Changes in ribonuclease activity in Ribes leaves and pine tissue culture infected with blister rust, Cronartium ribicola

Physiological

Plant Pathologv

(1974)

4, 359-371

Changes in ribonuclease activity in Ribes leaves and pine tissue culture infected with blister rust, Cronartium ribicola A. E. HARVEY,?

A. K. CHAKRAVORTY, MICHAEL SHAW and Laboratory of Plant Biochemistry and Pathologv, Department of Plant Science, Faculty The University of British Columbia, Vancouver 8, B.C., Canada (Accepted for publication,

December

L. A.

SCRUBB of Agricultural Sciences,

1973)

Ribes leaves (Ribes nigrum L.) and pine tissue cultures (Pinw monticola Dougl.) were inoculated with blister rust aeciospores (Cronartium ribicola J. C. Fisch. ex Rabenh.) and mycelium grown in axenic culture respectively. The ribonuclease activity in the cell-free extracts of the infected tissues were determined at various times after inoculation. In both cases the ribonuclease activity increases in a biphasic manner. The early phase of ribonuclease increase coincides with a significant decline in the stability of the enzyme at higher temperatures, and the later phase coincides with the appearance of comparatively thermostable and diethylpyrocarbonate-insensitive ribonuclease molecules. The second phase of increase in activity is also accompanied by substantial changes in the substrate specificities of the enzyme from infected Rib@ leaves and pine tissue culture, indicating the formation of enzyme molecules with unique catalytic properties. Sectioning and incubation of the healthy (uninoculated) host tissues result in an increase in the ribonuclease activity but mechanical injury does not cause any detectable changes in the properties of the enzyme. The quantitative and qualitative changes in the ribonuclease activity of rust-infected flax cotyledons, Ribes leaves and pine tissue culture were compared to gain an insight into (a) the specificity of the changes in various host-rust combinations and (b) the role of the host plant and the rust fungus in determining the changes in the catalytic properties of the enzyme.

INTRODUCTION

Rohringer et al. [S] observed increases in RNase activity in rust-infected wheat leaves. Recent studies in this laboratory have revealed a positive correlation between the growth of the rust and the accumulation of qualitatively different RNase molecules in inoculated wheat leaves [Z]. Quantitative and qualitative changes in the RNase activity of a susceptible variety of flax following inoculation with flax rust have also been reported [7]. That these changes are specifically elicited by compatible host-parasite interactions is indicated by the findings that (a) the later phase of RNase increase and the accompanying changes in properties of the enzyme have not been detected in an incompatible flax rust combination and (b) while mechanical injury causes a significant increase in the level of RNase in the healthy cotyledons, the enzyme remains strictly “host-type” as indicated by the lack of demonstrable changes in its physical and catalytic properties [7]. The experiments described in this paper were designed to determine whether similar changes occur t Present address: U.S.D.A., Forestry Sciences Laboratory,

Forest Missoula,

Service, Intermountain Forest Montana 59801, U.S.A.

and Range

Experiment

Station,

360

A. E. Harvey

et al.

in blister rust-infected tissues. The levels and properties of RNase from blister rust-Ribes and blister rust-pine combinations were studied to examine the changes that may occur in two different hosts, Ribes (Ribes nigrum L.), a dicotyledonous angiosperm, and pine (Pinus monticola Dougl.), a coniferous gymnosperm, inoculated with the diploid and the haploid phases respectively of the same rust, Cronartium ribicola J. C. Fisch. ex Rabenh. In addition to the plants inoculated in viva, an artificial host-rust culture has been established with pine cortical tissue culture and rust mycelium grown on host-free medium. The results of these experiments suggest that a new class of RNase molecules with unique catalytic properties is produced in all these host-rust combinations. MATERIALS AND Biological materials,

METHODS

in vivo

Open-pollinated seeds of pine (P. monticola) collected in the St. Joe National Forest, Idaho, U.S.A., were germinated and the seedlings were grown in nursery beds. Some of the plants were inoculated with blister rust (C. ribicola) according to the following procedure. Five-year-old plants were enclosed in a wooden framing which extended about 25 cm beyond the growing tips of the plants. The framing was covered with a coarse wire mesh. The plants were fogged with tap water and teliospore-bearing leaves of Ribes (Ribes petiolare L.) were placed on the wire mesh, spore side down. These, in turn, were covered with several layers of burlap cloth kept moist throughout a 72-h inoculation period. Under these conditions of inoculation in the greenhouse, leaf spots became visible 45 to 60 days after inoculation and stem canker appeared about six months following the appearance of leaf spots. When infection was well established, cortical tissue from healthy (uninoculated) and infected plants of corresponding age was excised and used either for the extraction of RNase or to initiate tissue cultures in vitro [4, 51. Ribes. Ribes plants (a susceptible clone of R. nigrum L.) were maintained in controlled environmental growth chambers (16 h at 20 “C and under 1000 ft-c fluorescent light and 8 h in the dark at 5 “C, each day). The plants were treated by spraying the abaxial leaf surfaces with water and by lightly dusting with blister rust aeciospores. They were then covered with plastic bags for 48 h. The healthy (control) plants were treated under identical conditions except that they were not inoculated. Under these conditions of inoculation, leaf spots appeared about 6 to 8 days after inoculation and pustules were visible after 10 to 14 days.

Pine.

Biological

materials,

in vitro

of blister rust. Blister rust was grown on host-free media under conditions similar to those described for flax rust ([3] and Harvey & Grasham, unpublished) using aerial hyphae derived from infected host tissue [5] as inoculum. Pine tissue culture. Primary explants of healthy and naturally infected pine cortex were grown in chemically defined media [4] containing, in g/l, the following components: glucose, 35.0; Noble agar, 8.0; Ca(NO,)s.H,O, 0.5; MgSO,.SH,O, 0.14; KHsPO,, O-14; (NH,),SO,, 2.5 x 10m2; Fe,(SO,),, 1.4 x 10e2; MnSO,.4H,O, 3-5 x 10-s; 2,4-dichlorophenoxyacetic acid, I.0 x 10e4; and kinetin, 1-Ox 10e5.

Axenic culture

RNase

changes

in rust

infected

R&es

and

361

pine

Both healthy and infected primary explants were grown of pH of the media (6-O), temperature and photoperiod fluorescent light and 8 h at 5 “C). Host-rust

under identical conditions (16 h at 21 “C, 1000 ft-c

culture

In these experiments, 4-month-old host tissue cultures (primary explants from healthy pine cortex) were directly exposed to rust mycelium. The blister rust, grown for 30 days on a chemically defined medium, was placed adjacent to and in contact with the host tissue culture as shown diagrammatically in Fig. 1. Microscopic examination revealed that the rust hyphae invaded the host tissue within the first 7 days after inoculation under these conditions.

FIG. 1. Diagram culture. extraction.

R, rust

of host-rust culture initiated in Z&O with mycelium; H, host tissue; S, shaded portion

blister rvst, mycelia and pine tissue of the host tissue used for RNase

Processing of samples for RNase extraction Ribes. Inoculated Ribes leaves at 2,4,8, 16 and 32 days after inoculation and healthy leaves of corresponding age were collected, divided into 5.0-g lots and frozen quickly in liquid nitrogen. These samples were stored frozen for 1 to 2 weeks prior to extraction. Pine cortex. Explants of healthy and infected pine cortex grown on artificial media were gently removed from the flasks and thoroughly rinsed to dislodge any adhering medium. One whole, undamaged, 6-month-old culture of either healthy or infected tissue (weighing approximately 2.0 g) was extracted immediately after rinsing at 0 to 4 “C. Host-rust culture. The samples from the host-rust tissue culture system [Fig. 1 (a)] were prepared by excising a segment of the host tissue within 1 to l-5 cm from the contact point (shown by a shaded area in Fig. l), at 4, 8, 16, 32, 64 and 140 days after the rust mycelium had been placed adjacent to the host tissue culture. The samples were washed thoroughly with distilled water and extracted immediately. Mechanical

injury

Ribes leaves. Healthy (uninoculated) which were incubated under aseptic gramicidin D (Sigma Chemical) for were rinsed with glass-distilled water,

Ribes leaves were sectioned into 5-ems discs conditions in a solution containing 6 pg/ml 6 h at 20 “C. After incubation, the sections frozen quickly and stored until extraction.

362

A. E. Harvey

et

al.

Pine tissue culture. Individual tissue cultures were sectioned into 1 x 2 x 20 mm strips under sterile conditions. The strips were incubated on a moist filter paper placed over a dialysis membrane covering the standard tissue culture medium, for 6 h at 20 “C. After incubation, RNase was extracted from the strips as well as from the filter paper to determine any possible loss of the enzyme from the cut surfaces. R.Nase extraction and assay and other methods. All procedures were as described earlier [7]. One unit of RNase is defined as the quantity of enzyme catalysing an increase in A 260 nm of 1-O under the standard conditions of assay and specific activity of RNase is units/mg protein. The specific activities of other hydrolases are expressed as AA at the appropriate wavelength/mg protein. RESULTS

Changes in the RNase actiuity of Ribes following inoculation with blister rust

leaves and pine tissue culture

The RNase activities in the cell-free extracts from healthy and inoculated Ribes leaves were determined at various stages after inoculation with blister rust aeciospores. The results are presented in Fig. 2. The specific activity of RNase (units/mg protein) from the healthy leaves does not change appreciably during the experimental period. However, the specific activity of the RNase of the inoculated leaves increases substantially between 2 to 6 days after inoculation, drops down to the same level as that of the healthy leaves at 8 days and then increases again to a value about four times higher than that of the healthy leaves, between 16 to 20 days after inoculation. The resulting biphasic curve (Fig. 2) is very similar to those observed in flax cotyledons [7] and wheat leaves [Z, S] following inoculation with the appropriate species of rust. The only difference between the results presented in Fig. 2 and those reported before is that in flax and wheat the changes occur much earlier (within the first 6 to 8 days) after inoculation.

z B O

FIG. 2.

Changes

l ---•----:------•’ I I 2

I 8 4 Time after inoculation (days)

in the RNase activity of Ribes leaves at various aeciospores of blister rust fungus. ( l ) Healthy,

I 16

times after ( 0) infected.

inoculation

with

In order to examine the changes in the RNase activity of the pine tissue culture, several batches of host-rust cultures were prepared as illustrated in Fig. 1, by

RNase changes in rust infected Ribes and pine

363

placing rust mycelium (previously grown on a chemically defined solid medium) adjacent to and in contact with the host tissue culture. The RNase activity of the host tissue sample (the shaded area in Fig. 1) was estimated at different time intervals following inoculation.The results presented in Fig. 3 show that a biphasic curve similar to those observed in inoculated Ribes leaves (Fig. 2) and other host species [Z, 6, 71 is obtained. The bimodal increase in the RNase activity of the artificially produced host-rust combination with pine tissue culture and blister rust mycelia occurs over a very long period of time, although the overall pattern of RNase increase in this in vitro combination is strikingly similar to the pattern observed in all other naturally inoculated host-rust combinations.

Time after inoculation (days) FIG. 3. Changes in the RNase activity of pine tissue blister rust mycelium. ( l ) Healthy pine tissue culture, (shaded portion of pine tissue as shown in Fig. 1).

Levels of Ma-se and other hydrolytic and pine tissue culture

culture following ( 0) “inoculated”

‘cinoculation” pine tissue

with culture

enzymes in infected Ribes leaves

In these experiments, the cell-free extracts from healthy and infected pine tissue culture (explants of healthy and naturally infected pine cortex, see “Materials and Methods”) and Ribes leaves were assayed for the following enzymes (substrates are indicated in parentheses) : RNase (high molecular weight yeast RNA), DNase (calf thymus DNA, heat denatured), non-specific phosphodiesterase (Ca-bis(pnitrophenyl) phosphate) as well as acid and alkaline phosphatase (carboxyphenyl phosphate) activities [7]. The results, presented in Table 1, show that the RNase activity is substantially higher (3 to 4 times) in the infected samples than in the corresponding healthy tissues. The specific activities of some of the other hydrolases in the infected Ribes leaves and pine tissue culture are the same as those of the healthy samples (DNase of pine and alkaline phosphatase of Ribes), much lower (acid phosphatase of pine and Ribes and alkaline phosphatase of pine), or slightly higher (DNase and phosphodiesterase of Ribes) than those of the corresponding healthy samples. These results corroborate our previous finding that of all the hydrolases examined only the RNase activity of flax cotyledons increase remarkably in response to rust infection [7]. 24

364

A. E. Harvey TABLE

Levels of RNase, &erase activities

Specific RibeP

RNase DNase Acid phosphatase Alkaline phosphatase Phosphodiesterase Ribes leaves The healthy tissue grown

Effect of mechanical

1

Dflase, acid phosphatase, aNcal& phosphatase and sugar non-specijc phosphodiin the cell-free extracts from healthy and blister rust-infected Ribes leaves and pine tissue culture.

Enzyme

a Infected spores. cortical

et al

activity

(~A/mg

protein) Pine

Ratios (infected/healthy) Ribes Pine

Healthy

Infected

Healthy

Infected

26.0 40.0 4.8 9.6 4.4

100.0 60.0 2.0 10.5 6.2

19.0 18.4 6.6 11.4 0

110.0 19.2 3.2 4.8 0

were harvested and infected on chemically

16 days after inoculation with blister rust aeciopine samples were the primary explants of pine defined media for six months [4].

3.9 1.5 0.4 1.1 1.4

5.8 1.0 0.5 0.4 -

injury

The effect of mechanical injury on the RNase activities of healthy Ribes leaves and pine tissue culture was studied by sectioning and incubating these samples as followed by extraction and the estimation described in “Materials and Methods”, of RNase activity. There was only a very slight increase (10 to 20%) in the RNase activity of the healthy pine tissue culture as a result of this treatment. The RNase activity of healthy Ribes leaves increased by l-5- to 2*0-fold in response to mechanical injury [unpublished observation]. Also, as shown below, the RNases of the sectioned Ribes leaves and pine tissue culture exhibit properties that are identical to those of the corresponding healthy samples, indicating that the increased RNase is strictly “host-type”. These results are similar to those observed with mechanically injured flax cotyledons [7]. Properties

of RNase

Thermal stability. The stability of RNase from healthy, mechanically injured and blister rust-infected pine tissue culture was estimated by preincubating the extract at various temperatures for 10 min, cooling to 0 “C and then incubating under standard assay conditions to estimate the residual RNase activity. The RNase from blister rust grown on host-free, chemically defined media was also included in these experiments for the sake of comparison. The infected tissue in this case is a primary explant from rust-infected pine stem cortex grown in the culture medium for six months. The healthy tissue represents a similar explant from healthy pine stem cortex grown under identical conditions and for the same length of time. The results of these experiments are presented in Fig. 4. The RNases from both healthy and mechanically injured pine tissue culture are quite stable at higher temperatures and lose no activity even at 100 “C. The rust enzyme is comparatively unstable. It loses 50 to 60% of the original activity at 80 and 100 “C. The RNase from infected pine tissue culture is the most temperature labile of all, being increasingly inactivated at temperatures above 20 “C and losing 90% of the original activity at 80 to 100 “C. These results show that the RNase from infected tissue

RNase

culture healthy Similar

changes

in rust

365

Ribes and pine

infected

is much more unstable at higher temperatures than that from either the host tissue culture of comparable age or the rust grown on host-free media. results were obtained with Ribes leaves inoculated in vivo.

Q

I

I

I

I

I

0

20

40

60

80

I 100

Temperature (“Cl

FIG. 4. Thermal stability curves for the RNase from healthy, mechanically injured and infected pine tissue culture and from blister rust mycelium. Healthy and infected pine tissue cultures represent the primary explants of pine cortical tissue from healthy and rust-infected pine, grown for six months on chemically defined media under identical conditions. Blister rust mycelium was grown on host-free media as described in “Materials and Methods”. (0) Healthy pine tissue, (m) mechanically injured pine tissue, ( 0) rust-infected pine tissue and (A) rust grown on chemically defined media.

The production of a highly temperature-labile RNase component in pine tissue culture and Ribes leaves was explored further by determining the relative temperature stability of the enzyme at different times after inoculation. The results are presented in Fig. 5. In pine tissue culture, the formation of a temperature-sensitive RNase begins within the first 2 to 3 days after the rust mycelium has been placed in contact with the tissue culture (Fig. 1) and the RNase extracted from the host tissue becomes increasingly labile at 80 “C with time (days after inoculation). Before inoculation, lOOoh of the RNase activity is recovered after heat treatment (Fig. 4). The amounts of original activity recovered after 10 min at 80 “C, at 8, 16 and 32 days after inoculation are 60, 50 and 40%, respectively. At 64 days after inoculation, however, the RNase appears to become more stable than the enzyme at 32 days after inoculation, as revealed by the finding that 75% of the activity is recovered after 10 min at 80 “C. There is no further change in temperature stability for up to 140 days after inoculation (data not included in Fig. 5). A comparison of the temperature stability of RNase (Fig. 5) with the changes in the specific activity of the enzyme from inoculated tissue culture at various times after inoculation (Fig. 3) reveals a striking correlation between changes in specific activity and the stability properties of the enzyme. The early phase of RNase increase in the inoculated tissue which occurs between 4 and 32 days after inoculation (Fig. 3)

366

A.

E. Harvey

et al.

coincides with an increasing thermolability of the enzyme (Fig. 5). The later phase of RNase increase (32 to 128 days after inoculation) coincides with a relative increase in the stability of the enzyme at 80 “C. However, the RNase that accumulates at 64 to 128 days after inoculation is not quite as stable as enzyme from the healthy tissue (Fig. 4). The results of the experiment with the infected tissue culture ipzvitro (Fig. 4), have suggested a much greater thermolability of the RNase than that observed in the artificially produced host-rust combination (Fig. 5) at the later stages (64 to 128 days) after inoculation. The reason for this difference is not clear.

FIG. 5. Changes in the thermal stability of RNase from pine tissue culture and Ribes leaves following inoculation with blister rust mycelia and aeciospores, respectively. The cell-free extracts from pine tissue culture (shaded portion in Fig. 1) and Ribes leaves, at different times after inoculation, were treated by holding at 80 “C for 10 min, cooling to 0 ‘C and then assaying for residual RNase activity. ( 0) Pine tissue culture, ( l ) Ribes leaves.

A similar correlation between the early phase of RNase increase and a concomitant decrease in thermal stability of the enzyme from inoculated Ribes leaves is also apparent in Fig. 5. Thus, only 30% of the RNase activity extracted from Ribes leaves at two days after inoculation is lost when the enzyme preparation is heated at 80 “C for 10 min. At eight days after inoculation, 85 to 90% of the activity is lost under these conditions. The enzyme is considerably more stable at 16 days after inoculation. The observations on the changes in RNase activity (Figs 2 and 3) and a parallel change in the stability of the enzyme (Fig. 5), characterized at first by a decrease followed by an increase in stability at 80 “C, suggest that the RNase molecules formed during the two phases (early and later) are different with respect to their temperature sensitivity. A comparison between Ribes leaves inoculated in vivo and the pine tissue culture inoculated in vitro (Fig. 5) shows that despite a difference in the time of appearance (in days after inoculation), the overall pattern of the changes in temperature stability of the enzyme in these two systems are remarkably similar. PH optima. The pH-activity profiles of RNase extracted from the primary explants of healthy and rust-infected pine stem cortex are shown in Fig. 6. The enzymes from

RNase

changes

in rust

Ribes and pine

infected

367

both healthy and infected tissues exhibit similar pH optima, being at pH 4.5.

the peak of activity

160 -

25

35

, 45

, 55

, 6.5

-.7.5

8.5

PH

FIG. 6. pH Activity curves for the RNase from healthy and rust-infected pine tissue culture. The materials were the same as those described in the legend of Fig. 4. Buffers used are: Na,HPO,-citric acid, pH 2.5 to 4.5; K.HsPO,-NaOH, pH 5-5 to 7.0, and Tris-HCl, pH 8.0 to 8.5. (0) Healthy pine tissue culture, (0), infected pine tissue culture.

Fig. 7 shows the pH-activity curves obtained with the RNase from healthy and infected Ribes leaves. The enzyme from infected leaves shows a sharp peak of activity at pH 5.0 while that of the RNase from healthy leaves is rather broad, there being only a slight change in specific activities measured at pH values of 4.5 to 6.0.

0

: 2-5

3,5

45

5-5

6-5

75

8.5

PH

FIG. 7. inoculation) Fig. 6. (i)

pH Activity profiles for the RNase from healthy and rust-infected Ri6es leaves. The buffers used were the same as those described Healthy Ribes leaves, (0) infected Ribes leaves.

(16 days after in the legend of

(DEP) sensitivity. The effect of DEP on the activity of RNase extracted from various sources has been estimated by studying the 14C-labelled poly(I)$

Diethylpyrocarbonate t Abbreviations polycytidilic and

used are: poly(A), polyinosinic acids (also

poly (U), poly(C) and poly(I), referred to as PA, pU, PC, PI),

polyadenilic, respectively.’

polyuridilic,

368

A. E. Harvey

et al.

hydrolysing activity of these enzymes in the absence or the presence of a saturating concentration of DEP. The results presented in Table 2 reveal that the RNases of the healthy pine tissue culture and Ribes leaves are highly sensitive to DEP, their activities being inhibited by 80 to 90% in the presence of this compound. The rust mycelial enzyme is completely inactivated by DEP. In contrast, the RNases from rust-infected pine tissue culture and Ribes leaves are significantly less sensitive to DEP than the enzymes from the corresponding healthy tissue. Similar results were obtained when the enzymic activity was assayed with RNA as substrate. TABLE Effect blister

of diethylpyrocarbonate rust mycelium

Source

2

(DEP) on the 14C-labelledpoly(I) and from healthy and infected Ribes

of RNasea

Blister rust mycelia Healthy pine tissue Infected pine tissue Healthy Ribes leaves Infected Ribes leaves

degrading activity of the RNasesfrom leaves and pine tissue culture

Hydrolysis of poly(1) (d/min/mg protein X 10m3) -DEP +DEP 16.0 19.2 509.0 490.0 510.0

0 1.5 226.0 102.9 204.0

Inhibition

(“/o)

100 92 55.6 79 60

a Blister rust was grown on a completely defined medium. Samples of pine and Ribes were the same as those used in the experiment of Table 1. DEP was added to the enzyme solution and mixed thoroughly at 0 “Cl, 5 min prior to the addition of poly(1) and incubation at 37 “C. The final incubation mixture (1.0 ml) contained 4.2 mg DEP and 36 to 45 Erg protein.

The relative rates of hydrolysis of various homoribopolymers by the RNases from healthy and infected Ribes leaves and pine tissue culture (host-rust with mechanically culture) are presented in Table 3. The results of experiments injured Ribes leaves and the rust mycelium grown on host-free media are also presented, for comparison. The RNase from mechanically injured Ribes leaves hydrolyses various polyribonucleotides at a rate 5 to 25% greater than that of the enzyme from healthy leaves, but its substrate preference is quite similar to that of the RNase from healthy leaves. The most striking difference between the substrate specificities of the enzymes from healthy and infected Ribes leaves is that the rate of poly(C) hydrolysis of the enzyme from infected leaves is 4.5 times greater than that of the RNase from healthy leaves. The RNase from infected leaves also exhibits a slightly higher rate of poly(A) and poly(1) degradation compared with that of the healthy leaves, whereas the activity against poly(U) after inoculation. These d ecreases slightly results demonstrate that while mechanical injury of Ribes leaves causes a general increase in the rate of hydrolysis of all the polynucleotides tested, the effect of rust infection on the hydrolysis of these polynucleotide substrates is a great deal more specific. The effect of rust infection on the relative ability of the RNase from pine tissue culture to depolymerize the various polynucleotide substrates is more dramatic. Thus, the enzyme from healthy pine tissue hydrolyses poly(U) at a rate greater than the Substrate

speczjkity.

RNase

changes

in rust

infected

R&es

and

369

pine

rate of hydrolysis of poly(A) and poly(C). The RNase from the infected tissue, on the other hand, is incapable of depolymerizing poly(U). Compared with the RNase from healthy pine tissue culture, the enzyme from infected tissue exhibits lower rates of poly(A) and poly(C) hydrolysis, the decrease in poly(A) degrading activity being considerably greater than that of poly(C). TABLE

Substrate

spec$icities

3

of RNase from healthy and infected Ribes leaves and pine tissue culture from blister rust mycelium grown in host-free media

Sample

PONY (4

Ribes leaves Healthy Mechanically injured Infected (16 d)

3015.5 3219.0 3563.1

Pine tissue culture Healthy Infected (64 d) (host-rust culture) Blister rust (axenic culture) a During the progress available.

Relative rate of hydrolysis (d/min/mg protein x 10-s) POlY CC) POlY VJ) 1628.0 2075.2 1450.4

114.7 138.7 503.2

143.5 36.2

194.3 0

42.0 31.6

222.8

130.7

118.0

radiolabelled

poly(1)

of this work,

of POlY (1) 489.6 543.6 510.0

became

Substrate preference

pA>pU>pI>pC pA>pU>pI>pC pA>pU>pI

1”

pU>pA>pC pAmpC

16.0

pA>pU>pC>pI

no longer

and

mpC

commercially

The substrate specificities of the RNase from blister rust differ from those of the enzyme from infected Ribes leaves in that the poly(C) degrading activity of the rust enzyme is seven times greater than its ability to hydrolyse poly(1) while the enzyme from infected Ribes leaves hydrolyses poly(C) and poly(1) at similar rates. The rust enzyme differs from that of the infected pine tissue in that it is capable of hydrolysing poly(U). Thus, in either case, the RNases from infected Ribes leaves and pine tissue culture have catalytic properties that are quite different from those of the corresponding healthy samples on the one hand and the rust mycelial enzyme on the other. DISCUSSION The results presented in this paper demonstrate that the changes in RNase activity during rust infection, previously observed in inoculated flax cotyledons [7] and wheat leaves (2, S], also occur when Ribes leaves are inoculated with blister rust and when an artificial host-rust complex is initiated with blister rust mycelium and pine tissue culture. These changes namely, (i) a characteristic bimodal increase in RNase activity with the progress of the disease and (ii) the appearance of new RNase molecules with unique catalytic properties, take place in all host-pathogen combinations studied thus far with taxonomically different rust fungi and such divergent host plants as flax, Ribes, pine and wheat. It was concluded from some observations of the flax-rust system that the early phase of increase in RNase activity may primarily represent a non-specific host response since it occurs in susceptible as well as resistant varieties of flax following

370

A. E.Harvey

eta/,

inoculation with flax rust [7]. The results of the experiments on the thermal stability of RNase in Ribes and pine (Fig. 5) indicate that the early phase of RNase increase is accompanied by the appearance of increasingly temperature-labile RNase molecules which are detectable within the first 2 to 4 days after inoculation. These results suggest that unlike the increase in RNase activity in response to mechanical injury which is caused by a rapid synthesis of host type RNase [I, 71, the early phase of RNase increase in the inoculated tissue is at least in part due to the formation of new, highly temperature-sensitive RNase molecules. This conclusion has also been supported by the finding that in inoculated wheat leaves, the early phase of RNase increase is associated with the appearance of new catalytic properties as revealed by a significant change in the substrate specificities of the enzyme [Z]. Thus, at least a part of the early phase of RNase increase is elicited specifically by host-pathogen interactions. The results of the experiments on the blister rust mycelium-pine tissue culture combination suggest that quantitative and qualitative changes that occur in this in vitro system closely follow the pattern observed in various other naturally infected host plants [Z, 71. While tissue cultures offer many advantages over the morphologically complex whole plants as experimental material, it should be pointed out that we have included this artificially grown combination in this study simply as a model system to monitor the changes and compare these with those observed in naturally infected flax cotyledons and Ribes leaves. It is also necessary to emphasize that the effects observed in the artificial host-rust complex may or may not reflect the changes that presumably occur during blister rust-pine interactions in vivo. In these studies, the pine tissue is grown on culture media that contain 2,4-dichlorophenoxyacetic acid and kinetin. Some growth regulators have been reported to cause a decrease in the RNase levels of plant tissues [S, 91. A summary of the data on qualitative changes in the RNase activity of rustinfected flax [7j, Ribes and pine tissue culture is presented in Table 4. A comparison of the RNases from healthy and infected plants of each combination reveals TABLE A comparison

4

of the qualitative changes in the R.No.re of $a+rust, R&es-blister pine (tissue culture)-blister rust combinations Flaxa

Properties

of RNase

Stability (%of original activity destroyed at 80 “Cl, 10 mm) DEP sensitivity (% inhibition by DEP) Relative rates of hydrolysis (disint/min/mg protein x 10m3) of PONY (A) POlY WJ) POlY 0

rust and white

Ribes

Healthy

Infected

15

50

24

305 366 247

Pine Infected

Healthy

25

50

0

25

77

79

60

92

55

320 370 1700

3015 1628 115

3563 1450 503

144 194 42

36 0 31

Healthy

a Infected samples were: flax and Ribes, 8 and 16 days after inoculation pine tissue culture, 64 days after the host-rust culture had been initiated. cell-free supernatant fraction was used for assaying the RNase activity.

respectively; In all cases, the

Infected

RNase changes in rust infected Ribes and pine

371

the properties of the new RNase. The data show that the new RNase molecules in all these combinations are characterized by a decrease in stability at 80 “C. Other changes in the properties of this enzyme in the various combinations are: (a) flax-rust: increases in DEP sensitivity and poly(C) depolymerizing activity; (b) Ribesrust: a decrease in DEP sensitivity and an increase in the rate of poly(C) hydrolysis and (c) pine-rust: a decrease in DEP sensitivity, a decrease in the rates of poly(A)and poly(C) hydrolysis and the complete loss of poly(U) hydrolysing activity. These findings suggest that the exact nature of the changes in properties of RNase is unique to each host-rust combination, although the pattern of a biphasic increase in the activity of this enzyme in various combinations is quite similar. The observed changes in the properties of RNase brought about in two different hosts (Ribes and pine tissue) by the diploid and haploid phases respectively of the same rust (Cronartium) indicate that it may be the host plant that plays a major role in determining the properties of the new RNase molecules that are formed in the infected tissue. This possibility will be explored in a subsequent paper. This study was supported by grants from the National Research Council of Canada to M. S. A. E. H. was a visiting research associate during the tenure of this work. REFERENCES 1. BAGI, G. & FARKAS, G. L. ( 1967). On the nature of increase in ribonuclease activity in mechanically damaged tobacco leaf tissue. P&ochemistry 6, 161-169. 2. CHAKRAVORTY, A. K., SHAW, M. & SCRUBB, L. A. (1974). Ribonuclease activity of wheat leaves and rust infection. .ivature, 247, 577-580. 3. COFFEY, M. D., BOSE, A. & SHAW, M. (1970). In vitro culture of the flax rust, Melarnpsora lini. Canadian Journal of Botany 48, 773-776. 4. HARVEY, A. E. (1967). Tissue culture of Pinus monticola on a chemically defined medium. Canadian 30uml of Botany 45, 1783-l 787. of the rust fungus Cronartium ribicola in tissue 5. HARVEY, A. E. & GRASHAM, J. L. (1969). G rowth cultures of Pinus monticola. Canadian 3ournd of Botany 47, 663-666. 6. ROHRINCER, R., SAMBORSKI, D. J. & PERSON, C. 0. (1961). Ribonuclease activity in rusted wheat leaves. Canadian Journal of Botany 39, 775-784. 7. SCRUBB, L. A., CHAKRAVORTY, A. K. & SHAW, M. (1972). Changes in the ribonuclease activity of flax cotyledons following inoculation with flax rust. Plant Physiulogy 50, 73-79. 8. SRIVASTAVA, B. I. S. (1968). Acceleration of senescence and of the chromatin associated nucleases in excised barley leaves by abscisin II and its reversal by kinetin. Biochimica at biophysics Acta 169,

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20, 1112-l

Indoleacetic 119.

acid-induced

decrease

of ribonuclease

activity.

Physidogia