 2E and2F). Necrosis then spread to other nearby cells, such as theoutside cortical (Figure 2G) and inside pith (Figure 2H) cells.No necrotic cells were observed in the plants inoculatedwith Fny-CMV only (Figures 2A and 2B) or in the mock-inoc-ulated plants (data not shown).
Chromatin condensation is a typical feature of apoptosis.4
9
,6-Diamidino-2-phenylindole (DAPI) dihydrochloride, aUV-excitable dye that can specifically bind double-strandedDNA in the nucleus, was used to assess the structure of thechromatin, as shown in Figure 3. The stems from infectedand mock-inoculated tomato plants were fixed, sectioned,stained with DAPI dihydrochloride, and observed under afluorescence microscope. No abnormal structures were ap-parent in the nuclei of the mock-inoculated or Fny-CMV–infected tomato (data not shown). Within the stems of plantsinfected with Fny-CMV and D4-satRNA, necrosis occurredalong one side (Figure 3B). The nuclei in the pith cells adja-cent to the collapsed dead cells along the necrotic side ofthe stem showed condensed or marginalized chromatin(Figure 3A), whereas the nonnecrotic side of the stem dis-
SatRNA-Induced PCD in Tomato 1081
played uniform fluorescence, which is characteristic of nor-mal, noncondensed nuclear DNA (Figure 3C).
Detection of Nuclear DNA Fragmentation
Nuclear DNA fragmentation often occurs with PCD and canbe detected by the banding pattern of total DNA seen afteragarose gel electrophoresis and by TUNEL analysis used todetect free 3
9
-OH groups in DNA in situ. Total DNA was ex-tracted from prenecrotic, necrotic, and nonnecrotic tissuesof the infected tomato plants and analyzed by agarose gelelectrophoresis, with visualization by ethidium bromide,shown in Figure 4. Prenecrotic tissue was from tomatoplants infected with Fny-CMV and D4-satRNA before theonset of visual symptoms, generally at
z
8 DPI. DNA frag-ments of
z
180 and 360 bp (one or two nucleosome lengths)were observed in the total DNA extracted from prenecroticand necrotic tissues. Additional bands of
z
100, 150, and300 bp were also detected. No conspicuous degradationwas found in the nonnecrotic tissues (Figures 4A and 4B).
The TUNEL procedure was performed with serial sectionsof tomato stems; results are shown in Figure 5. Sectionsfrom Fny-CMV–infected plants were treated with DNase I asa positive control, and all the nuclei were stained using theTUNEL procedure (Figure 5B). In the experimental sections,a positive signal was found in a few cells along one side ofthe stem in the plants infected by Fny-CMV and satRNA butnot in mock- or Fny-CMV–inoculated tomato plants (Figure5). No positive signal was noted in sections from which theterminal deoxynucleotidyltransferase enzyme was omitted(data not shown).
SatRNA Concentrations and Localization in Prenecrotic and Necrotic Tissues
Cross-sections of tomato stems used for TUNEL were an-nealed with nonradioactive minus- or plus-strand-specific
probes of satRNA, shown in Figures 6 to 8. The localizationpatterns of D4-satRNA in tissues were observed by bright-field microscopy, and the TUNEL signal was observed underfluorescence optics. In prenecrotic or necrotic stems, theplus strand of satRNA was distributed in most of the cellsoutside of the vascular cylinder and in some vascular bun-dles and nearby pith cells (Figures 6A to 6C). The localiza-tion patterns of plus- and minus-strand satRNA in theprenecrotic (data not shown) and necrotic stems were thesame (Figure 7). Necrosis appeared at the side of the stemwith plus- or minus-strand satRNAs in the vascular bundlecells (Figures 6B and 7A to 7D). There was no satRNA in thevascular cells at the nonnecrotic side of the stem (Figures6C, 7E, and 7F).
Staining by using the TUNEL procedure revealed nuclearDNA fragmentation, starting from the infected vascular cellsin the stems in the area of the second node, including pri-marily the external phloem cells (Figures 8A and 8B), the in-ternal phloem cells, and a few vascular contact cells in theprenecrotic tissue (data not shown). Later, some nearby in-fected cells (vascular, pith, and cortical cells) becameTUNEL positive, as seen in the staining of necrotic tissue(Figures 8C and 8D).
Total nucleic acids were extracted from the apices in thearea of the second node below the meristem of infectedplants over a period from 3 to 11 DPI; they were analyzed byRNA gel blotting (Sambrook et al., 1989) with strand-specificprobes. The accumulation of plus-strand satRNA increasedrapidly and remained high throughout this period, whereasthe accumulation of minus-strand satRNA in this tissue wasrelatively slow until the initiation of necrosis, at which timethe minus-strand satRNA accumulated rapidly (Figure 9).
Inoculation Tests of the Necrotic Initiation Sites in Tomato Plants at Different Developmental Stages
Necrosis in tomato has a regular temporal and spatial pat-tern of cell death under the usual greenhouse conditions
Figure 1. Symptoms in Systemic Virus–Infected Tomato at 10 DPI.
(A) Mock-inoculated plant.(B) Fny-CMV–inoculated plant showing mild mosaic symptoms.(C) and (D) Fny-CMV– and D4-satRNA–inoculated plant showing systemic necrosis and leaf epinasty at the apex. (D) provides a magnified viewof the apex of the plant from (C), showing necrosis in the petiole and stem (boxed).
1082 The Plant Cell
used for plant propagation. When the first leaves of tomatoseedlings at the three-leaf stage were inoculated, cell deathusually started with the prevascular cells (or with the youngvascular cells) along one side of the stem in the area of thesecond node, indicating a role for developmental processesof the infected cell during necrosis. When the first leaves oftomato plants at different developmental stages (from two-leaf to eight-leaf stages) or when leaves at different posi-tions from five-leaf-stage tomato plants were inoculated, thetemporal and spatial patterns of necrosis shifted; nonethe-less, in all the cases tested, necrosis initiated at the stem inthe area of the second node below the meristem (Table 1).Necrosis was also observed starting from the veins or mid-ribs of inoculated leaflets, the second or third leaflets abovethe inoculated leaves, and the stems below the inoculated
leaves when the plants were inoculated at later develop-mental stages (five-leaf to eight-leaf stages).
DISCUSSION
PCD Is Involved in the D4-satRNA–Induced Systemic Necrosis in Tomato
Host cell death is one of the consequences of plant–patho-gen interactions. It can lead to either necrosis disease(Wang et al., 1996a; Navarre and Wolpert, 1999) or diseaseresistance, such as extreme resistance or systemic acquiredresistance (Greenberg et al., 1994; Mittler et al., 1995;
Figure 2. Fresh Stem Sections from the Second Node below the Meristem of Virus-Infected Tomato Plants.
Bright-field illumination ([A], [C], and [E]) shows nonnecrotic tissues (A) and brown necrosis (arrows in [C] and [E]). UV illumination ([B], [D], and[F] to [H]) shows red autofluorescence from chlorophyll ([B], [D], and [F]), green autofluorescence in the xylem cells ([D] and [F] to [H]), and yel-low autofluorescence from necrotic cells (arrows in [D] and [F] to [H]). Necrotic cells are cambium and external phloem cells ([E] and [F]), corti-cal and vascular cells (G), and pith and phloem cells (H). Plants were infected with Fny-CMV ([A] and [B]) or with Fny-CMV and D4-satRNA ([C]to [H]).(A) to (D) Whole-stem sections at 10 DPI.(E) and (F) Cambium layer and phloem cells at 9 DPI.(G) Cortical cells at 10 DPI.(H) Pith cells at 10 DPI.Bars in (A) to (D) 5 0.04 mm; bars in (E) to (H) 5 0.005 mm.
SatRNA-Induced PCD in Tomato 1083
Bendahmane et al., 1999). Disease resistance usually resultsfrom a response to incompatible pathogens. During extremeresistance, the inoculated cells commit suicide as well asdisrupt the replication of the pathogen or promote the deg-radation of viral RNA. In the HR resulting in systemic ac-quired resistance, not all of the infected cells die, and theHR type of viral resistance is thought to be a tissue-relatedphenomenon requiring cell-to-cell contact (Graca and Martin,1976). Necrosis disease is usually caused by compatiblepathogens that use dead tissue as a nutrient source. Thecells are either killed by the action of pathogen-derived tox-ins or are induced to die at a late stage of infection.
Necrosis caused by viral pathogens is unusual becausethe pathogen is a biotroph, which means the necrosis is sui-cidal for the pathogen. In no known case could the satRNAbe considered a mutualist for the virus; rather, it behaveslike a mutualist for the plant. Perhaps the satRNA originatedas a defense molecule in some unknown host plant, devel-oped the ability to be replicated by the virus, and evolvedinto a selfish RNA, parasitizing the viral machinery. Clearly,the satRNA did not evolve in a host such as tomato, where itultimately destroys both itself and its helper virus. These in-teractions most likely represent the accidental triggering ofthe cell-death pathway by the satRNA.
Whether the host cells utilize similar molecular or cellularmechanisms in processing the cell death induced by diversepathogens that have different effects on host plants is notknown. Studies show that the HR is uncoupled from the re-sistance mechanism in at least some cases, indicating thatthe host responses of cell death and resistance may use dif-ferent pathways (del Pozo and Lam, 1998; Yu et al., 1998;Bendahmane et al., 1999). PCD is involved in some cases ofboth pathogen-induced resistance and disease cell death(Greenberg et al., 1994; Ryerson and Heath, 1996; Wang et
al., 1996a, 1996b; Kosslak et al., 1997; Navarre andWolpert, 1999). Here, cell death occurring during the sys-temic necrosis induced by D4-satRNA shares cellular fea-tures similar to those of PCD. The nuclear chromatin in thecells undergoing cell death was obviously condensed. Nu-clear DNA fragmentation was detected in situ by the TUNELprocedure. The temporal and spatial pattern of the nuclearDNA fragmentation in the tissue matched the necrosis pat-tern, appearing along one side of the stem when the tomatoplants were inoculated on a single leaf. Although the com-plete typical DNA ladder of apoptosis was not observedin total DNA, the DNA fragments of
z
180 and 360 bp areconsistent with fragments being one and two nucleosomeslong. Moreover, detection of the DNA ladder may be diffi-cult for technical reasons (Groover et al., 1997; Gao andShowalter, 1999). The cells are not synchronized in intacttissues during the development of systemic necrosis. In thisstudy, only a few cells were processing nuclear DNA frag-mentation at the prenecrotic stage, whereas the later stageshowed substantial DNA degradation (Figure 4). In addition,using this system, some other endonucleases may havebeen activated with specific digestion sites that produce thesmaller 100- and 150-bp fragments. In virus-induced PCDin plants, no typical DNA ladders of apoptosis have beenfound; however, Ca
2
1
-activated DNA endonuclease activityhas been detected, and an endonuclease implicated in DNAfragmentation during the HR has been purified from tobaccoinfected with tobacco mosaic virus (Mittler and Lam, 1995,1997).
The similarities of cell morphology during pathogen-induced resistance and disease cell death suggest thatcommon mechanisms might exist in the execution of celldeath but might have multiple, distinct signaling pathways.The HR may result from a PCD pathway that is induced by
Figure 3. DAPI Staining of Cell Nuclei from Stem of Tomato Plant Infected with Fny-CMV and D4-satRNA.
Stained nuclei show condensed chromatin ([A] and [D]) or uniform chromatin ([C] and [E]).(A) and (B) Nuclei from the necrotic side of the stem tissue (the upper box shown in [B]) under bright-field microscopy.(C) Nuclei from the nonnecrotic side of the stem tissue (the lower box shown in [B]).(D) and (E) Magnified nuclei indicated by the arrows in (A) and (C).Bars in (A) and (C) 5 0.04 mm; bar in (B) 5 0.1 mm; bars in (D) and (E) 5 0.01 mm.
1084 The Plant Cell
the specific interaction between pathogen avirulence geneproducts and host resistance gene products, but the recog-nition of pathogens and the activation of the PCD pathwayare poorly understood. Alternaria tomato canker disease re-sults from the
A. a. lycopersici
toxin, which can also induceapoptosis in monkey kidney cells, but the molecular mecha-nism in tomato has not been determined (Wang et al., 1996a).The oat blight disease induced by the toxin victorin involvesthe specific cleavage of ribulose-1,5-bisphosphate carboxyl-ase/oxygenase and may interact with senescence mecha-nisms (Navarre and Wolpert, 1999). The types and numberof pathways, whether they are activated separately or havecross-talk, and how they converge to result in the similar cellmorphology and ultimately cell death are still unknown.
The generation of reactive oxygen species (ROS) hasbeen suggested as a key trigger or mediator to PCD accom-panying the HR (Jabs et al., 1996; May et al., 1996; Mittler etal., 1996; Bestwick et al., 1997; Lamb and Dixon, 1997;Desikan et al., 1998). Hydrogen peroxide also accumulatesin lettuce leaf tracheary elements (Bestwick et al., 1997) andsenescing pea leaves (Pastori and del Río, 1997). Thus, ROSmight be a general trigger for PCD in plants, although theymay be insufficient to induce PCD (Glazener et al., 1996).
Here, hydrogen peroxide correlated closely with the D4-satRNA–induced rapid cell death (data not shown), whichsuggests the possibility of a common pathway for hydrogenperoxide
to induce host cell death during both the incompat-ible and susceptible reactions.
The accumulation of hydrogen peroxide involves the acti-vation of a plasma membrane–associated NAD(P)H oxidase(Jabs et al., 1996) and the inhibition of catalase expressionand suppression of cytosolic ascorbate peroxidase duringthe response of plants to pathogens (Chamnongpol et al.,1996; Takahashi et al., 1997; Mittler et al., 1998). Recently,cysteine proteases were found to be activated by oxidativestress in soybean cells and inhibition of cysteine proteasescould block PCD (Solomon et al., 1999). Although special-ized cysteine proteases known as caspases are active dur-ing animal apoptosis, no caspase has yet been identified inplants; however, caspase activity has been detected in to-bacco mosaic virus–induced HR in tobacco, and caspaseinhibitors can block PCD induced by bacteria in tobacco(del Pozo and Lam, 1998).
A Large Amount of Minus-Strand satRNA Is Correlated with the Tomato Systemic Necrosis
The decisive role of minus-strand D-satRNA in inducing ne-crosis in tomato was shown by its expression from a potatovirus X vector (Taliansky et al., 1998). Here, the localizationpattern of the minus strand was the same as that of the plusstrand in the tissues that were processing cell death. ThesatRNA was seen in the prenecrotic tissues
z
1 week beforethe onset of necrosis (data not shown). Therefore, the spatialdistribution of viral RNAs is insufficient to account for thespecific cell death pattern in tomato. Taliansky et al. (1998)suggested that a certain threshold of minus-strand D-sat-RNA might be required in the syndrome in tomato. In thepresent study, RNA gel blot analysis shows a large amountof minus-strand satRNA accumulating during necrosis and achange in the ratio of minus- and plus-strand satRNAs oc-curring during the initiation of necrosis. The accumulation ofminus-strand satRNA increased rapidly during the rapidspread of cell death. The same analysis was performed forthe infected plants when the plants were inoculated at dif-ferent leaf stages. Although the temporal patterns for theappearance of systemic necrosis were shifted, the tendencyof the minus-strand satRNA accumulation was similar in allcases tested (data not shown). These results support the im-portant role of the amount of minus-strand satRNA in theformation of the specific temporal pattern of cell death, al-though double-stranded satRNA possibly is also involved inthe pathogenesis. The amounts of minus- or plus-strand sat-RNAs in nonnecrotic tissues show a timing tendency similarto that in the tissue in which PCD is initiated (data notshown). Therefore, the initiation of cell death not only in-volves the amount of minus-strand satRNA in the tissue butalso is associated with the cell type and developmental
Figure 4. Total DNA from Tomato Tissues Infected by Fny-CMV andD4-satRNA.
(A) and (B) Lanes 1 contain the 1-kb DNA ladder (Gibco); lanes 2,victorin-treated oat leaf DNA ladder marker.(A) Lane 3 contains prenecrotic tissue at 9 DPI; lane 4, nonnecrotictissue at 9 DPI.(B) Lane 3 contains nonnecrotic tissue at 10 DPI; lane 4, necrotic tissue.Arrows from top to bottom indicate 360, 300, 180, and 100 bp (A) or180, 150, and 100 bp (B), respectively.
SatRNA-Induced PCD in Tomato 1085
stage, as shown by the results from the inoculation testsand
in situ hybridization.
Developmental Regulation Is Involved in Initiation of PCD Induced by D4-satRNA
Tomato plants infected with CMV and D-satRNA were char-acterized by necrosis throughout the plants at the late stageof infection (Kaper and Waterworth, 1977). The extent of ne-crosis in the plants was influenced by environmental factors,such as temperature, and infected plants at a particular de-velopmental stage were more susceptible to cell death(Kaper et al., 1995). In this study, our inoculation test resultsand fresh tissue sections show that necrosis appeared in aspecific temporal and spatial pattern that varied dependingon the developmental stage of the inoculated leaf and thetomato plant. Necrosis started from the vascular cells in thearea of the second node below the meristem and spreadvery rapidly to the meristem, the petiole of the leaflet, andother nearby cells in the stem below the node. This develop-ment of necrosis indicates that the cells in the area of the
second node are susceptible to cell death. When the plantswere inoculated at approximately the five- to eight-leafstage, the necrosis, in addition to sharing a common initia-tion site, also started from several secondary sites, includingthe petioles of the inoculated leaflet and of the second orthird leaflet above the inoculated leaf and in the stems be-low those leaflets. Newly formed vascular cells in the sec-ondary growth of the tomato stem are produced at thosesites, and these may be in a physiologic state similar to thevascular tissue in the area of the second node.
The vascular cells in the stem became necrotic first, asobserved in the fresh tissue sections. Sections double-labeled for D4-satRNA localization and nuclear DNA frag-mentation initially were found only in the developing vascu-lar cells, principally the phloem cells.
After the death of these vascular cells, some nearby in-fected cells were triggered very quickly to PCD. The rapidspread of the cell death caused a visible brown necrosis thatdeveloped only along the side of the stem or petiole thatcontained satRNA in the vascular bundles. Hence, the spa-tial pattern of necrosis is associated with both the develop-ment of vascular cells and satRNA localization in the tissues.
Figure 5. TUNEL Assay of Longitudinal Sections from the Second Node of Infected Tomato Stems.
(A) Fny-CMV–infected tomato.(B) Fny-CMV–infected tomato treated with DNase I (positive control).(C) and (D) Tomato infected with Fny-CMV and D4-satRNA at 9 DPI (prenecrotic stage), with (D) showing a magnified view of a portion in (C). Ar-rows in (D) indicate TUNEL-positive nuclei with nuclear DNA fragmentation.The dark green fluorescence in (A) to (D) is the autofluorescence of nuclei in the normal cells. Bars in (A) to (C) 5 0.02 mm; bar in (D) 5 0.005 mm.
1086 The Plant Cell
In the tomato fruit necrosis caused by another CMV satRNA,the necrosis was thought to be caused by incomplete differ-entiation of the vascular tissue of the fruit stalk (Crescenzi etal., 1993). These results indicate that satRNA might alter thenormal vascular cell development. PCD is involved in vascu-lar cell differentiation such as xylogenesis (Fukuda, 1997).Perhaps D-satRNA interacts with the developmental regula-tion pathway in the vascular cells in the area of the secondnode below the meristem and induces PCD similar to thatinvolved in xylogenesis or senescence.
In conclusion, our study shows that in the complicated tri-lateral interaction among D-satRNA, helper virus, and to-mato, PCD is involved in the lethal systemic necrosisdisease. The infected prevascular cells close to the mer-istem and the phloem and cambium cells in the area of thesecond node below the meristem may be more susceptibleto the accumulation of minus-strand satRNA and thus mayinitiate the process of PCD, which leads to the rapid deathof the other nearby infected cells. This system provides uswith an opportunity to study both the molecular regulation ofPCD in plants and the potential role of RNA in the inductionof PCD and may ultimately lead to development of improvedplant resistance to the lethal necrosis of tomato induced bysatRNA.
METHODS
Viruses, Plants, and Plant Inoculations
Plasmid pDsat4 was described previously (Kurath and Palukaitis,1989). Plasmid pDsat4SP6 was generated by polymerase chain re-
action amplification of pDsat4 with the primers 5
9
-GGGAATTCATTT-AGGTGACACTATA
GTTTTGTTTG
-3
9
and 59-GGGGTCTAGACC-CGGGTCCTG-39 (satellite RNA [satRNA] sequences are shown inboldface). The amplified product was digested with EcoRI and XbaI(underlined in the respective primers) and cloned into the analo-gous sites in pBluescript KS1 (Stratagene, La Jolla, CA). Lineariza-tion of the resulting clone with SmaI and transcription with bacte-riophage SP6 polymerase (Ambion, Austin, TX) to generate precisesatRNA transcripts were then possible. Zucchini squash (Cucurbitapepo cv Elite) was infected by the transcripts generated in vitrofrom cDNA clones of the three viral RNAs (Roossinck et al., 1997)and pDsat4SP6. Virus was purified, and viral RNAs were extractedby the protocol previously described by Roossinck and White(1998).
Tomato (Lycopersicon esculentum cv Rutgers) seedlings used forinoculation were grown to two- to eight-leaf stages under green-house conditions. Fny–cucumber mosaic virus (CMV) RNAs or Fny-CMV and satRNAs were inoculated onto the first leaves of tomato ata total concentration of 500 mg/mL in 50 mM Na2HPO4. The controlplants were inoculated with inoculation buffer. In the inoculation test,eight seedlings were inoculated at each developmental stage, fromtwo- to eight-leaf. All of the infected plants were kept under green-house conditions.
The nonradioactive and radioactive RNA probes were generatedfrom pDsat4 and pDsat4SK. Plasmid pDsat4SK, generated by in-serting the full-length cDNA of D satRNA sequences from pDsat4(cutting with SmaI and BamHI) into the pBluescript SK1 vector,was used to generate the minus-strand probe by T7 RNA poly-merase.
Light Microscopy of Stem Sections
When the symptoms in tomato induced by Fny-CMV with satRNAfirst appeared, stems of infected and control plants were excisedand then sectioned serially by hand with a razor blade. All of the sec-tions were examined under a Nikon Microphot photomicroscope
Figure 6. Localization of Plus-Strand D4-satRNA in Stems of Infected Tomato.
(A) Prenecrotic tissue at 9 DPI.(B) and (C) Stem section at 10 DPI, showing the necrotic side (B) and the nonnecrotic side (C).Arrows indicate vascular bundles with positive signal for satRNA ([A] and [B]) and without signal (C). Bars 5 0.01 mm.
SatRNA-Induced PCD in Tomato 1087
equipped with filters specific for epifluorescence and fluoresceinisothiocyanate, and photographs were taken using Kodak 160T film(Eastman Kodak, Rochester, NY).
Isolation of Nuclear DNA for Fragmentation Analysis
Approximately 100 mg of the nonnecrotic, prenecrotic, and necrotictissues from tomato plants infected by Fny-CMV and D4-satRNA
were harvested, flash-frozen in liquid nitrogen, and ground; the pow-der was then transferred to 750 mL of extraction buffer (0.1 M Tris, 50mM EDTA, 500 mM NaCl, and 1% SDS), followed by vortex mixingand incubation at 658C for 20 min. To this was added 250 mL of 5 Mpotassium acetate; the contents of the tubes were mixed well, incu-bated on ice for 30 min, and centrifuged at 18,000g for 10 min in amicrocentrifuge. The supernatant was removed to a fresh tube. Anequal volume of isopropanol was added to the supernatant, and themixture was centrifuged at 18,000g for 5 min. The precipitates were
Figure 7. Distribution of the Plus- and Minus-Strand D4-satRNAs in Serial Sections of Necrotic Stem.
(A) to (D) Necrotic side of the stem. The same positive-staining vascular bundle indicated by the arrows in (A) and (C) is magnified in (B) and (D).(E) and (F) Nonnecrotic side of the stem. The arrows indicate a nonstaining vascular bundle.Plus strands are shown in (A), (B), and (E); minus strands are shown in (C), (D), and (F). Bars in (A), (C), (E), and (F) 5 0.02 mm; bars in (B) and(D) 5 0.0025 mm.
1088 The Plant Cell
dissolved in 100 mL of Tris-EDTA (TE) buffer (Sambrook et al., 1989)and extracted with TE buffer–saturated phenol:chloroform (1:1 [v/v]).The nucleic acid was reprecipitated with one volume of isopropanol.The total nucleic acid was dissolved in 50 mL of TE buffer containing100 mg/mL RNase A (Sigma) and incubated for 20 min. The DNA wasanalyzed on 1.5% agarose gel in Tris-acetate/EDTA electrophoresisbuffer (Sambrook et al., 1989) and visualized by staining with ethidiumbromide.
Fixation, Dehydration, and Embedding of the Tissues
Just before and after necrosis was visible in the tomato plants in-fected by Fny-CMV and D4-satRNA, the stem or meristem tissueswere cut, as were the counterparts from Fny-CMV– and mock-inoc-ulated plants. The excised tissues were placed in glass vials containinga fixative solution of 3% paraformaldehyde and 1% glutaraldehyde in0.1 M cacodylate buffer, pH 7.4. The tissues were microwaved for 3sec and allowed to set in fixative for 3 to 4 hr at room temperature.After fixation, the tissues were washed in 0.1 M cacodylate bufferthree times and dehydrated in a graded series of ethanol solutionsconsisting of 20:80, 40:60, 60:40, 80:20, 95:5, and 100:0 ethanol inwater (v/v), followed by infiltration in low-melting-point wax (Vitha et
al., 1997). The components of the wax were nine parts polyethyleneglycol distearate and one part 1-hexadecanol (Sigma). Infiltrationwas done at 378C by transferring tissues to a 1:1 (v/v) mixture of waxand ethanol for 4 hr, a 2:1 (v/v) mixture overnight, and to pure wax for8 hr. The tissues were transferred into plastic weighing boats at roomtemperature and then embedded.
Nuclear Staining and TerminalDeoxynucleotidyltransferase-MediateddUTP Nick End Labeling
The embedded tissues were sectioned with a microtome (ReichertJung, model 2050, Cambridge Instruments, Heidelberger) to be 10mm thick and transferred to slides coated with 0.1% polylysine. Theserial sections on the slides were treated with 5% acetone and 2%1-butanol three times, followed by serial solutions of concentratedethanol in 20 mM Tris base, 0.5 M NaCl, and 0.85% Tween 20, pH7.5. The slides were rinsed with H2O and PBS, treated with protein-ase K for 30 min at 378C, washed in 0.2% glycine PBS solution for 2min, rinsed with PBS and water several times, and finally air dried.
For 49,6-diamidino-2-phenylindole (DAPI) staining, the sectionswere incubated for 20 min with a 0.5-mg/mL solution of the stain in
Figure 8. In Situ Hybridization for satRNA and TUNEL Analysis in Fny-CMV– and D4-satRNA–Infected Stem Tissue.
(A) and (B) Portion of the stem cross-section containing external phloem cells at 9 DPI (prenecrotic).(C) and (D) Portion of the stem cross-section containing phloem and pith cells at 10 DPI (necrotic).The cells stained in brownish purple are satRNA-containing cells. (B) and (D) were visualized under a fluorescence field exposed in blue light. Ar-rows indicate the cells positive both for satRNA and TUNEL. Black and white arrows indicate phloem cells ([A] to [D]); yellow arrows indicatepith cells ([B] and [D]). Bars in (A) and (B) 5 0.005 mm; bars in (C) and (D) 5 0.01 mm.
SatRNA-Induced PCD in Tomato 1089
H2O, washed with H2O, and mounted with a solution of 50% glycerinin H2O. The stained nuclei emitted blue fluorescence when excitedby UV light.
An in situ cell death detection kit (Boehringer Mannheim) was usedto detect the nuclear DNA fragmentation according to the protocolprovided by the manufacturer. The air-dried sections were incubatedat 378C for 1.5 hr with the reaction mixture containing terminal deox-ynucleotidyltransferase and fluorescein-labeled dUTP and then wererinsed with PBS. Some slides were sealed by mounting solution with50% glycerine and 1% p-phenylenediamine in H2O. Some were usedfor subsequent RNA in situ hybridization. The positive signal showedthe bright green fluorescence excited by blue light. All the photo-graphs were taken with 1600T film by microscopic photography witha Nikon Microphot-FX camera.
RNA in Situ Hybridization for Detecting the Localization of Minus- and Plus-Strand satRNA in Tissues
Previously published protocols for RNA in situ hybridization (Coen etal., 1990; Ding et al., 1996) were modified and used here for detect-ing the localization of plus-strand or minus-strand satRNA. Theprobes of plus- and minus-strand satRNAs were labeled by tran-scription of pDsat4 or pDsat4SK with the inclusion of digoxygenin-UTP. The in vitro hybridization buffer contained salts (0.3 M NaCl,0.01 M Tris-HCl, pH 6.8, 0.01 M Na3PO4, and 5 mM EDTA), 50%deionized formamide, 1.25 mg/mL tRNA, Denhardt’s solution (0.002g/L each Ficoll 400, polyvinylpyrrolidone, and BSA), and 12.5% dex-tran sulfate. The probes were denatured at 808C for 5 min and werekept in 50% formamide solution. The probes were mixed with hybrid-ization buffer and loaded on the sections.
The sections were incubated at 508C overnight for hybridization,washed first with 50% formamide in 2 3 SSC (1 3 SSC is 0.15 MNaCl and 0.017 M sodium citrate) solution at 508C for 2 hr and thenwith H2O five times at room temperature, and digested with 20 mg/mL RNase A in H2O for 20 min at 378C. The sections on the slideswere washed completely with PBS, buffer 1 (0.1 M Tris-HCl, pH 7.5,and 0.15 M NaCl solution), and buffer 2 (0.5% blocking reagent [Boeh-ringer Mannheim] in buffer 1); prehybridized with buffer 3 (1% BSAand 0.3% Triton X-100 in buffer 1); and hybridized with anti–digoxi-genin–alkaline phosphatase, 1:3000 in buffer 1, for 1 hr. The slides werewashed with H2O five times, buffer 1 for 5 min, and buffer 4 (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, and 0.05 M MgCl2) for 5 min. Later, they wereincubated with 0.15 mg/mL nitroblue tetrazolium salt and 0.75 mg/mL5-bromo-4-chloro-3-indolyl phosphate in buffer 4 for 6 to 8 hr, afterwhich the sections were observed under the microscope.
Total RNA Extraction and RNA Gel Blot Analysis
One gram of the tissues in the area of the apex, including the secondnode, the inoculated leaves and stems in the area of the nodes, andthe cotyledons and the stems to which the cotyledons were attached,were harvested from mock-, Fny-CMV–, and Fny-CMV– plus D4-sat-RNA–inoculated tomato at 3, 5, 7, 9, and 11 days postinoculation(DPI). The tissues were ground in liquid nitrogen and mixed with 2 mLof extraction buffer (0.1 M NaCl, 0.01 M Tris, pH 8, 0.1 mM EDTA,and 1% SDS). The mixture was extracted with phenol:chloroform(1:1 [v/v]) twice and precipitated with 0.3 M sodium acetate andethanol. The nucleic acids were resuspended in Tris-EDTA buffer(Sambrook et al., 1989) and digested with RNase-free DNase (RQ1;
Figure 9. RNA Gel Analysis and Quantitation of Relative Amounts of Plus- and Minus-Strand D4-satRNAs at Apices of Infected Tomato Plants.
(A) and (B) Plus-strand satRNA.(C) and (D) Minus-strand satRNA.In (B) and (D), lanes 1 contain the total RNAs from mock-inoculated plants; lanes 2, those from the Fny-CMV–infected plants; and lanes 3 to 7,those from the Fny-CMV– and D4-satRNA–infected plants at 3, 5, 7, 9, and 11 DPI, respectively.
1090 The Plant Cell
Promega) and then reextracted with phenol:chloroform and reprecip-itated with ethanol. The final RNA pellets were resuspended in 150mL of H2O. Five microliters of each denatured sample (or of a 1:10 di-lution for the 11-DPI sample) was added to 15 mL of denaturingbuffer (formamide:formaldehyde [37% solution]:10 3 Mops buffer[Sambrook et al., 1989], 500:162:100 [v/v/v]), heated to 658C for 10min, and loaded onto a 1.5% agarose gel. After electrophoresis, thesamples were bidirectionally transferred to two Hybond N1 (Amer-sham) membranes. The probes were a-P32-UTP–labeled minus- orplus-strand satRNAs generated by in vitro transcription as describedabove. After hybridization, band density was determined by using aPhosphorImager (Molecular Dynamics, Sunnyvale, CA). Probes werestripped in boiling 0.1% SDS for 2 hr, and the blots were rehybridizedwith an 18S rRNA cDNA probe. The hybridization density was againmeasured using the PhosphorImager. The relative amounts of theplus and minus strands were measured as a ratio with the 18S rRNA.
ACKNOWLEDGMENTS
We thank Dr. Tom Wolpert for providing the DNA ladder from oat,Drs. Elison Blancaflor and NingHui Cheng for careful reading of themanuscript, and Cuc Ly for assistance with images. This work wassupported by the S.R. Noble Foundation.
Received January 7, 2000; accepted May 11, 2000.
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Seven- to eight-leaf stage First 15–19 Inoculated leaflet; andsecond or third leafletsabove the inoculated leaf,and surrounding stem
Five-leaf stage Third 12 Inoculated leafletFourth 10–12 Inoculated leaflet
a All plants had primary initiation sites at the stem in the area of the second node below the meristem.
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DOI 10.1105/tpc.12.7.1079 2000;12;1079-1092Plant Cell
Ping Xu and Marilyn J. RoossinckInduced Programmed Cell Death in Tomato−Cucumber Mosaic Virus D Satellite RNA
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