PHYSIOL. PLANT. 58: 523-526. Copenhagen 1983

Mechanism of parasitization by Cuscuta reflexa: RNase activityin the haustorial region of Cuscuta and infected host tissues

Anjali Gupta and Madhav Singh

Gupta, A. and Singh, M. 1983. Mechanisni of parasitization by Cuscuta reflexa:RNase activity in the haustorial region oi Cuscuta and infected host tissues. - Physiol.Plant. 58: 523-526. „. , • , - ...

In a study of host-parasite interrelationship and the mechanism of parasitization,ribonuclease activity was determined in Cuscuta reflexa Roxb, and in infected andcontrol host plants oi Lantana catnara L. In the haustoria-bearing region oi Cuscuta,the concave half of the vine bearing the haustorial site, always showed signifieantlyhigher RNase activity than the convex half, irrespective of the differing enzymeactivity of the parasite tissue growing on different hosts - Brassica catnpestris L.,Helianthus annutts L., Lantana catnara L., Medicago sativa L. and Solattwit nigrutn L.The uninfected host branch of L. catnara showed the maximum specific RNaseactivity in the apical region which decreased toward the base, while the infected hostbranch showed minimum specific RNase activity in the apical region, gradually in-creasing towards the infected region.

Additiottal key words - Angiosperm parasite, N'-benzyladenine, host-parasite in-terrelationship, Lantana catnara.

A. Gupta and M. Singh (reprint requests), Dept. of Bioehetnistry, Luckttow Univ.,Lttcknow-226007, Uttar Pradesh, India.

Introduction

The mode of haustorial contact by Cusctita with the hostsieve elements has been discussed by Dorr (1972). Thetip of such a hypha has dense cytoplasm and an abun-dance of organelles such as ribosomes, dietyosomes andmitochondria, indicative of a metabolically very aetivecell (Dorr 1969).

The eytokinin benzyladenine (BA) has been found tostimulate the eoiling and formation of haustoria on free-hanging vines oi Cuscuta chinensis (Paliyath et al. 1978)and Cuscuta catnpestris (Tsivion 1978) and also tostimulate nuelease and protease activities in bean leaves(Naito et al. 1979). Sinee BA may be regarded as aderivative of nueleotides, nucleases might play somerole during the development of haustoria or subsequentevents. Changes in the aetivity of RNase in the haus-toria-bearing region of Cuscuta reflexa and the infectedregions of the host plant are reported herein.

Received 1 February, 1983; revised, 8 April, 1983

Physiol. Plant. 58, 1983

Abbreviations - BA, N^-benzyladenine; RNase, ribonuclease;tRNA, transfer-ribonucleic acid.

Materials and methods

Three dozen of each host plant were grown in the De-partmental garden under similar natural environmentduring August to October (day temperature, from30-35°C). Half the number of plants were infected withCuscuta and the other half served as the control. Ac-tively growing branches of Lantana camara were in-fected with Cuscuta. The infected braneh (about 40 em)of the host was eut and divided into several parts,namely, apical, near-apical, near-infected, infected(bearing haustorial connections), below infected andlower regions as shown in Fig. 1. A similar braneh of theeontrol host plant was used for comparison. The parasi-tic curls were collected separately from several hostplants (L. camara, Helianthus annuus, Brassica cam-

523

pestris, Solanum nigrum, and Medicago sativa) and di-vided into two nearly equal longitudinal halves. Theinner half, which was in contact with the host and alsothe site of haustorial emergence was termed the "con-cave half" and the other as the "convex half".

In another set of experiments Cuscuta, growing on L.camara, was allowed to form curls and develop haus-toria-like structures on glassrods and was used forRNase assay in the eoneave and convex halves. The hosttissue used for RNase assay eonsisted only of thebranehes without the leaves.

Data represent the means of three sets of experimentswith three replieates in each.

Chemicals. RNA (Torula yeast, type VI Sigma, USA)was used as the substrate for RNase after purifieationwith ethanolic preeipitation. Other chemieals used wereof analytieal grade.

Preparations of RNase (EC 3.1.27.1). A 10% homoge-nate (w/v) was made in a Virtis homogenizer in the cold(0^°C) in a medium consisting of 0.1 M sodium acetatebuffer, pH 5.2, (experimentally determined optimumfor RNase aetivity) containing 0.015 M freshly neu-tralized cysteine hydrochloride. The homogenate wasfiltered through muslin eloth and centrifuged at 18 000g for 30 min in the cold (0-4°C) and the resultingsupernatant was used as the source of the enzyme.

RNase assay. RNase was assayed aeeording to themethod of Reddi (1966). One unit was that amount ofenzyme whieh eaused an increase of one absorbanceunit at 260 nm in 20 min at 37°C. The speeific activitywas expressed as the units of enzyme activity (mg pro-tein)"'. Protein was determined by the method of Lowryet al. (1951).

Results

RNase activity in different regions of infected and uninfectedbranches of L. camara

Figure 1 shows the RNase activity in the various regionsof the control and Cuscuta-infected branehes. On afresh weight basis, the control host branch showedmaximal enzyme aetivity in the apical region, decreasingtowards the base, but in the infeeted braneh, maximumaetivity oeeurred below the infected region, and wasnearly five times as much as that in the apical region.The enzyme activity deereased both towards the mor-phological apex and the base. The activities in the basaland apical regions were almost equal, whereas in theeontrol branch, the apical region had about six timesmore aetivity than that in the most basal region tested.

The specific activity of RNase in the infeeted hostshowed a similar pattern, with maximum aetivityoccurring below the infected region. Contrariwise, thecontrol host branch showed maximum speeifie activity

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Fig. 1. RNase activity in the infected and control host branch ofL. camara. Each value is the average of three separate samplesand bars indicate the SD.

in the apieal region, gradually decreasing towards themorphological base.

The activity of RNase in the two longitudinal halves ofhaustoria-bearing region of Cuscuta

On a fresh weight basis, the RNase activity was signifi-cantly higher in the concave half than in the eonvex halfof Cuscuta growing on the different hosts analysed(Tab. 1).

When Cuscuta growing on L. camara was allowed toeurl on a glass-rod, it developed struetures resemblinghaustoria. Again the concave half showed significantlyhigher RNase activity (both total and specific) than the

524 Physiol. Plant, 58, 1983

Tab. 1. The activity of RNase in the two longitudinal halves of haustoria-bearing region of Cuscuta, curling on different hosts andalso while curling around a glass-rod with L. camara. Values are means of three sets of experiments with three replicates in eachset, ±SD.

Host plants

L. catnara

Curled around aglass-rod(Host L. catnara)

H. annuus

B. campestris

S. nigrutn

M. sativa

The concave half of haustoria-bearingregion of C. reflexa

Activityunit (g FW)-'

322±10.70

305± 5.10

500±11.50

490± 6.10

520± 7.10

560± 10.50

mg protein(g FW)-'

11.1

8.906.40

5.30

5.30

7.50

Sp. aetivityunit mg-'

29.0±0.96

34.3±0.57

78.0+1.79

93.0±1.1598.0±1.33

75.0± 1.40

The convex half of haustoria-bearingregion of C. reflexa

Activityunit (g FW)-'

154± 8.10

249± 4.80

345±14.80

332±10.50370±11.60

430± 9.60

mg protein(g FW)-'

9.30

8.40

5.504.70

4.10

6.30

Sp. activityunit mg-'

16.6±0.87

29.6±0.57

63.0±2.6571.0±2.23

90.0±2.83

68.0 + 1.52

eonvex half, though the difference in the two halves wassomewhat lower when the parasite was curling on aglass-rod than when on a living host branch.

It is interesting to note that the protein eontent was inall eases higher in the eoneave halves than in the convexhalves. This lowered the magnitude of the differenee inthe specific activity between the two halves.

Discussion

Host-dependent variations in the bioehemieal eomposi-tion, including some enzymes in Cuscttta, 1 are wellknown. Singh and Krishnan (1971), while showing var-iation in nuclease activity along the length of Cuscutavine, overlooked the signifieance of the length of thevines. The present data, based on more careful experi-mentation, show greater RNase aetivity in those regionsof Cuscuta bearing haustoria as compared to the haus-toria-free regions. The physiological significance of thehigher RNase activity in the eonvave half of the parasi-tic curls, the site of haustorial emergenee, is unclear.However, sinee the transmission of virum (Hollings etal. 1976) and of mycoplasma (Cazelles 1978) throughthe Cuscuta bridge is well established, the low-molecu-lar weight host RNA may easily be translocated to theparasite as such or in the form of oligonucleotides afterdegradation by RNase. In case the host RNA is notcompatible with the parasite, the high RNase aetivity inthe haustorial region may degrade the host RNA, pro-viding precursors for the parasite.

When the host tissue was freed of parasitic curls, partof the haustoria embedded in the host was invariablyleft behind and this could have been responsible, at leastpartly, in contributing to the high RNase activity in thisregion of the host. Therefore, this high RNase activitywould have helped in degradation and translocation of

host RNA to the parasite during its live-contact with thehost.

Lower RNase activity in the apical region of the in-fected host, compared to the control host plant, mightbe due to the lower metabolic activity of this regionbecause of the transfer of xylem constituents to theparasite including eytokinin (Tsivion 1978). Cuscuta haslower endogenous eytokinin levels than the host plants(Jacob et al. 1975) and the haustoria have higher adap-tational demand for eytokinin (Tsivion 1978). There-fore, acropetally translocated eytokinin (Ramina et al.1979) may be transferred from host to parasite throughthe xylem (Kuijt and Toth 1976, Tsivion 1978). Thetransfer of eytokinin (or even tRNA) from the host toparasite is likely to decrease the protein synthesis in theinfected host shoot. The flow of sugars, amino aeids andother nutrients to the region of high eytokinin concen-tration (Mothes and Engelbrecht 1961) will also affectthe metabolic aetivity of the host shoot-apex.

The greater RNase activity in the concave halves ofCuscuta vine, eurling either on a host plant or on aglass-rod, may be the result of a higher eoneentration ofeytokinin in this region. Because of the acropetal trans-location of eytokinin from host to parasite, eytokininmight be transloeated from the first haustoria-bearingregion, on a living support, to the second haus-toria-bearing region, on the non-living support (viz. aglass-rod), in the same vine of Cuscuta.

Though varying from host to host, the RNase activityof the parasite was always higher in the eoneave than inthe convex half.

Acknowledgements - One of the authors (A.G.) is a recipientof a Junior Research fellowship of I.C.A.R., New Delhi, undera research scheme. The generous aid of the UNDP/UNESCOand UGC, New Delhi, under the Programme of Special Assis-tance to this department is gratefully acknowledged. The au-

Physiol. Plant. 58, t983

thors are also grateful to Professor G. G. Sanwal for his keeninterest and helpful discussion.

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Edited by R. Rajagopal

526 Physiol. Plant. 58. 1983