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Activated synthesis of poly(A)-containing messenger RNA in soybean hypocotyls inoculated with Phytophthora megasperma var. sojae
Physiological
Plant Pathology
(1977)
10, 125-138
Activated synthesis of poly(A)-containing messenger RNA in soybean hypocotyls inoculated with Phyfophthora megasperma var. sojae M. YOSHIKAWA-~,H. MASAGO Laboratory of Plant Pathology, Fanclty of Agriculture, Kyoto Pre$ectural University, Kyoto 606, Japan
and N. T. KEEN Department of Plimt Pathology, University Riverside, California 92502, U.S.A. (Acceptedfor
publication
Decembm
of California,
1976)
rH]Uridine pulse-labelled RNA isolated from Harosoy 63 soybean hypocotyls infected with incompatible and compatible races of Phytopthora megasfierma var. sojae was analyzed by poly(U)-Sepharose aihnity chromatography and by poly(U)-iilter binding assayfor polyriboadenylic acid holy(A)]-containing messenger RNA (mRNA). Poly(A)tontaining mRNA constituted about 1% of the total pulse-labelled RNA in healthy hypocotyk and the remainder of the radioactivity was distributed among heavy and light ribosomal RNA (rRNA) and soluble RNA (sRNA). Plants inoculated with an incompatible race of the fungus synthesized poly(A)-containing mRNA about 6 times more rapidly than uninoculated control plants, as early as 4 h after inoculation. A similar increase occurred in plants inoculated with a compatible race of the fungus but it was of lesser magnitude. Growth of the incompatible fungus was not inhibited until about 10 h after inoculation. Following the early increase, the rate of synthesis of poly(A)-containing mRNA declined after 6 to 8 h in the incompatible interaction and reached the same rate as in the compatible interaction after 12 h. Sucrose density gradient analysis indicated that the synthesis of certain 18 S species of poly(A)-containing mRNA increased at the early stages of both the incompatible and compatible interactions, although the rate of synthesis was again higher in the former interaction. Synthesis of rRNA and sRNA also increased early after inoculation to higher degrees in the incompatible interaction than in the compatible, but the increases did not exceed about twice the rate in uninoculated control plants.
INTRODUCTION
It remains largely unknown whether or not the initiation of resistance responses is mediated by alterations at the gene transcriptional level. Although several authors [Z, 8, 15, 21, 22, 24, 281 have shown increased ribosomal RNA (rRNA) synthesis in various host-pathogen complexes, no one has demonstrated increased synthesis of messenger RNA (mRNA) in plants inoculated with incompatible pathogen races. Tani et al. [23] demonstrated a slight increase in mRNA content in a crown rustinoculated susceptible oat cultivar, but no increase was detected in the corresponding resistant one. These reports, however, generally deal with rather late stages of infection and do not assesspossible changes in DNA transcription that may occur in advance of the establishment of incompatible or compatible interactions. t To whom 12
all correspondence
should
be directed.
126
M. Yoshikawa,
H. Masago and N. T. Keen
The general lack of information on possible alterations of mRNA synthesis during and/or preceding the expression of resistance has been due to the technical difficulty of isolating mRNA from plants. Such experiments are now possible, however, due to the discovery that most eukaryotic mRNA and heterogenous nuclear RNA (hnRNA), possibly a mRNA precursor, contain polyriboadenylic acid [poly (A)] sequences 50 to 200 nucleotides long covalently linked to the 3’-OH terminus [see 71. This important finding allows the isolation of these mRNA species by relatively simple procedures such as affinity chromatography on oligo(dT)-cellulose [I], poly(U)-Sepharose [4] or poly(U)-cellulose [19] columns, or by binding to poly(U)-filters [19] or Millipore filters [13]. Using these techniques, poly(A)containing mRNA has now been isolated from a number of higher plant species [e.g. 251 as well as from other eukaryotic organisms [see 71. We have employed these techniques to investigate the rates of synthesis of mRNA in the hypocotyls of soybean plants inoculated with incompatible or compatible races of Phytophthora megasperma var. sojae. Considerable evidence suggests that glyceollin [16] and related phytoalexins are responsible for resistance in this host-parasite system, the most salient feature being that they are synthesized several times more rapidly in incompatible soybean-P. megasperma var. sojae interactions than in compatible ones [5, 6, 9-12, Yoshikawa, unpublished data]. This indicates that the expression of resistance is an active process, presumably involving more rapid synthesis of the enzymes required for phytoalexin biosynthesis [16] and possibly also increased transcription during early stages of the interaction. In this communication we report that both incompatible and compatible interactions indeed result in increased synthesis of poly(A)-containing mRNA but that the increase is greater in the incompatible interaction. MATERIALS
AND
METHODS
Chemicals
Polyriboadenylic acid [poly (A)] and pancreatic ribonuclease were obtained from Sigma Chemical Co. Polyribouridylic acid [poly (U)] was purchased from Boehringer Mannheim GmbH, poly(U)-Sepharose 4B from Pharmacia Fine Chem., deoxyribonuclease from Worthington Biochemical Corp. and diethylpyrocarbonate from Nakarai Chemical Ltd. [G-sH]Uridine was obtained from The Radiochemical Centre, Amersham. Plant
and fungus
culture methodr
Seeds of the soybean (Glycine max L. Merr.) cultivar Harosoy 63 (resistant to race 1 and susceptible to race 4 of Phytophthora megasperma Drechs. var. sojae A. A. Hildb.) were planted in plastic pans containing a mixture of 75% vermiculite and 25% Sumirin Yuki Derma (Sumitomo Forestry Ltd). Plants were grown in a greenhouse maintained at 25 “C. Races 1 (P900, ATCC 34 000, [14]) and 4 (kindly supplied by D. Schwenk, [1&j) were maintained on V-8 juice agar. For inoculum production, the fungi were grown for 3 to 4 days in a pea broth medium [rl] and rinsed with deionized water prior to inoculation.
Activated
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RNA
127
Seven- to 8-day-old plants were cut at the base of the hypocotyls and placed in deionized water in a beaker. A longitudinal slash wound (c. 1 cm long) was made with a razor blade on each hypocotyl, approximately 1 cm below the cotyledonary node, and a small piece of mycelium was placed into the wound. A piece of cotton wool immersed in deionized water was placed into the wounds of control plants. Plants were maintained at a relative humidity of 100% after inoculation in a growth chamber at 25 “C under fluorescent lighting (c. 2000 lx). [3H] Uridine feeding
and RNA
extraction
At various times after inoculation, the fimgal inoculum or cotton wool was removed from the hypocotyl wounds of 20 plants prior to isotope feeding to minimize contamination of the extracted RNA with fungal RNA. Ten pl of [3H]uridine solution (50 yCi/ml, 6.5 Cm/ I mmol) was placed in each wound of the hypocotyls and the feeding was continued for 2 h, during which the isotope solution was almost completely taken up by the hypocotyls. rH]Uridine labelled RNA was isolated by a modification of the procedure described by Aviv & Leder [I]. After feeding, 20 wounded portions (1 cm long, c.lg fresh weight) of the hypocotyls were harvested, quickly rinsed with deionized water and then immediately pulverized in a mortar under liquid N,. The resulting powder was suspended in 5 ml of 100 nm Tris-HCl (pH 9.0) containing 0.4 M NaCl, 1 y0 sodium lauryl sulphate (SLS), 1 mu MgCl, and O-1 ml diethylpyrocarbonate at 4 “C. The suspension was vigorously stirred after addition of 3 ml of phenol : chloroform : isoamyl alcohol (50 : 50 : 2, v/v) for 15 min at room temperature, chilled to 4 “C, and the phases were separated by centrifugation at 5000 g for 10 min. The residue after removal of the aqueous phase was re-extracted with 3 ml of the same extraction buffer, and the combined aqueous phases were re-extracted with 5 ml of the phenol mixture. The radioactivity in a 0.05 ml aliquot of the aqueous phase was determined in 10 ml of Triton X-100 scintillation mixture consisting of 2 vols of toluene scintillation mixture (5 g PPO, O-2 g POPOP per litre of toluene) and 1 vol. of Triton X-100, and referred to as the total uptake fraction; less than 5% of the total radioactivity remained in the combined phenol phases. Crude RNA was precipitated from the aqueous phase with 2.5 vols of ethanol at - 20 “C overnight. The RNA was collected by centrifugation at 12 000 g at 0 “C for 5 min. The RNA pellet was washed twice with ethanol-O-2 M NaCl (2 : l), dissolved in 1 ml of NETS buffer (10 nm Tris-HCl (pH 7*4), 100 mM NaCl, 10 mM EDTA and 0.2% SLS) unless otherwise specified, and centrifuged at 20 000 g to remove any insoluble material. The crude RNA thus extracted had Az60/A280 and Az6,,/A230 ratios in excess of 2.2 and was not significantly contaminated with DNA, based on DNase treatment followed by sucrose density gradient analysis. Isolation
of poly(A)-containing
mRNA
by poly( U)-Sphe arose column and poti(Jilter
Poly(A)-containing mRNA was isolated from the crude RNA by affinity chromatography on poly(U)-Sepharose 4B columns according to the method of Eiden & Nichols [4] with slight modifications. The crude RNA solution (0.8 ml) was mixed with 1.2 ml of NETS buffer and applied to a microcolumn containing 2 ml of a poly(U)-Sepharose slurry which had been washed with 50 ml of NETS buffer.
I28
M. Yoshikawa,
H. Masago
and
N. T. Keen
Unbound RNA was washed through the column with 6 ml of NETS buffer and fractions of 2 ml were collected. The column was then washed with 6 ml of 10 mM Tris-HCl (pH 7.4) containing O-1 M NaCl. The RNA which remained bound to the column was then eluted with 6 ml of deionized water at 50 “C in an incubator maintained at the same temperature. The radioactivity in each 2 ml fraction was determined by counting a 0.5 ml aliquot in the Triton X-100 scintillation mixture. Poly(U)-fibre glass filters (Whatman GF/C, 2.4 cm diameter) were prepared by the method described by Sheldon et al. [19] except that the filters treated with poly(U) were irradiated for 4 mm on each side at a distance of 20 cm from a 15 W National germicidal lamp. Under these conditions, about 84% of the applied poly(U) was retained per filter. One-tenth ml of the crude RNA solution was diluted in 20 vols of ice-cold binding buffer [ 10 mM Tris-HCl (pH 7*5), 0~2 M NaCl]. After 10 min in the cold, the solution was passed at about 1 ml/min through a poly(U)-filter that had been equilibrated with the same buffer. The filters were washed twice with 10 ml of the binding buffer and then dried. The filters were directly counted in 10 ml of the toluene scintillation mixture. Thus, mRNA isolated in this study refers to those mRNAs that contain poly(A) sequences. Sucrose density gradient
centrifugation
[sH]Uridine labelled, poly(A) -containing mRNA isolated by poly(U)-Sepharose chromatography [fractions 7 and 8 in Fig. 2(b)] was precipitated by addition of onetenth vol. of 20% potassium acetate, O-5 mg unlabelled carrier RNA from soybean hypocotyls, and 2.5 vols of cold ethanol. The precipitated RNA was collected by centrifugation and dissolved in 0.5 ml of NETS buffer. The RNA solutions were layered onto linear 5 to 20% sucrose gradients in the same buffer and centrifuged in an SW 25 rotor at 22 500 rev/mm for 15 h at 25 “C. Fractions of 1.5 ml were collected from the bottom of the tube and the radioactivity in 1 ml portions of each fraction was determined in the Triton X-100 scintillation mixture. Glassware
All glassware used for RNA extraction and isolation was carefully hand-washed and autoclaved, and all solutions were autoclaved unless they contained ethanol or SLS. Microsco$ic
observations
The inoculated hypocotyls were sampled at intervals after inoculation and longitudinal hand-sections were made at right angles to the inoculated surfaces. The sections were stained with Rose Bengal and the hyphal growth was determined under the microscope by measuring the distance between the penetration points and the margins of invading hyphae. At least five hypocotyls were sampled at intervals and about ten penetration points per hypocotyl were observed. RESULTS
of RNA binding to pob( U)-jilter and poly( 7J)-Sepharose column To assessthe specificity of binding, various RNAs were applied to poly(U)-filters or poly(U)-Sepharose columns and the fractions remaining bound to the filter or column were analysed. Poly(U)-filter specifically bound poly(A) but did not bind Characteristics
Activated
synthesis
of poly(A)-containing
messenger
5
IO
Input
RNA
15
20
RNA
I!?9
’
50
(pg)
FIG. 1. Binding of various RNAs to poly(U)-filters. l , poly(A); A, poly(U) ; A, unlabelled RNA extracted from soybean hypocotyls. Different amounts of each RNA in 2 ml of 10 mM Tris-HCl (pH 7.5) containing 200 mu NaCl were passed through the filters and the amounts of the bound RNA were calculated from the differences in optical density of the input solution and the filtrate. Data are means of two replicate experiments. TABLE Some chara&ri.&s
of thz binding
Treatment Control RNase DNase Poly(A) Poly(U)
(10 pg/ml)c (10 pg/ml)c (100 pg/ml)d (100 pg/ml)d
of [3H]widine
1 labelled
3H-radioactivity (ct/min) in RNA bound to poly(U)-filter” 1818 57 1838 214 1728
soybean RNA
to poly(U)-Jilters
% of [W-JRNA bound/ [3H]crude RNA appliedb 1.10 0.03 1.11 0.13 l-05
@ Data are means of two replicate analyses. b [sHJUridine labelled crude RNA (165 100 ct/min) extracted from healthy soybean hypocotyls was applied to the filter for each treatment. G The extracted crude RNA was dissolved in 10 n-m Tris-HCl (pH 7.5), divided into portions and then the buffer concentrations were brought to 10 n-m Tris-HCl (pH 7.5) + 200 mu NaCl for RNase treatment or to 10 mM Tris-HCl (pH 7.5) + 2 rnM MgCl, for DNase treatment by adding concentrated NaCl or MgCls, respectively. The treatments were performed at 30 “C for 60 min in 2 ml reaction mixtures. NaCJ concentration in the reaction mixture after DNase treatment was brought to 200 no prior to the poly(U)-filter assay. d Two ml of unlabelled poly(A) or poly(U) solution at the indicated concentration was passed through the poly(U)-filter before the [sH]uridine labelled crude RNA solution.
detectable poly(U) or unlabelled crude RNA from soybean hypocotyls (Fig. 1). When rH]uridine labelled soybean crude RNA was applied, the filter retained about 1-1o/o of the radioactivity (Table 1). The binding was almost completely abolished by pretreatment of the crude RNA solution with RNase, but DNase had no effect. Pre-washing of the filter with poly(A) but not poly(U) also reduced the radioactivity subsequently bound to the filter. Thus, it appeared that the fraction bound
130
2
4 Fraction
M. Yoshikawa,
H. Masago
6
IO
0
and
N. T. Keen
number
FIG. 2. Affinity chromatography of RNA on poly(U)-Sepharose columns. The arrows indicate the points of addition of buffer [lo mM Tris-HCl (pH 7.4), 100 nm NaCl] (1) and 50 ‘C water (2). (a) Approximately 30 pg of poly(A) (o), poly(U) (A) or 40 pg of unlabelled RNA (A) extracted from soybean hypocotyls was loaded onto three identical columns. Two ml fractions were collected for measurement of optical density, D. (b) pH]Uridine labelled RNA extracted from healthy soybean hypocotyls was chromatographed. The vertical closed bars indicate the radioactivity that was subsequently m-bound to poly(U)-filters. The re-binding assay was carried out as follows: RNA eluted in each fraction was precipitated by ethanol, dissolved in 10 mM Tris-HCI (pH 7.5) containing 200 mM NaCl and then subjected to the poly(U)-filter binding assay. The representative data obtained from three and two replicate experiments are shown in (a) and (b), respectively.
to the filter was RNA and not DNA, and that the RNA was bound to the filter through poly(A) sequences. Similarly, poly(U)-Sepharose columns retained only poly(A) [Fig. 2(a)], and the RNA fractions of C3H]uridine labelled soybean RNA bound to the column could subsequently be bound to poly(U)-filters [Fig. 2(b)]. However, almost no radioactivity was detected on the filters through which were passed RNA fractions that did not bind to the column. This low background ensured the reliability of the filter assay for detecting the low percentage (about 1%) of poly(A)-containing RNA present in the crude RNA. We henceforth will call the RNA unbound to poly(U)Sepharose columns “bulk RNA”, and the RNA bound to either the columns or poly(U)-filters “poly(A) -containing mRNA” since evidence obtained from various
Activated
synthesis
of poly(A)-containing
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RNA
131
eukaryotic cells has conclusively shown that these poly(A)-containing RNAs are indeed mRNAs [e.g. 7, 25-J. Sucrose density gradient analysis demonstrated that the profile of rH]uridine labelled bulk RNA was identical to that of the cold RNA from soybean hypocotyls; thus bulk RNA consisted of 25 and 18 S rRNA and 4 S soluble RNA (sRNA) (Fig. 3). However, poly(A)-containing mRNA isolated by poly(U)-Sepharose chromatography was heterogenous and very different from bulk RNA in its size distribution. The fraction of poly(A) -containing mRNA that sedimented near the bottom of the tube may be either hnRNA, a possible precursor of mRNA [e.g. 31, or possibly aggregated cytoplasmic mRNA [17], but further characterization of the fraction was not done. I
500
-
B II II
5
- 25
IO Fraction
15
20
number
FIG. 3. Sucrose density gradient profiles of RNA fractionated by poiy(U)-Sepharose chromatography. Fractions 1 and 2 (unbound RNA, bulk RNA) and 7 and 8 [bound RNA, poly(A)-containing mRNA] from Fig. 2(b) were pooled separately and the ethanol-precipitated RNA was dissolved and run in parallel gradients. l , bound RNA; 0, unbound RNA. The arrows indicate the position of marker unlabelled RNA from soybean hypocotyls. The profiles are from a typical experiment, representative of several replicate experiments.
Cytological
observation
Since our objective was to assessmRNA metabolism at early stages of infection before the establishment of incompatible or compatible interactions, the time of resistance expression in Harosoy 63 soybean hypocotyls against the incompatible race of P. megaspermavar. sojae was first evaluated (Fig. 4). Both the incompatible race (race 1) and the compatible race (race 4) grew in the hypocotyls at a similar rate until 8 h after inoculation; however, growth of the incompatible race was arrested withii the subsequent 2 h. Thus, in terms of inhibited fungal growth, resistance was expressed between 8 and 10 h after inoculation. No visible disease symptoms were apparent before 12 h after inoculation.
132
M.Yoshikawa,H.Masago
and
N.T.Keen
I500 -
-
4
S
12
16
20
Hours after
24
‘-
inoculation
FIG. 4. Growth of the two races of P. mega@erma var. sojae in Harosoy 63 soybean hypocotyls. Wound areas of the hypocotyls were inoculated with the incompatible race (race 1, l ) or the compatible race (race 4, o), and hyphal growth was measured after making thin sections and staining them by Rose Bengal. Points and brackets are means and standard errors, respectively, obtained from three replicate experiments. Five hypocotyls and about ten infected sites per hypocotyl were observed in each experiment.
Changes in @y(A)-containing
mRNA
synthesis after inoculation
Slightly increased synthesis of poly(A)-containing mRNA was noted as early as 2 h after the inoculation of hypocotyls with the incompatible or compatible races of P. megasperma var. sojae, and greater increases were observed at 4 h (Table 2). The TABLE
2
Incorporation of [3H’Juridine into poly (A)-contuining mR.NA of Harosoy 63 soybeanh.ypocotylsat the early stages after wounding only (Cont.) and inoculation with the incompatible race (race 1, Incomp.) or the compatibb race (race 4, Comb.) of P. megasperma var. sojae
Time after inocuiationb
3H-radioactivity
(ct/min
x lo-s)/20
hypocotyls
Poly(U)-Sepharose
affinity
chromatography
(c. 1 g fresh wt)@ Poly(U)-filter =w
(C) (A)
Inoculation
Total uptake
(B)
Bulk (rRNA+
RNAC sRNA)
2h
Cont. Incomp. Comp.
48401 44 725 46 233
3291 4262 3471
4h
Cont. Incomp. Comp.
46 142 42 113 45 649
3309 6348 4941
a b c d e
Data The The The The
% of B/A 6.8 (1.0)8 9.5 (1.4) 7.5 (1.1) 7.2 (1.0) 15.1 (2.1) 10.8 (l-5)
Poly(A)containing r&NAG
bimlmg
(“1
% of C/A
Poly(A)containing mRNA”
% of D/A
30.1 81-8 46.7
0.062 0.18 O-10
(I-O)@ (2.9) (1.6)
24-8 52.8 30.5
0.051 0.12 0.066
(1-O)' (2-4) (l-3)
34-l 177.7 94.9
0.074 0.42 O-21
(1.0) (5.7) (2.8)
32.0 164.2 86.3
0,069 040 0.19
(l-0) (5.8) (2.8)
are from a typical experiment, representative of four replicate experiments. time when [sK]uridine feeding was initiated and the isotope was fed for tbe subsequent 2 h. RNA fractions unbound (B) and bound (C) to poly(U)-Sepharose columns. RNA fraction bound to poly(U)-filters. values in parentheses indicate the ratios (inoculated/uninoculated) in o/0 of B/A, C/A or D/A.
Activated
synthesis of poly(A)-containing
messenger
133
RNA
incompatible interaction, however, resulted in greater synthesis than the correspondFurthermore, the degree of the increase in poly(A)ing compatible interaction. containing mRNA synthesis was greater than that for bulk RNA (rRNA+sRNA) synthesis. Therefore, it appeared that the incompatible interaction resulted in a rather specific activation of mRNA synthesis at relatively early stages of infection. Figure 5 indicates that the highly activated synthesis of poly(A)-containing mRNA in the incompatible interaction decreased after 6 or 8 h, and reached the same rate as that in the compatible interaction after 12 h. Table 3 indicates that the increases in the poly(A)-containing mRNA synthesis observed at early stages of the incompatible interaction mainly reflect activated
,, 4
8
12
”
I 24
Hours after inoculation
FIG. 5. Synthesis of bulk RNA and poly(A)-containing mRNA after infection of Harosoy 63 soybean hypocotyls with the incompatible race (race 1, l ) or the compatible race (race 4, 0) of P. mego$ermu var. sojae. (a) Bulk RNA unbound to poly(U)-Sepharose that consists of rRNA and sRNA. (b) Poly(A)-containing mRNA bound to poly(U)-Sepharose. (c) Poly(A)-contaming mRNA bound to poly(U)-filter. The time for initiation of rH]uridine feeding is indicated on the abscissa and the isotope was fed for 2 h. Means and standard errors are indicated where four replicate experiments were run, and other points are means of three replicate experiments. rH]Uridme incorporation was corrected for differences in total rH]uridine uptake by the hypocotyls.
M. Yoshikawa,
134
H. Masago and N. T. Keen
synthesis by the host cells and not synthesis by either the fungal inoculum or contaminating bacteria. Although P. megaspermavar. sojae can synthesize poly(A)-contaming mRNA [see Treatment 3(b) in Table 31, the increased synthesis of mRNA by inoculated hypocotyls in which the fungal inoculum was removed before or after the isotope feeding [Treatments 3(a), 4 and 5 in Table 31 occurs at a sufficiently early (4 h) time to preclude significant amounts of invading hyphae in the host (Fig. 4). Furthermore, treatment throughout the inoculation and isotope feeding procedures with a combination of antibiotics, which almost completely suppressed the growth of soil bacteria, did not reduce the observed increase in mRNA synthesis (Treatment 5 in Table 3).
TABLE
3
Host dependence of the enhanced poly(A)-containing mR.M synthesis at the ear& stage (4 h after inoculation) of the incomfiatible interaction between Harosoy 63 soybean hypocotyls and race 1 of P. megasperma var. sojae sH-radioactivity hypocotyls
Treatment8
Inoculation
Source for RNA extraction
(1)
None
Host
22;
Race
1
Host
Race
1
Host (Fungal Host Host
Race 1 Race 1 + rifampicin + ampicillin
f fungal moculum inoculum)
(4 Total uptake
(ct/min x lo-s)/ZO (c. 1 g fresh wt.)@
(B) Poly(A)containing mRNA bound to poly(U)-filter
%of B/A
Ratio in % of B/A
43 a72
26.1
O-059
1
40 654
193.5
0.48
8.1
32 628 (6786) 42 818 44 615
117.5 (34.8) 154.1 165.7
0.36
6.1
0.33 0.35
5.6 5.9
a Data are from a typical experiment, representative of three replicate experiments. b Treatment. (1) The hypocotyls received only wounding. (2) After feeding with rH]uridine, both the host and fungal inoculum together were subjected to RNA extraction. (3) After the isotope feeding, the fungal inoculum was removed from the host and RNA of the host and fungal inoculum was separately extracted. For the extraction of fungal labelled RNA, 1 g of unlabelled soybean hypocotyls was homogenized together with the labelled fungal sample. (4) This treatment is the same as that described in Materials and Methods and was employed throughout the present study, in which the fungal inoculum was removed from the host before the isotope feeding. (5) The same as Treatment (4) but mycelial suspension for inoculation and the isotope solution contained rifampicin (10 pg/ml) and ampicillin (500 pg/ml) to suppress bacterial growth.
Sucrose density gradient analysis of poly(A)-containing mRNA isolated by poly(U)-Sepharose chromatography revealed that the synthesis of certain 18 S species of mRNA was predominantly enhanced at the early stage (4 h) of the incompatible interaction, and a lesser degree of enhancement occurred in the compatibIe interaction [Fig. 6(a) and (b)]. A similar trend was observed in poly(A)-containing mRNA isolated at 2 or 6 h after inoculation.
Activated
synthesis
of poly(A)-containing
messenger
5
I5
IO Fraction
135
RNA
20
number
FIG. 6. (a) Sucrose density gradient profiles of poly(A)-containing mRNA extracted from Harosoy 63 soybean hypocotyls at 4 h after only wounding (A) and inoculation with the incompatible race (race 1, 0) or the compatible race (race 4: 0) of P. megusparma var. sojae. Poly(A)-containing mRNA was isolated by poly(U)-Sepharose chromatography and run in parallel gradients. The arrows indicate the positions of marker unlabelled RNA from soybean hypocotyls. The profiles are from a typical experiment, representative of four replicate experi(b) Ratios of inoculated/uninoculated sH-radioactivity were calculated for each fraction ments. of the gradients. Each value was obtained from Fig. 6(a). l , the incompatible host-fungus combination; 0, the compatible host-fungus combination.
DISCUSSION
The most significant finding of this study is that the rate of mRNA synthesis markedly increased as soon as 4 h after inoculation in the soybean-P. megasperma var. sojae system (Fig. 5). Thus the interaction between the host and pathogen resulted in a rapid alteration of host metabolism at the gene transcriptional level. These increases in mRNA synthesis clearly precede the expression of resistance in the incompatible reaction which occurs at about 10 h after inoculation, based on the observed inhibition of fungal growth (Fig. 4).
136
M. Yoshikawa,
H. Masago and N. T. Keen
The rates of mRNA synthesis in the early stage of the incompatible interaction were more rapid than in the corresponding stage of the compatible interaction (Fig. 5). Therefore, the two interactions appear to be distinguished by a quantitative difference in gene activation rather than by an all or none process. However, further approaches are necessary to more critically determine whether any qualitative differences exist between the mRNAs synthesized during early stages in the incompatible and compatible interactions and to clarify the role of the new mRNAs in determining ultimate rates of phytoalexin production. Our results indicating an association of increased mRNA synthesis and resistance expression are also in agreement with other investigations showing that RNA and protein synthesis are required for the expression of disease resistance [e.g. 20, 26, 27, 291. Our preliminary studies with P. megasjerma var. sojaesystem have also supported this interpretation, since actinomycin D and blasticidin S, RNA and protein synthesis inhibitors, respectively, negated both the ability of soybean plants to make phytoalexins rapidly and their resistance to the incompatible race of the fungus. It is often difficult to determine whether observed physiological changes in infected plants are due to either the metabolism of host, pathogen or contaminating organisms such as bacteria, especially where the metabolism concerned is commonly shared among the possible organisms. The following lines of evidence suggest that the increased mRNA synthesis observed during early stages of the soybean-P. mega.sjw-ma var. sojae interaction is a product of host metabolism: (a) The fungal inoculum was routinely removed before isotope feeding. (b) The contribution of mRNA by invading hyphae must be negligible since rates of mRNA synthesis were much higher at 4 to 6 h after inoculation, when the host cells were not yet extensively colonized by the fungus in both the incompatible and compatible combinations, than at 12 to 24 h, when fungal growth was extensive in the compatible combination. (c) At 4 to 6 h, furthermore, mRNA synthesis was much higher in the incompatible combination. It is unlikely that the two races of P. megasjwrma var. sojae possesssuch differential ability to synthesize mRNA. (d) Possible bacterial contribution can be eliminated by the fact that treatment with antibiotics throughout the inoculation and isotope feeding procedures did not reduce the observed enhancement in mRNA synthesis and, furthermore, significant numbers of bacteria could not be isolated from the surface sterilized-infected hypocotyls within 12 h after inoculation. Although the present study has dealt with only those mRNAs that contain poly(A) sequences, there is some eukaryotic mRNA that is not associated with poly(A) sequences. Such mRNA containing no poly(A) sequence, however, constitutes only a minor portion of total eukaryotic mRNA, and only histone mRNA [see 71 has thus far been shown to be one. Further characterization of the poly(A)-containing mRNA isolated in this study, for example, by testing its template activity in the synthesis of protein, is now under way.
We are grateful to Dr J. D. Paxton for kindly supplying soybean seeds. We also acknowledge Drs T. Tani and S. Ouchi and Mr H. Yamamoto for valuable discussion of the work.
Activated
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RNA
137
REFERENCES 1. AVIV, H. & LEDER, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proceedings of the National Academy of Sciences U.S.A. 69, 1408-1412. 2. CHAKRAVORTY, A. K. & SHAW, M. (197 1). Changes in the transcription pattern of flax cotyledons after inoculation with flax rust. Biochemical Journal 123, 551-557. 3. EDMONDS, M., VAUGHAN, M. H. JR & NAKAZATO, H. (1971). Polyadenylic acid sequences in the heterogenous nuclear RNA and rapidly-labeled polyribosomal RNA of Hela cells: possible evidence for a precursor relationship. Proceedings of the .National Academy of Sciences U.S.A. 68, 1336-1340. 4. E~DEN, J. J. & NICHOLS, J. L. (1973). Ch aracterization of poly(riboadenylic acid) segments in L-cell messenger ribonucleic acid. Biochemistry 12, 3951-3955. 5. FRANK, J. A. & PAXTON, J. D. (1970). T ime sequence for phytoalexin production in Harosoy and Harosoy 63 soybeans. Phytopathology 68, 3 15-318. 6. GRAY, G., KLARMAN, W. L. & BRIDGE, M. A. (1968). Relative quantities of antifungal metabolites produced in resistant and susceptible soybean plants inoculated with Phytophthora megasfierma var. sojae and closely related non-pathogenic fungi. Canadian Journal of Botany 46, 285-288. 7. GREENBERG, J. R. (1975). Messenger RNA metabolism of animal cells. Possible involvement of untranslated sequences and mRNA-associated proteins. Journal of Cell Biology 64,269-288. 8. Harr~~uss, R. & BAUER, M. (1969). Der Einfluss von 5-Fluorouracil auf den Nukleinsaurestoffwechsel von Phaseolus vulgaris nach Intektion mit Uromyces phaseoli. Phytopathologische
138 24. 25. 26. 27.
28. 29.
M. Yoshikawa, H. Masago and N. T. Keen TANI, T., YOSHIICAWA, M. & NAITO, N. (1975). Selective enhancement ofribosomal RNA synthesis of crown rust-infected oat leaves by stem excision. Physiological Plant Patlwlogy 5, 193-199. TOBIN, E. M. & KLEIN, A. 0. (1975). Isolation and translation of plant messenger RNA. Plant Physiology 56, 88-92.. . VANCE, C. P. & SHERWOOD, R. T. (1976). Cycloheximide treatments implicate papilla formation in resistance of reed canarygrass to fimrci. Phvtobatho1w.v 66,498~502. VON BROEMBSEN, S. L. & HA&IGER, L. As (1972) .& Cha&terization of disease resistance responses in certain gene-for-gene interactions between flax and Melampsora lini. Physiologkal Plant Pathology 2,207-215. WOLF, G. (1968). On the incorporation of 3aP into various nucleic acid fractions of rust-infected primary leaves of wheat. NetherlandsJournal of Plant Pathology 74 (Suppl. l), 19-23. ZUCKER, M. & EL-ZAYAT, M. M. (1968). The effect of cycloheximide on the resistance of potato discs to invasion by a fluorescent Pseudomonas spp. Phytopatholopy 58, 329-334.
Plant Pathology
(1977)
10, 125-138
Activated synthesis of poly(A)-containing messenger RNA in soybean hypocotyls inoculated with Phyfophthora megasperma var. sojae M. YOSHIKAWA-~,H. MASAGO Laboratory of Plant Pathology, Fanclty of Agriculture, Kyoto Pre$ectural University, Kyoto 606, Japan
and N. T. KEEN Department of Plimt Pathology, University Riverside, California 92502, U.S.A. (Acceptedfor
publication
Decembm
of California,
1976)
rH]Uridine pulse-labelled RNA isolated from Harosoy 63 soybean hypocotyls infected with incompatible and compatible races of Phytopthora megasfierma var. sojae was analyzed by poly(U)-Sepharose aihnity chromatography and by poly(U)-iilter binding assayfor polyriboadenylic acid holy(A)]-containing messenger RNA (mRNA). Poly(A)tontaining mRNA constituted about 1% of the total pulse-labelled RNA in healthy hypocotyk and the remainder of the radioactivity was distributed among heavy and light ribosomal RNA (rRNA) and soluble RNA (sRNA). Plants inoculated with an incompatible race of the fungus synthesized poly(A)-containing mRNA about 6 times more rapidly than uninoculated control plants, as early as 4 h after inoculation. A similar increase occurred in plants inoculated with a compatible race of the fungus but it was of lesser magnitude. Growth of the incompatible fungus was not inhibited until about 10 h after inoculation. Following the early increase, the rate of synthesis of poly(A)-containing mRNA declined after 6 to 8 h in the incompatible interaction and reached the same rate as in the compatible interaction after 12 h. Sucrose density gradient analysis indicated that the synthesis of certain 18 S species of poly(A)-containing mRNA increased at the early stages of both the incompatible and compatible interactions, although the rate of synthesis was again higher in the former interaction. Synthesis of rRNA and sRNA also increased early after inoculation to higher degrees in the incompatible interaction than in the compatible, but the increases did not exceed about twice the rate in uninoculated control plants.
INTRODUCTION
It remains largely unknown whether or not the initiation of resistance responses is mediated by alterations at the gene transcriptional level. Although several authors [Z, 8, 15, 21, 22, 24, 281 have shown increased ribosomal RNA (rRNA) synthesis in various host-pathogen complexes, no one has demonstrated increased synthesis of messenger RNA (mRNA) in plants inoculated with incompatible pathogen races. Tani et al. [23] demonstrated a slight increase in mRNA content in a crown rustinoculated susceptible oat cultivar, but no increase was detected in the corresponding resistant one. These reports, however, generally deal with rather late stages of infection and do not assesspossible changes in DNA transcription that may occur in advance of the establishment of incompatible or compatible interactions. t To whom 12
all correspondence
should
be directed.
126
M. Yoshikawa,
H. Masago and N. T. Keen
The general lack of information on possible alterations of mRNA synthesis during and/or preceding the expression of resistance has been due to the technical difficulty of isolating mRNA from plants. Such experiments are now possible, however, due to the discovery that most eukaryotic mRNA and heterogenous nuclear RNA (hnRNA), possibly a mRNA precursor, contain polyriboadenylic acid [poly (A)] sequences 50 to 200 nucleotides long covalently linked to the 3’-OH terminus [see 71. This important finding allows the isolation of these mRNA species by relatively simple procedures such as affinity chromatography on oligo(dT)-cellulose [I], poly(U)-Sepharose [4] or poly(U)-cellulose [19] columns, or by binding to poly(U)-filters [19] or Millipore filters [13]. Using these techniques, poly(A)containing mRNA has now been isolated from a number of higher plant species [e.g. 251 as well as from other eukaryotic organisms [see 71. We have employed these techniques to investigate the rates of synthesis of mRNA in the hypocotyls of soybean plants inoculated with incompatible or compatible races of Phytophthora megasperma var. sojae. Considerable evidence suggests that glyceollin [16] and related phytoalexins are responsible for resistance in this host-parasite system, the most salient feature being that they are synthesized several times more rapidly in incompatible soybean-P. megasperma var. sojae interactions than in compatible ones [5, 6, 9-12, Yoshikawa, unpublished data]. This indicates that the expression of resistance is an active process, presumably involving more rapid synthesis of the enzymes required for phytoalexin biosynthesis [16] and possibly also increased transcription during early stages of the interaction. In this communication we report that both incompatible and compatible interactions indeed result in increased synthesis of poly(A)-containing mRNA but that the increase is greater in the incompatible interaction. MATERIALS
AND
METHODS
Chemicals
Polyriboadenylic acid [poly (A)] and pancreatic ribonuclease were obtained from Sigma Chemical Co. Polyribouridylic acid [poly (U)] was purchased from Boehringer Mannheim GmbH, poly(U)-Sepharose 4B from Pharmacia Fine Chem., deoxyribonuclease from Worthington Biochemical Corp. and diethylpyrocarbonate from Nakarai Chemical Ltd. [G-sH]Uridine was obtained from The Radiochemical Centre, Amersham. Plant
and fungus
culture methodr
Seeds of the soybean (Glycine max L. Merr.) cultivar Harosoy 63 (resistant to race 1 and susceptible to race 4 of Phytophthora megasperma Drechs. var. sojae A. A. Hildb.) were planted in plastic pans containing a mixture of 75% vermiculite and 25% Sumirin Yuki Derma (Sumitomo Forestry Ltd). Plants were grown in a greenhouse maintained at 25 “C. Races 1 (P900, ATCC 34 000, [14]) and 4 (kindly supplied by D. Schwenk, [1&j) were maintained on V-8 juice agar. For inoculum production, the fungi were grown for 3 to 4 days in a pea broth medium [rl] and rinsed with deionized water prior to inoculation.
Activated
synthesis
of poly(A)-containing
messenger
RNA
127
Seven- to 8-day-old plants were cut at the base of the hypocotyls and placed in deionized water in a beaker. A longitudinal slash wound (c. 1 cm long) was made with a razor blade on each hypocotyl, approximately 1 cm below the cotyledonary node, and a small piece of mycelium was placed into the wound. A piece of cotton wool immersed in deionized water was placed into the wounds of control plants. Plants were maintained at a relative humidity of 100% after inoculation in a growth chamber at 25 “C under fluorescent lighting (c. 2000 lx). [3H] Uridine feeding
and RNA
extraction
At various times after inoculation, the fimgal inoculum or cotton wool was removed from the hypocotyl wounds of 20 plants prior to isotope feeding to minimize contamination of the extracted RNA with fungal RNA. Ten pl of [3H]uridine solution (50 yCi/ml, 6.5 Cm/ I mmol) was placed in each wound of the hypocotyls and the feeding was continued for 2 h, during which the isotope solution was almost completely taken up by the hypocotyls. rH]Uridine labelled RNA was isolated by a modification of the procedure described by Aviv & Leder [I]. After feeding, 20 wounded portions (1 cm long, c.lg fresh weight) of the hypocotyls were harvested, quickly rinsed with deionized water and then immediately pulverized in a mortar under liquid N,. The resulting powder was suspended in 5 ml of 100 nm Tris-HCl (pH 9.0) containing 0.4 M NaCl, 1 y0 sodium lauryl sulphate (SLS), 1 mu MgCl, and O-1 ml diethylpyrocarbonate at 4 “C. The suspension was vigorously stirred after addition of 3 ml of phenol : chloroform : isoamyl alcohol (50 : 50 : 2, v/v) for 15 min at room temperature, chilled to 4 “C, and the phases were separated by centrifugation at 5000 g for 10 min. The residue after removal of the aqueous phase was re-extracted with 3 ml of the same extraction buffer, and the combined aqueous phases were re-extracted with 5 ml of the phenol mixture. The radioactivity in a 0.05 ml aliquot of the aqueous phase was determined in 10 ml of Triton X-100 scintillation mixture consisting of 2 vols of toluene scintillation mixture (5 g PPO, O-2 g POPOP per litre of toluene) and 1 vol. of Triton X-100, and referred to as the total uptake fraction; less than 5% of the total radioactivity remained in the combined phenol phases. Crude RNA was precipitated from the aqueous phase with 2.5 vols of ethanol at - 20 “C overnight. The RNA was collected by centrifugation at 12 000 g at 0 “C for 5 min. The RNA pellet was washed twice with ethanol-O-2 M NaCl (2 : l), dissolved in 1 ml of NETS buffer (10 nm Tris-HCl (pH 7*4), 100 mM NaCl, 10 mM EDTA and 0.2% SLS) unless otherwise specified, and centrifuged at 20 000 g to remove any insoluble material. The crude RNA thus extracted had Az60/A280 and Az6,,/A230 ratios in excess of 2.2 and was not significantly contaminated with DNA, based on DNase treatment followed by sucrose density gradient analysis. Isolation
of poly(A)-containing
mRNA
by poly( U)-Sphe arose column and poti(Jilter
Poly(A)-containing mRNA was isolated from the crude RNA by affinity chromatography on poly(U)-Sepharose 4B columns according to the method of Eiden & Nichols [4] with slight modifications. The crude RNA solution (0.8 ml) was mixed with 1.2 ml of NETS buffer and applied to a microcolumn containing 2 ml of a poly(U)-Sepharose slurry which had been washed with 50 ml of NETS buffer.
I28
M. Yoshikawa,
H. Masago
and
N. T. Keen
Unbound RNA was washed through the column with 6 ml of NETS buffer and fractions of 2 ml were collected. The column was then washed with 6 ml of 10 mM Tris-HCl (pH 7.4) containing O-1 M NaCl. The RNA which remained bound to the column was then eluted with 6 ml of deionized water at 50 “C in an incubator maintained at the same temperature. The radioactivity in each 2 ml fraction was determined by counting a 0.5 ml aliquot in the Triton X-100 scintillation mixture. Poly(U)-fibre glass filters (Whatman GF/C, 2.4 cm diameter) were prepared by the method described by Sheldon et al. [19] except that the filters treated with poly(U) were irradiated for 4 mm on each side at a distance of 20 cm from a 15 W National germicidal lamp. Under these conditions, about 84% of the applied poly(U) was retained per filter. One-tenth ml of the crude RNA solution was diluted in 20 vols of ice-cold binding buffer [ 10 mM Tris-HCl (pH 7*5), 0~2 M NaCl]. After 10 min in the cold, the solution was passed at about 1 ml/min through a poly(U)-filter that had been equilibrated with the same buffer. The filters were washed twice with 10 ml of the binding buffer and then dried. The filters were directly counted in 10 ml of the toluene scintillation mixture. Thus, mRNA isolated in this study refers to those mRNAs that contain poly(A) sequences. Sucrose density gradient
centrifugation
[sH]Uridine labelled, poly(A) -containing mRNA isolated by poly(U)-Sepharose chromatography [fractions 7 and 8 in Fig. 2(b)] was precipitated by addition of onetenth vol. of 20% potassium acetate, O-5 mg unlabelled carrier RNA from soybean hypocotyls, and 2.5 vols of cold ethanol. The precipitated RNA was collected by centrifugation and dissolved in 0.5 ml of NETS buffer. The RNA solutions were layered onto linear 5 to 20% sucrose gradients in the same buffer and centrifuged in an SW 25 rotor at 22 500 rev/mm for 15 h at 25 “C. Fractions of 1.5 ml were collected from the bottom of the tube and the radioactivity in 1 ml portions of each fraction was determined in the Triton X-100 scintillation mixture. Glassware
All glassware used for RNA extraction and isolation was carefully hand-washed and autoclaved, and all solutions were autoclaved unless they contained ethanol or SLS. Microsco$ic
observations
The inoculated hypocotyls were sampled at intervals after inoculation and longitudinal hand-sections were made at right angles to the inoculated surfaces. The sections were stained with Rose Bengal and the hyphal growth was determined under the microscope by measuring the distance between the penetration points and the margins of invading hyphae. At least five hypocotyls were sampled at intervals and about ten penetration points per hypocotyl were observed. RESULTS
of RNA binding to pob( U)-jilter and poly( 7J)-Sepharose column To assessthe specificity of binding, various RNAs were applied to poly(U)-filters or poly(U)-Sepharose columns and the fractions remaining bound to the filter or column were analysed. Poly(U)-filter specifically bound poly(A) but did not bind Characteristics
Activated
synthesis
of poly(A)-containing
messenger
5
IO
Input
RNA
15
20
RNA
I!?9
’
50
(pg)
FIG. 1. Binding of various RNAs to poly(U)-filters. l , poly(A); A, poly(U) ; A, unlabelled RNA extracted from soybean hypocotyls. Different amounts of each RNA in 2 ml of 10 mM Tris-HCl (pH 7.5) containing 200 mu NaCl were passed through the filters and the amounts of the bound RNA were calculated from the differences in optical density of the input solution and the filtrate. Data are means of two replicate experiments. TABLE Some chara&ri.&s
of thz binding
Treatment Control RNase DNase Poly(A) Poly(U)
(10 pg/ml)c (10 pg/ml)c (100 pg/ml)d (100 pg/ml)d
of [3H]widine
1 labelled
3H-radioactivity (ct/min) in RNA bound to poly(U)-filter” 1818 57 1838 214 1728
soybean RNA
to poly(U)-Jilters
% of [W-JRNA bound/ [3H]crude RNA appliedb 1.10 0.03 1.11 0.13 l-05
@ Data are means of two replicate analyses. b [sHJUridine labelled crude RNA (165 100 ct/min) extracted from healthy soybean hypocotyls was applied to the filter for each treatment. G The extracted crude RNA was dissolved in 10 n-m Tris-HCl (pH 7.5), divided into portions and then the buffer concentrations were brought to 10 n-m Tris-HCl (pH 7.5) + 200 mu NaCl for RNase treatment or to 10 mM Tris-HCl (pH 7.5) + 2 rnM MgCl, for DNase treatment by adding concentrated NaCl or MgCls, respectively. The treatments were performed at 30 “C for 60 min in 2 ml reaction mixtures. NaCJ concentration in the reaction mixture after DNase treatment was brought to 200 no prior to the poly(U)-filter assay. d Two ml of unlabelled poly(A) or poly(U) solution at the indicated concentration was passed through the poly(U)-filter before the [sH]uridine labelled crude RNA solution.
detectable poly(U) or unlabelled crude RNA from soybean hypocotyls (Fig. 1). When rH]uridine labelled soybean crude RNA was applied, the filter retained about 1-1o/o of the radioactivity (Table 1). The binding was almost completely abolished by pretreatment of the crude RNA solution with RNase, but DNase had no effect. Pre-washing of the filter with poly(A) but not poly(U) also reduced the radioactivity subsequently bound to the filter. Thus, it appeared that the fraction bound
130
2
4 Fraction
M. Yoshikawa,
H. Masago
6
IO
0
and
N. T. Keen
number
FIG. 2. Affinity chromatography of RNA on poly(U)-Sepharose columns. The arrows indicate the points of addition of buffer [lo mM Tris-HCl (pH 7.4), 100 nm NaCl] (1) and 50 ‘C water (2). (a) Approximately 30 pg of poly(A) (o), poly(U) (A) or 40 pg of unlabelled RNA (A) extracted from soybean hypocotyls was loaded onto three identical columns. Two ml fractions were collected for measurement of optical density, D. (b) pH]Uridine labelled RNA extracted from healthy soybean hypocotyls was chromatographed. The vertical closed bars indicate the radioactivity that was subsequently m-bound to poly(U)-filters. The re-binding assay was carried out as follows: RNA eluted in each fraction was precipitated by ethanol, dissolved in 10 mM Tris-HCI (pH 7.5) containing 200 mM NaCl and then subjected to the poly(U)-filter binding assay. The representative data obtained from three and two replicate experiments are shown in (a) and (b), respectively.
to the filter was RNA and not DNA, and that the RNA was bound to the filter through poly(A) sequences. Similarly, poly(U)-Sepharose columns retained only poly(A) [Fig. 2(a)], and the RNA fractions of C3H]uridine labelled soybean RNA bound to the column could subsequently be bound to poly(U)-filters [Fig. 2(b)]. However, almost no radioactivity was detected on the filters through which were passed RNA fractions that did not bind to the column. This low background ensured the reliability of the filter assay for detecting the low percentage (about 1%) of poly(A)-containing RNA present in the crude RNA. We henceforth will call the RNA unbound to poly(U)Sepharose columns “bulk RNA”, and the RNA bound to either the columns or poly(U)-filters “poly(A) -containing mRNA” since evidence obtained from various
Activated
synthesis
of poly(A)-containing
messenger
RNA
131
eukaryotic cells has conclusively shown that these poly(A)-containing RNAs are indeed mRNAs [e.g. 7, 25-J. Sucrose density gradient analysis demonstrated that the profile of rH]uridine labelled bulk RNA was identical to that of the cold RNA from soybean hypocotyls; thus bulk RNA consisted of 25 and 18 S rRNA and 4 S soluble RNA (sRNA) (Fig. 3). However, poly(A)-containing mRNA isolated by poly(U)-Sepharose chromatography was heterogenous and very different from bulk RNA in its size distribution. The fraction of poly(A) -containing mRNA that sedimented near the bottom of the tube may be either hnRNA, a possible precursor of mRNA [e.g. 31, or possibly aggregated cytoplasmic mRNA [17], but further characterization of the fraction was not done. I
500
-
B II II
5
- 25
IO Fraction
15
20
number
FIG. 3. Sucrose density gradient profiles of RNA fractionated by poiy(U)-Sepharose chromatography. Fractions 1 and 2 (unbound RNA, bulk RNA) and 7 and 8 [bound RNA, poly(A)-containing mRNA] from Fig. 2(b) were pooled separately and the ethanol-precipitated RNA was dissolved and run in parallel gradients. l , bound RNA; 0, unbound RNA. The arrows indicate the position of marker unlabelled RNA from soybean hypocotyls. The profiles are from a typical experiment, representative of several replicate experiments.
Cytological
observation
Since our objective was to assessmRNA metabolism at early stages of infection before the establishment of incompatible or compatible interactions, the time of resistance expression in Harosoy 63 soybean hypocotyls against the incompatible race of P. megaspermavar. sojae was first evaluated (Fig. 4). Both the incompatible race (race 1) and the compatible race (race 4) grew in the hypocotyls at a similar rate until 8 h after inoculation; however, growth of the incompatible race was arrested withii the subsequent 2 h. Thus, in terms of inhibited fungal growth, resistance was expressed between 8 and 10 h after inoculation. No visible disease symptoms were apparent before 12 h after inoculation.
132
M.Yoshikawa,H.Masago
and
N.T.Keen
I500 -
-
4
S
12
16
20
Hours after
24
‘-
inoculation
FIG. 4. Growth of the two races of P. mega@erma var. sojae in Harosoy 63 soybean hypocotyls. Wound areas of the hypocotyls were inoculated with the incompatible race (race 1, l ) or the compatible race (race 4, o), and hyphal growth was measured after making thin sections and staining them by Rose Bengal. Points and brackets are means and standard errors, respectively, obtained from three replicate experiments. Five hypocotyls and about ten infected sites per hypocotyl were observed in each experiment.
Changes in @y(A)-containing
mRNA
synthesis after inoculation
Slightly increased synthesis of poly(A)-containing mRNA was noted as early as 2 h after the inoculation of hypocotyls with the incompatible or compatible races of P. megasperma var. sojae, and greater increases were observed at 4 h (Table 2). The TABLE
2
Incorporation of [3H’Juridine into poly (A)-contuining mR.NA of Harosoy 63 soybeanh.ypocotylsat the early stages after wounding only (Cont.) and inoculation with the incompatible race (race 1, Incomp.) or the compatibb race (race 4, Comb.) of P. megasperma var. sojae
Time after inocuiationb
3H-radioactivity
(ct/min
x lo-s)/20
hypocotyls
Poly(U)-Sepharose
affinity
chromatography
(c. 1 g fresh wt)@ Poly(U)-filter =w
(C) (A)
Inoculation
Total uptake
(B)
Bulk (rRNA+
RNAC sRNA)
2h
Cont. Incomp. Comp.
48401 44 725 46 233
3291 4262 3471
4h
Cont. Incomp. Comp.
46 142 42 113 45 649
3309 6348 4941
a b c d e
Data The The The The
% of B/A 6.8 (1.0)8 9.5 (1.4) 7.5 (1.1) 7.2 (1.0) 15.1 (2.1) 10.8 (l-5)
Poly(A)containing r&NAG
bimlmg
(“1
% of C/A
Poly(A)containing mRNA”
% of D/A
30.1 81-8 46.7
0.062 0.18 O-10
(I-O)@ (2.9) (1.6)
24-8 52.8 30.5
0.051 0.12 0.066
(1-O)' (2-4) (l-3)
34-l 177.7 94.9
0.074 0.42 O-21
(1.0) (5.7) (2.8)
32.0 164.2 86.3
0,069 040 0.19
(l-0) (5.8) (2.8)
are from a typical experiment, representative of four replicate experiments. time when [sK]uridine feeding was initiated and the isotope was fed for tbe subsequent 2 h. RNA fractions unbound (B) and bound (C) to poly(U)-Sepharose columns. RNA fraction bound to poly(U)-filters. values in parentheses indicate the ratios (inoculated/uninoculated) in o/0 of B/A, C/A or D/A.
Activated
synthesis of poly(A)-containing
messenger
133
RNA
incompatible interaction, however, resulted in greater synthesis than the correspondFurthermore, the degree of the increase in poly(A)ing compatible interaction. containing mRNA synthesis was greater than that for bulk RNA (rRNA+sRNA) synthesis. Therefore, it appeared that the incompatible interaction resulted in a rather specific activation of mRNA synthesis at relatively early stages of infection. Figure 5 indicates that the highly activated synthesis of poly(A)-containing mRNA in the incompatible interaction decreased after 6 or 8 h, and reached the same rate as that in the compatible interaction after 12 h. Table 3 indicates that the increases in the poly(A)-containing mRNA synthesis observed at early stages of the incompatible interaction mainly reflect activated
,, 4
8
12
”
I 24
Hours after inoculation
FIG. 5. Synthesis of bulk RNA and poly(A)-containing mRNA after infection of Harosoy 63 soybean hypocotyls with the incompatible race (race 1, l ) or the compatible race (race 4, 0) of P. mego$ermu var. sojae. (a) Bulk RNA unbound to poly(U)-Sepharose that consists of rRNA and sRNA. (b) Poly(A)-containing mRNA bound to poly(U)-Sepharose. (c) Poly(A)-contaming mRNA bound to poly(U)-filter. The time for initiation of rH]uridine feeding is indicated on the abscissa and the isotope was fed for 2 h. Means and standard errors are indicated where four replicate experiments were run, and other points are means of three replicate experiments. rH]Uridme incorporation was corrected for differences in total rH]uridine uptake by the hypocotyls.
M. Yoshikawa,
134
H. Masago and N. T. Keen
synthesis by the host cells and not synthesis by either the fungal inoculum or contaminating bacteria. Although P. megaspermavar. sojae can synthesize poly(A)-contaming mRNA [see Treatment 3(b) in Table 31, the increased synthesis of mRNA by inoculated hypocotyls in which the fungal inoculum was removed before or after the isotope feeding [Treatments 3(a), 4 and 5 in Table 31 occurs at a sufficiently early (4 h) time to preclude significant amounts of invading hyphae in the host (Fig. 4). Furthermore, treatment throughout the inoculation and isotope feeding procedures with a combination of antibiotics, which almost completely suppressed the growth of soil bacteria, did not reduce the observed increase in mRNA synthesis (Treatment 5 in Table 3).
TABLE
3
Host dependence of the enhanced poly(A)-containing mR.M synthesis at the ear& stage (4 h after inoculation) of the incomfiatible interaction between Harosoy 63 soybean hypocotyls and race 1 of P. megasperma var. sojae sH-radioactivity hypocotyls
Treatment8
Inoculation
Source for RNA extraction
(1)
None
Host
22;
Race
1
Host
Race
1
Host (Fungal Host Host
Race 1 Race 1 + rifampicin + ampicillin
f fungal moculum inoculum)
(4 Total uptake
(ct/min x lo-s)/ZO (c. 1 g fresh wt.)@
(B) Poly(A)containing mRNA bound to poly(U)-filter
%of B/A
Ratio in % of B/A
43 a72
26.1
O-059
1
40 654
193.5
0.48
8.1
32 628 (6786) 42 818 44 615
117.5 (34.8) 154.1 165.7
0.36
6.1
0.33 0.35
5.6 5.9
a Data are from a typical experiment, representative of three replicate experiments. b Treatment. (1) The hypocotyls received only wounding. (2) After feeding with rH]uridine, both the host and fungal inoculum together were subjected to RNA extraction. (3) After the isotope feeding, the fungal inoculum was removed from the host and RNA of the host and fungal inoculum was separately extracted. For the extraction of fungal labelled RNA, 1 g of unlabelled soybean hypocotyls was homogenized together with the labelled fungal sample. (4) This treatment is the same as that described in Materials and Methods and was employed throughout the present study, in which the fungal inoculum was removed from the host before the isotope feeding. (5) The same as Treatment (4) but mycelial suspension for inoculation and the isotope solution contained rifampicin (10 pg/ml) and ampicillin (500 pg/ml) to suppress bacterial growth.
Sucrose density gradient analysis of poly(A)-containing mRNA isolated by poly(U)-Sepharose chromatography revealed that the synthesis of certain 18 S species of mRNA was predominantly enhanced at the early stage (4 h) of the incompatible interaction, and a lesser degree of enhancement occurred in the compatibIe interaction [Fig. 6(a) and (b)]. A similar trend was observed in poly(A)-containing mRNA isolated at 2 or 6 h after inoculation.
Activated
synthesis
of poly(A)-containing
messenger
5
I5
IO Fraction
135
RNA
20
number
FIG. 6. (a) Sucrose density gradient profiles of poly(A)-containing mRNA extracted from Harosoy 63 soybean hypocotyls at 4 h after only wounding (A) and inoculation with the incompatible race (race 1, 0) or the compatible race (race 4: 0) of P. megusparma var. sojae. Poly(A)-containing mRNA was isolated by poly(U)-Sepharose chromatography and run in parallel gradients. The arrows indicate the positions of marker unlabelled RNA from soybean hypocotyls. The profiles are from a typical experiment, representative of four replicate experi(b) Ratios of inoculated/uninoculated sH-radioactivity were calculated for each fraction ments. of the gradients. Each value was obtained from Fig. 6(a). l , the incompatible host-fungus combination; 0, the compatible host-fungus combination.
DISCUSSION
The most significant finding of this study is that the rate of mRNA synthesis markedly increased as soon as 4 h after inoculation in the soybean-P. megasperma var. sojae system (Fig. 5). Thus the interaction between the host and pathogen resulted in a rapid alteration of host metabolism at the gene transcriptional level. These increases in mRNA synthesis clearly precede the expression of resistance in the incompatible reaction which occurs at about 10 h after inoculation, based on the observed inhibition of fungal growth (Fig. 4).
136
M. Yoshikawa,
H. Masago and N. T. Keen
The rates of mRNA synthesis in the early stage of the incompatible interaction were more rapid than in the corresponding stage of the compatible interaction (Fig. 5). Therefore, the two interactions appear to be distinguished by a quantitative difference in gene activation rather than by an all or none process. However, further approaches are necessary to more critically determine whether any qualitative differences exist between the mRNAs synthesized during early stages in the incompatible and compatible interactions and to clarify the role of the new mRNAs in determining ultimate rates of phytoalexin production. Our results indicating an association of increased mRNA synthesis and resistance expression are also in agreement with other investigations showing that RNA and protein synthesis are required for the expression of disease resistance [e.g. 20, 26, 27, 291. Our preliminary studies with P. megasjerma var. sojaesystem have also supported this interpretation, since actinomycin D and blasticidin S, RNA and protein synthesis inhibitors, respectively, negated both the ability of soybean plants to make phytoalexins rapidly and their resistance to the incompatible race of the fungus. It is often difficult to determine whether observed physiological changes in infected plants are due to either the metabolism of host, pathogen or contaminating organisms such as bacteria, especially where the metabolism concerned is commonly shared among the possible organisms. The following lines of evidence suggest that the increased mRNA synthesis observed during early stages of the soybean-P. mega.sjw-ma var. sojae interaction is a product of host metabolism: (a) The fungal inoculum was routinely removed before isotope feeding. (b) The contribution of mRNA by invading hyphae must be negligible since rates of mRNA synthesis were much higher at 4 to 6 h after inoculation, when the host cells were not yet extensively colonized by the fungus in both the incompatible and compatible combinations, than at 12 to 24 h, when fungal growth was extensive in the compatible combination. (c) At 4 to 6 h, furthermore, mRNA synthesis was much higher in the incompatible combination. It is unlikely that the two races of P. megasjwrma var. sojae possesssuch differential ability to synthesize mRNA. (d) Possible bacterial contribution can be eliminated by the fact that treatment with antibiotics throughout the inoculation and isotope feeding procedures did not reduce the observed enhancement in mRNA synthesis and, furthermore, significant numbers of bacteria could not be isolated from the surface sterilized-infected hypocotyls within 12 h after inoculation. Although the present study has dealt with only those mRNAs that contain poly(A) sequences, there is some eukaryotic mRNA that is not associated with poly(A) sequences. Such mRNA containing no poly(A) sequence, however, constitutes only a minor portion of total eukaryotic mRNA, and only histone mRNA [see 71 has thus far been shown to be one. Further characterization of the poly(A)-containing mRNA isolated in this study, for example, by testing its template activity in the synthesis of protein, is now under way.
We are grateful to Dr J. D. Paxton for kindly supplying soybean seeds. We also acknowledge Drs T. Tani and S. Ouchi and Mr H. Yamamoto for valuable discussion of the work.
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137
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