Nuclear changes associated with the host-parasite interaction between Fusarium solani and peas

Physiological

Plant

Pathology

(1978)

12, 63-72

Nuclear changes associated with the host-parasite interaction between Fusarium solani and peas LEE

A.

HADWIGER

and

MICHAEL

Department of Plant Pathology, Washington Pullman, Washington 99164, U.S.A. (Accepted

for publication

August

J. ADAMS Statz

University,

1977)

Structural changes were observed within the nuclei of pea cells in contact with Fusarium solani macroconidia. The strands, which constitute the major visible structures of the pea nucleus when viewed in cross-section with the scanning electron microscope, progressively thicken and aggregate within the first 6 h after inoculation. The heavily stained chromatin fibrils of pea nuclei, viewed in thin section with the transmission electron microscope, tend to coalesce within 15 to 20 min following inoculation. The fibril coalescence observed in pea cells inoculated with F. sokzni f. sp. p;i was more complete than that observed in comparable cells inoculated with F. solani f. sp. phaseoli. The sedimentation velocity of the nucleoprotein from inoculated tissue was also characteristically altered within the first 20 min following inoculation with either fungus.

INTRODUCTION

Macroconidia ofFusarium solani (Mart.) Appel & Wr. f. sp. @is-i(F. R. Jones) Snyd. & Hans., a pathogen of peas, infect both pea seedling tissue and pea pod endocarp tissue when inoculated as a suspension [4]. When pea tissues are inoculated with F. solani f. sp. phaseoli (Burk.) Snyd. & Hans., a pathogen of beans, fungal growth is inhibited and only pinpoint lesions develop. Since essentially all cells of the pea endocarp surface respond to F. solani f. sp. phaseoli, this tissue was utilized to study the cell-to-cell interactions which are associated with resistance. Although the fungus, F. solani f. sp. pisi, is considered a “compatible fungus”, the pea tissue does in fact respond to the presence of this fungus. Responses to the incompatible fungus, F. solani f. sp. phaseoli, which include (i) increased protein synthesis [a], (ii ) increased activity of phenylalanine ammonia lyase [S] and (iii) de novo pisatin synthesis, are more immediate and initially more intense [ZO] than are the corresponding responses to the compatible F. solani f. sp. pisi. Consequently the compatible fungus is able to proliferate and infect pea tissue. Inhibitors of RNA and protein synthesis can block fungal-induced responses of the pea tissue and thus enable the incompatible fungus to grow on pea tissue [7, 11, 20-j. This suggests that the resistance response of the plant is dependent on RNA and protein synthesis. It was therefore possible to utilize these inhibitors to delimit the period following inoculation when the resistance response is being initiated. If RNA synthesis is not inhibited within 4 h and protein synthesis is not inhibited within 6 h the disease resistance response occurs [II]. Various aspects of the influence of pathogenic fungi on plant nuclei have been examined. The migration of nuclei in plant cells in the vicinity of germinating plant

64

L. A. Hadwiger and M. J. Adams

pathogenic fungi [ 17, 18, 201 can be viewed in living tissue with the light microscope. Microspectrophotometric measurements of the chemical content of nuclei within Puccinia graminis t&i&infected wheat cells has indicated that within 2 days there are no major changes in DNA content [2]. However, there appears to be a decrease in histones and an increase in RNA and acidic protein content within 2 days after inoculation. Changes in the nucleus or in nucleoprotein are of interest in that they may relate to the transcriptional changes which accompany resistance responses [9]. For example, many of the compounds which induce phytoalexin synthesis are DNAspecific and are known to change the physical properties of DNA in vitro [22, 231 and of nucleoprotein in vivo [ 10, 131. Herein we describe changes in the nuclear material of the cell as determined by sucrose gradient centrifugation and electron microscopy in the early hours following the inoculation of pea endocarp tissue with compatible and incompatible F. solani. MATERIALS AND METHODS Inoculum and tissue preparation: single-spore cultures of F. solani f. sp. pi& (clone P-A) [4] and f. sp. phaseoli (clone W-8) [20] were grown in pea-pod-supplemented (10 g/l) potato-dextrose agar. Immature pea pods (2 cm long) were developed on greenhouse-grown plants. The only pods utilized were those still enclosed by the blossom so that microbial contamination was minimized. Ultrastructural analysis of inoculated tissue Pea pod tissue was inoculated by overlaying the endocarp surface of each pod half with 5 l.~lof a spore suspension of such concentration that one spore was retained per two to five surface cells of the pea pod. The pod was allowed to air dry, so that the suspended spores came to rest on the endocarp surface in the absence of free water, prior to being placed in a moist environment. The inoculated pods were incubated at 22 “C and were protected from the direct radiation of the laboratory lights. After incubation for the designated period, pods were diced by clean crosssectional cuts with a razor blade. The freshly cut pieces (N 1 mm2) from the center of the pod were allowed to drop directly into a buffered fixing solution (3.5% glutaraldehyde, 4% formaldehyde in O-2 M cacodylate, pH 7.2) prepared according to Karnovsky [14]. After 2 h at 4 “C the pieces were washed in O-2 M cacodylate buffer, post-fixed in 2% 0~0, for l-& h and washed successively in 0.2 M cacodylate buffer and distilled water. The pieces were then transferred to 15% ethanol and then to a 30% ethanolic solution containing 1% uranyl acetate for 3 h. The pieces were then dehydrated via a graded ethanol series and were then subjected to an ascending series of solutions containing propylene oxide and Spur-r’s resin [12]. After polymerization the tissue pieces were sectioned on a Porter-Blum MT-2B ultramicrotome equipped with a diamond knife. Sections were examined with a Hitachi HN- 125 E transmission electron microscope. The freeze-fracture, freeze-dry techniques for preparing tissue for examination in the scanning electron microscope were derived in part from those utilized in freezeetch preparations. Pod halves were directly immersed for 10 s in a well of liquid

Interaction

between

Fusarium

solani

and

65

peas

freon and then were transferred to liquid nitrogen. The frozen pods were subsequently fractured into 1 to 2 mm2 pieces. While still in liquid nitrogen, the samples were transferred directly to a small reservoir of liquid nitrogen in one arm of a Delmar Stumpf/Roth cryosorption pump [19] for lyophilization. While under vacuum both arms of the apparatus were continuously cooled in liquid nitrogen-containing thermos flasks until lyophilization was complete (a minimum of 3 days). The samples were removed and immediately gold coated. The fractured cross-sections of the pods and occasionally cross-sections of the germinating macroconidia on the pod were viewed in an ETEC auto-scanning electron microscope. For comparison, these pod tissues were also prepared with critical point drying techniques [5]. Cross-sectional views of the fungal hyphae grown in liquid media were obtained by collecting fungi with more than 24 h growth on a Millipore filter (0.45 pm pore size). The filter was then frozen, fractured and prepared for examination by the same process used for pod tissue. Sucrose gradient

sedimentation

of

DNA-containing

fractions

of the fea cell

DNA-containing fractions of pea cells were separated by sedimentation velocity revising the techniques of Ide et al. [13] as follows : to label cellular DNA, immature pods were partially opened while attached to greenhouse-grown plants and 10 l.tCi [sH]thymidine (0.048 pg) was administered into the endocarp-lined cavity 18 to 24 h prior to harvest. The halves of harvested pods were completely separated and the endocarp surface was treated with H,O or spore suspensions of F. solani. At the conclusion of the treatment, pod halves were washed extensively to remove surface label and spore suspensions. Uniform slices (one to six cell layers) of the endocarp surface were removed with a razor blade and immediately blended for 15 s in 1 ml of a 1o/o sucrose, 0.05 M Tris (pH 8.0) buffer. The 45 000 rev/min capacity Virtis homogenizer was operated at medium speed. Intact cells, tissue and debris were retained on a nylon organdy filter (42 threads/cm). The filtrate was overlaid directly on a solution of 3% sucrose in 0.05 M Tris (pH 8.0) buffer and was centrifuged precisely 2 min at 15 000 rev/min in a Spinco SW 50L rotor to pellet nuclei (the surface cells of the endocarp are essentially free of mature chloroplasts). The supernatant, containing the microsomal fraction and soluble compounds, was carefully removed. The pellet or “nuclear fraction” was resuspended in 0.5 ml buffer and stroked precisely 6 times with a Vitro smooth-walled dounce tissue homogenizer. This dispersed nuclear fraction, designated as the “nucleoprotein fraction”, was immediately overlaid on a 5 to 20% sucrose-based gradient with a O-2 ml 80% sucrose cushion. The 5% component of the gradient contained 5 g sucrose, 1.86 g EDTA, 5.8 g NaCl and 1 ml of 1.0 M Tris, pH 7.4 per 100 ml. The 20% component contained 20 g sucrose, 372 mg EDTA, 5.8 g NaCl and 1-Oml of 1-OM Tris, pH 7.4 per 100 ml. The nucleoprotein fraction was centrifuged 1 or 2 h at 36 000 rev/min in a Spinco SW 50L rotor. Four-drop fractions were immediately pumped through the bottom of the tube. Each fraction was resuspended in 10 ml of 5% trichloroacetic acid and was filtered with PHWP-025-00 Millipore filters (O-30 pm pore size). The filters were rinsed with 15 ml of chloroform and counted in a Triton X-100 based scintillation fluid. Counts retained on filters were greatly in excess of background counts. 5

L. A. Hadwiger

66

RESULTS

and M. J. Adams

AND DISCUSSION

The morphological changes in the freeze-fractured, freeze-dried pea cells in direct contact with spores of Fwarium solani were viewed in a scanning electron microscope (Plate 1). The cross-sectional scanning electron micrograph of the interphase nucleus in water-treated pea endocarp tissue [Plate 1 (a)] shows the reticular structure of the nucleus typicahy observed within the first 9 h after the pod is excised. Structural changes [Plate l(b), (c)] can be observed in the pea nucleus within 1 h after a spore of the “incompatible” Fusarium solani f. sp. phaseoli has contacted the pea cell. The stranded material constituting the nuclear structure appears to condense and there is a gradual disappearance or diminished resolution of the nuclear membrane. These subtle changes are often accompanied by an increased prominence of the cytoplasmic network. The structure of nuclei in non-inoculated pea endocarp cells is variable. Although most of the cells are in interphase, various stages of mitosis are observed. Occasionally some cells in control tissue have nuclei which resemble the condensed state of the nucleus seen in Plate 1(b) ; however, the fungal-induced condensation and distortion were consistently observed in all cells adjacent to fungal spores. Within 6 h the structure of the nucleus becomes severely distorted and strands which make up the nucleus aggregate into thicker units [Plate 1(d)]. Th is nuclear reorganization is prevalent in all the surface cell layers of endocarp tissue adjacent to the inoculum. The contact between the F. solani spores and the host cell appears to be adhesive. In some cross-sectional fractures the upper portion of a spore will break away leaving a portion of the spore in contact with the host, in preference to a clean break between host and parasite. Tiny hair-like strands interconnect host and fungus [Plate 1(b)]. Whether these structures involve functional interchanges between cells or are simply macromolecular debris is not clear. The hair-like structures are not visible in fixed and stained cross-sections prepared for transmission electron microscope viewing. In a fresh state most of the spores applied to a pea endocarp surface can be removed with a gentle water wash within 1 h but are securely attached within 6 h after inoculation. Therefore the tenacity of the spore to host adhesion appears to increase with time. The “compatible” pathogenic fungus, F. solani f. sp. pisi, incites an early morphological response in the pea cell [Plate 2(a)]. The reorganization of the cytoplasmic network in pea cells around the point of contact by the pathogen emphasizes the influence the pathogen can exert in the absence of visible penetration. The cytoplasmic network is possibly involved in the repositioning of the nucleus, which is readily observed in live endocarp cells infected with F. solani [20]. The “strandlike” organization of the pea cell traverses both the cytoplasm and nucleus, and suggests a potential for cell-to-cell communications. The organization of the pea nucleus is distorted within 1 h after the compatible fungal spore comes in contact with the pea cell [Plate 2(a)]. Within 6 h [Plate 2(b)] the nucleus as well as the adjoining cytoplasm is converted into a coarse net-Eke structure. Occasionally, a cross-sectional break occurred in the infecting fungal spore which enabled a comparison of the organization of its cytoplasm with the organization of fungal cytoplasm in hyphae, free from the influence of the host cell. The internal structure of a fungal hypha grown in liquid culture is shown in Plate 3(a)

PLATE 1. Scanning electron micrographs showing cross-sections of freeze-fractured, freezedried pea pod cells from tissue treated with HsO or inoculated with F. solani f. sp. phaseoli. (a) A cross-section of a pea cell 1 h after tissue was treated with HsO. The cross-sectional view of the organized texture of a nucleus is typical for control tissue for up to 9 h following excision. The nucleus is surrounded by a continuous margin we have designated as the nuclear envelope. (b) A cross-section of pea tissue 1 h after being inoculated. The spore is centered over a pea nucleus with a compact texture. The arrow points to tiny hair-like strands interconnecting host and fungus. (c) The cross-section of cells below the tip of a germinating spore 1 h after inoculation shows varying degrees of condensation in pea nuclei. The nuclear membrane is no longer clearly discernible. (d) A cross-section of cells 6 h after inoculation shows the thickening or aggregation of nuclear strands which results in the development of large interstrand cavities. Resolution of the nuclear envelope is further diminished. n, nucleus; c, chloroplast; f, fungus. PLATE 2. Scanning electron micrographs showing cross-sections of freeze-fractured, freezedried pea endocarp tissue after inoculation with F. solani f. sp. pisi (compatible reaction). (a) A germinating macroconidium on pea tissue 1 h after inoculation. Note the organization of the cytoplasmic network in the vicinity of the germinating tip of a Fusarium macroconidia and the condensed state of the pea nucleus. The spore has also established an auxiliary association with the plant surface through a lateral protrusion bordered with hair-like adhesions (arrow). (b) A pea cell one cell layer below the inoculated surface of the pod tissue, 6 h after inoculation. The structure of the nucleus, nuclear membrane and cytoplasmic network is severely distorted. f, fungus; cn, cytoplasmic network; n, nucleus. PLATE 3. Scanning electron micrographs showing cross-sectional views of Fuxarium solani f. sp. phaseoli in in vitro culture and on the surface of pea endocarp tissue. (a) A Fusarium hypha in cross-section shows the organization of the cytoplasmic network in fungi cultured in liquid media. (b) The cross-sectional contents of a macroconidia 6 h after being applied to the pea endocarp. f, fungus. PLATE 4. Transmission electron micrographs of the surface cells of pea endocarp tissue 15 min following treatment with HsO or macroconidia ofF. solani f. sp. phnreoli or F. solani f. sp. pisi. (a) Nuclear (see arrow) and cytoplasmic components from normal pea cells from watertreated tissue remain organized following the treatment. (b) There is a moderate but consistent coalescence of the heavily stained chromatin fibrils (see arrow) of the pea nucleus in cells adjacent to the F. solani f. sp. phuseoli spores. The membranes of the nuclear envelope appear to separate. The organization of the pea cell organelles deteriorates. Strand-like materials appear in the vacuole of the cell. (c) A pea nucleus in a cell adjacent to a F. solani f. sp. p&i spore. Note the intense coalescence of the heavily stained portion (see arrow) of the pea nucleus, separations of the membrane in regions of the nuclear envelope, and the strand-like material in the vacuole. (d) A cross-section of the pea cell and of the adjacent fungus, F. solani f. sp. phaseoli 15 min after inoculation are shown at a lower magnification. nm, nuclear membrane; n, nucleus; f, fungus. Scales indicate 1 pm. PLATE 5. Transmission electron micrographs of nuclear organization in control pod tissue or tissue inoculated with F. solani. (a) The nuclear envelope in the surface cell of the pea endocarp 14 h after treatment with HsO remains organized. (b) The chromatin fibrils (see arrow), of a pea nucleus, from a surface cell of pea endocarp 14 h after being inoculated with F. solani f. sp. phaseoli, remains somewhat dispersed. Also the nuclear membrane deteriorates. (c, d) Higher magnifications of the disorganization of chromatin fibrils and the nuclear membrane which has occurred within 6 h in tissue inoculated with F. solani f. sp. phaseoli or F. solani f. sp. pisi are shown in (c) and (d) respectively. The inserted photo in (c) shows a cross-section of a F. solani f. sp. pisi macroconidia 4 h after inoculation. nm, nuclear membrane ; n, nucleus ; mit, mitochondria.

[facing page 661

PLATE

1

-

_ PLATE

4

PLATE

5

Interaction

between Fusarium

so/ad

and peas

67

and the internal structure of a fungal spore after it comes in contact with pea tissue is shown in Plate 3(b). The portions of germinating macroconidia which lack rigidity often appear collapsed when in contact with host tissue. These changes may be due to a reciprocal influence of the host cell on the organization of cytoplasm in the fungal cell. Plant tissue prepared with the freeze-fracture, freeze-dry technique was also compared [IO] with tissue prepared by a critical-point drying technique [5] which employs a pre-treatment with ethanol prior to desiccation. The harshness of the latter technique usually resulted in gross distortions within the cell or the complete absence of cell contents. For comparative purposes the surface cells of pea endocarp tissue were also examined in thin section with the conventional fixing and staining techniques used with transmission electron microscopy. Although treatment with heavy metal salts [,?I] such as uranyl acetate alters the nucleoprotein, the organizational features of all nuclei examined are presumably subject to common artifactual effects. The nuclei from the control tissue [Plate 4(a)] contain well-defined chromatin fibrils and the nuclear membranes remain intact. A detectable change in nuclei of the pea cells adjacent to the fungus occurs within 15 min. The heavy-staining fibrils of the pea nucleus [Plate 4(b), (d)] in cells adjacent to a F. solani f. sp. phaseoli spore tend to coalesce and regions of the nuclear membrane appear to separate. Also the general organization of pea cell organelles deteriorates and strand-like materials appear to disperse in the vacuole of the cell. A more intense coalescence is observed in the heavy-stained portions of pea nuclei in cells adjacent to F. solani f. sp. pisi [Plate 4(c)] than in non-infected cells [Plate 4(a)]. The subtle dispersion and distortion of the stained chromatin fibrils which occur 6 h or more after inoculation can be seen in Plates 5(b) to (d). With extended periods there appears to be further deterioration of the nuclear membrane, but the contrast in microfibril appearance, between nuclei from inoculated [Plates 5(b) to (d)] and control cells [Plate 5(a)], is not visibly increased and is possibly decreased. The ultrastructures of pea nuclei prepared for electron microscope examination are somewhat affected by preparative techniques. Therefore, we have also evaluated nucleoprotein alterations with sedimentation-velocity techniques. Previously, Ide [13] has demonstrated that the changes in the conformation of “nucleoprotein” can influence the migration of this nucleoprotein in gradient centrifugations. Although the fractions obtained are not pure nucleoprotein, the nuclear material has experienced a minimum of the disruption which accompanies chromatin extraction. Two major thymidine-labelled peaks are routinely obtained in sucrose gradient separations of the nucleoprotein from control pea tissue (Fig. 1). The major high velocity peak (fractions 17 to 20) from tissue treated with F. solani f. sp. phaseoli inoculum does not migrate as far as a comparable peak from control tissue (fractions 8 to 15) which suggests that the nucleoprotein components in the former have become more dispersed. The high velocity peak (fractions 21 to 28) containing nucleoprotein from F. solani f. sp. pisi challenged pea tissue migrates less than the comparable peaks from either the F. solani f. sp. phaseoli-treated tissue or the control tissue. The high velocity nucleoprotein fraction may correspond to the heavily stained chromatin fib& seen in transmission electron micrographs (Plate 4). We have

L. A. Hadwiger

68

Fraciion

and

M. J. Adams

number

Fxo. 1. The relative sucrose-gradient migrations of [sH]thymidine labelled nucleoprotein components from pods inoculated with F. soluni. The DNA of each pea pod was prelabelled 16 h with 10 @i [sH]thymidine prior to pod excision. Excised pod halves were treated with water (0) or inoculated with F. solanif. sp. fiha.xoli (A) or F. solani f. sp. pin (B) for 25 min. The labelled nucleoprotein fraction (see Materials and Methods) was separated in a 5 to 20% sucrose-gradient by centrifugation at 36 000 rev/min in a Spinco SW5OL rotor for 1 h.

previously [II] examined pea nucleoprotein spreads from fractions obtained in the sucrose gradient centrifugations and found that fractions from the low velocity peak indeed contain the more dispersed chromatin while the high velocity peaks possess larger aggregates. The slower and more dispersed fractions, however, appear to be the more important in relation to transcription of RNA [16], therefore the slower peak was subjected to extended centrifugation periods to obtain additional fractionation. The fractionation of this peak by extended centrifugation is shown in Fig. 2. The high velocity peak has been forced to the bottom of the tube and the major low velocity peak has separated into multiple peaks. A portion of the uridine-labelled components of the cell migrate in the sucrose gradient

with

the nucleoprotein,

which

suggests

that

a residue

of the newly

trans-

cribed RNA is attached. Presumably this is the nascent RNA which is associated with the chromatin [3], since treatment with DNase or RNase prior to centrifugation allows nearly all of the incorporated rH] uridine to remain near the meniscus fraction. When the sedimentation velocity fractionation of nucleoprotein from cells prelabelled with [sH]thymidine and then inoculated with F. solani f. sp. phmeoli is compared with the fractionations of nucleoprotein from control and inoculated tissue pulse-labelled with [sH]uridine, most of the residual uridine-labelled RNA is found associated with the low velocity nucleoprotein fraction (Fig. 3). Co-fractionation of nascent RNA with chromatin fractions has been used as one criterion in establishing that the chromatin fraction is template active [3]. The proportion of this residual [sH]uridine-labelled material is consistently higher in the low-velocity nucleoprotein (fractions 26 to 28 in Fig. 3) from F. solani f. sp. phaseoli infected tissue than in the

Interaction

between

Fusarium

solani

and

69

peas

I

r L I 5

1 IO Fraclion

I I5

I

20

number

FIG. 2. An extended sedimentation and separation of components of the major, slowsedimenting peak presented in Fig. 1. The DNA of each pea pod was prelabelled 18 h witi 10 yCi PH’Jthymidine prior to pod excision. Excised pod halves were treated with H,O (0) or inoculated with F. solani f. sp. pllaseoli (A) or F. sohi f. sp. pisi (m for 30 min. The PHIthymidine-labelled nucleoprotein components from pea pod halves were overlaid on a 5 to 20% sucrose gradient (see Materials and Methods) and were centrifuged for 2 h at 36 000 rev/min in a SWSOL Spinco rotor.

comparable

peak in the control

tissue.

The percentage

of [3H]uridine

incorporated

into the nucleoprotein from F. solani f. sp. pisi treated tissue is usually comparable to that seen in the water control. The nucleoprotein fractions were also subjected to the DNA-specific dye, actinomycin D, as an additional means of examining their structural properties (Fig. 4). When [3H] act’momycin D was applied to the homogenate containing nucleoprotein for 30 min prior to density gradient centrifiugation, the radioactivity accumulated in fractions of low velocity. This result suggests that the very low velocity fractions contain DNA which is quite accessible to DNA-specific compounds. Except for migration bound by inoculated

differences and control

in minor dye-bound tissue was similar.

peaks the proportions

of dye

CONCLUSIONS

Changes in the texture of nuclei from tissue inoculated with F. solani are detectable in electron micrographs. These changes in the visible structures of the nucleus presumably reflect changes which occur in the condition of the constituent nucleoprotein. Changes in the nucleoprotein extracted from inoculated tissue are readily evident in sedimentation velocity studies.

70

L. A. Hadwiger

Fraction

and

M. J. Adams

number

FIG. 3. Relative migration and RNA synthesis associated with DNA containing fractions of pea cells in tissues inoculated with F. solaai f. sp. phaseoli. Two pod halves which each absorbed 20 t&i [*Hjuridine in 10 mm were treated with 5 ~1 of HsO (0) or inoculated with F. sobi f. sp. phaseoli macroconidia (A) for the remainder of a 4 h pulse label period. An additional reterence pod (H) was prelabelled with 10 pCi ~H]thymidine for 17 h and was then excised and inoculated with F. solani f. sp. pheoli for 3 h and 50 min. The nucleoprotein fractions of the endocarp tissue were separated in a 5 to 20% sucrose gradient (see Materials and Methods) following centrifugation for 2 h at 36 000 rev/mm in a Spinco SW5OL rotor. The percentage of precursor incorporated into recovered nucleic acid was based on the total isotope present in the initial extraction medium.

Fungal pathogens release both compounds which can elicit specific host responses [S, 15J and enzymes which can hydrolyze macromolecules in plant cells [24]. Therefore the effect of the pathogen on the nucleoprotein could be a specific regulatory effect or one which occurs by way of indirect effects such as membrane degeneration, deterioration of the cellular organelles, or selective hydrolysis of the nucleic acid or protein portions of the nucleoprotein. The effects on the nuclear material of the cell evaluated in this paper occur within the period following inoculation when RNA and protein synthesis are required for the disease-resistance response. The newly labelled RNA in both inoculated and control tissue is associated with the dispersed nucleoprotein which slowly sediments. Since the transcription of dispersed chromatin is known to be superior to that of condensed chromatin [16], we propose that the change in nucleoprotein of the infected pea cell is related to the initiation of the RNA transcriptional events associated with the disease-resistance response. Since no genetic function has been associated with the nucleoprotein fractions, our techniques do not distinguish cause from effect. That is, has the template activity of the dispersed chromatin

Interaction

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Fusarium

sohni

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71

peas

Fraction

number

FIG. 4. The relative

sedimentation velocity and dye-binding capacity of pea nucleoprotein fractions assayed with the DNA-intercalating dye, actinomycin D. [sH]actinomycin (10 &i, sp. act. = 1.2 mCi/mg) was added to the extraction media (see Materials and Methods) used to homogenize endocarp tissue from pea pods treated (35 min) with water ( l ) or tissue inoculated (35 min) with F. soluni f. sp. fh.woli (A). The [3H]actinomycin was allowed to complex with pea nucleoprotein for 30 min before the sucrose-gradient fractionation (see Materials and Methods) at 36 000 rev/min in a Spinco SW5OL for 2 h. A reference fractionation of pea nucleoprotein from [aH]thymidine-labelled endocarp tissue inoculated (35 min) with F. sokmi f. sp. phaceoli (m) was included to indicate the relative migration of the DNA-containing fractions.

become enhanced in the resistant tissue or has the template activity become enhanced because additional chromatin has become dispersed ? Nuclear and nucleoprotein changes occur within 15 to 30 min. Therefore it is reasonable to assume that fungal products are directly or indirectly responsible for these changes. Some of the microbial products which induce phytoalexin production do enter the cell directly and are localized in the nucleus [9]. However, work on animal cells has established that certain compounds which regulate cellular processes can remain outside the cell and still engineer internal changes both in cell organization or in cell regulation [I]. In either case, the host-parasite interactions which result in inhibited growth of the incompatible fungus occur in inoculated pea pod tissue prior to the visible penetration of the host tissue by the fungus. We thank A. L. Cohen for the facilities of the Electron Microscope Center and Janice Althoff and Karen Abe for their technical assistance.

72

L. A. Hadwiger

and

M. J. Adams

This work was financed in part by National Science Foundation Grant BMS 7414560 and U.S. Public Health Service Grant GM 18483. Scientific Paper No. 4789, College of Agriculture, Washington State University.

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