BIOLOGICAL CONTROLOF BLACKLINE DISEASE OF ENGLISH WALNUT

George Bruening, John Mircetich, Stephen D. Daubert, Adib RowhaniGale McGranahan,AbhayaM. Dandekar, Charles Leslie

ABSTRACT

The aim of research under project number 87 WMB 2 is to develop measures

for the biological control of cherry leafroll virus (CLRV), the causativeagent of walnut blackline (WBL). Researchers in the Department of Plant

Pathology and in the Department of Pomology, UCD, are collaborating to

explore control measures applicable to both newly planted walnuts and

existing stands. WBL results in the death of a narrow strip cambium and

phloem tissue at the rootstock-scion union of CLRV-infected English walnut

propagated on Northern California Black or Paradox rootstock. Our

principal approach to the control of CLRV is based on an earlierobservation that a small RNA molecule, the satellite RNA of tobacco

ringspot virus (STobRV RNA) , is able to interfere with CLRV in cells towhich STobRV RNA and CLRV were co-inoculated. Previous results imply thatSTobRV RNA introduced and maintained in all or most of the cells of walnut

tissue will make that tissue resistant to CLRV. We report recent results

on (1) the protection of walnut against WBL when STobRV RNA was co-inoculated with CLRV, (2) the first evidence of the expression ofSTobRV RNAs in transformed walnut tissue, and (3) progress developing

assay systems.

OBJECTIVE

The agent of walnut blackline (WBL) is a pollen-, seed- and graft-transmitted strain of cherry leafroll virus (CLRV; Mircetich et al.,

1980ab, Mircetich and Rowhani, 1984). New approaches to the control ofCLRV are suggested by (1) the recent development of procedures fortransforming and regenerating walnut and (2) recent observations of the

virus-restricting capabilities a satellite RNA molecule. Our three-yearobjective is to develop biological measures that can reduce or prevent

damage by CLRV. Most of our effort is concerned with controlling CLRVitself. We are attempting to introduce satellite RNA molecules into cells

that otherwise would be susceptible to the virus. Engineering walnut to

express satellite RNA from sequences installed in walnut DNA is likely tobe the most cost-effective way of accomplishing this. As indicated in our

research proposal, we also are attempting to use a virus to spreadsatellite RNA in walnut tissue and plan to test a natural variant ofwalnut rootstock which appears to tolerate CLRV better rootstocks now in

common use. It is our aim to design control measures that will beapplicable not only to new walnut stock but also to existing stands.

CLRV is a member of the nepovirus group of plant viruses. Another member,tobacco ringspot virus (TobRV), is sometimes found to be associated with a

small satellite RNA, the satellite RNA of tobacco ringspot virus, referredto hereafter as STobRV RNA. STobRV RNA is one of the most well-studied of

the plant virus satellite RNAs. TobRV is a supporting virus forSTobRV RNA: STobRV RNA replicates only in cells that are infected withTobRV.

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As was reported earlier (F. Ponz et al., 1987), CLRV does not support thereplication of STobRV RNA in the herbacious host cowpea (Vi~naun~uiculata). Nevertheless, STobRV RNA interfered with CLRV accumulation

in cowpea leaves co-inoculated with CLRV and STobRV RNA. CLRV was able tospread to and multiply in leaves that had not been inoculated withSTobRV RNA. This is expected since CLRV does not support the replication

and spread of STobRV RNA and therefore STobRV RNA does not reach any other

than the initially inoculated leaf cells.

An unusual activity of STobRV RNA, used in the constructions described

under RESULTS, is the ability of the RNA to cut itself at a particularphosphodiester bond, the "junction" or "J,I'of the diagrams belm,'. In the

upper diagram, "M" represents one "monomeric" sequence, that is, the 360

nucleotide residue sequence of STobRV RNA as is it produced in a TouRV-supported infection. The self-cutting reaction generates two copies of

RNA M and one copy each of the two terminal STobRV RNA fragments. P and D.

RNA P and RNA D, would together are equivalent to one, unpermuted RNA ~sequence if they were connected in the order RNA D-R~A P. Hence, the

starting sequence in the upper diagram is referred to as a trimericconstruction.

P M M DJ J J----------

P M M D-------------- -------------- ----------

The self-cutting reaction actually requires only a short sequence of

nucleotide residues near the junction (Buzayan et al., 1986). Thus, onepermuted monomer sequence, P-D, also cuts itself in a reaction thatgenerates one RNA P and one RNA D.

P DJ----------

P D-+ ----------

Not only was the accumulation of CLRV reduced when co-inoculated with

STobRV RNAj the translation of CLRV RNAs in vitro also was greatly reducedby STobRV RNA. This provides a possible mechanism for the anti-CLRV

action of the satellite RNA: interference with the production of CLRV-

specified proteins. Results from federally-supported research in theBruening laboratory in the past year show that RNA D alone is sufficientto interfere with the translation of CLRV RNAs.

Stated objectives for the second year were (1) to confirm the interference

STobRV RNA with CLRV in walnut, (2) to carry out initial transformation

experiments to determine the feasibility of generating STobRV RNA inwalnut cells, and (3) to continue to develop clones to detect CLRV and toimprove other assays.

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PROCEDUREand RESULTS

(1) Interference in walnut of STobRV RNA with CLRV. We presented lastyear preliminary results indicating reduced CLRV accumulation in walnutcambium tissue co-inoculated with CLRV and STobRV RNA. No informationwasavailableon the effects of STobRV RNA on induction of WBL.

Five 3-year old Hartley English walnut trees (Ju~lans re~ia) on NorthernCalifornia Black walnut ("Black," ~ hindsii) rootstock were inoculatedunder bark patches 10 cm above the graft union. One side of each trunkwas inoculated with CLRV alone, at 0.5 mg/ml. The other side wasinoculatedwith a mixtureof CLRV at 0.5 mg/ml and STobRV RNA at 0.3mg/ml. Inoculawere in 0.05 M sodium phosphate buffer, pH 6.5, containing26 mg/ml bentonite and 800 units/mlof the ribonucleaseinhibitorRNasin.

As is indicatedin Table 1, all five trees showed accumulationof CLRVbelow the patch inoculatedwith CLRV alone, as assessed by enzyme-linkedimmunosorbentassay (ELISA). At three months after inoculationfour ofthe five trees showed no significant increase of CLRV below the sitesinoculatedwith a mixture of CLRV and STobRV RNA.

Thus, at three months after inoculationsix of the ten inoculatedsitesresultedin infectionby CLRV. Taking the probability of infecting anysite as 0.60, the chance on a random basis of having the four uninfectedsites distributed entirely into the group of sites inoculated with CLRV +

STobRV RNA is only 0.01 according to a chi square test. The number ofdata points is considered to be adequatefor this test.

At nine months after inoculationone additionalsite had become infected

according to ELISA (Table 1). We found the same tally at the time of thefinal ELISA, 15 months after inoculation. The statistical analysis asdescribedin the previous paragraph indicatedthat, on the assumptionthatany site has a 0.70 chance of giving rise to a CLRV infection, theprobability of obtainingthe distributionseen in the last two columns ofTable 1 strictlyby chance is only 0.05.

When bark was removed from the regionof the graft union, we found noblackline symptom for any of the three sites that were negative for CLRVaccording to ELISA.. All sites positive for CLRV had WBL. Thus thecambium core samples taken at 9 and 15 months after inoculation predictedblackline symptoms found by inspection at 15 months. The abilityofSTobRV RNA to slow or prevent infectionof walnut by CLRV and to preventthe developmentof WBL symptomsappearsto be confirmed.

As we indicated in our report of last year walnut trees also wereinoculated with TobRV, with the intent of supporting STobRV RNAreplication. Our assays during the past year have confirmed that thesetrees had not become infected with TobRV.

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f

Table 1Protection of walnut trees against CLRV by satellite RNA of TobRV

Footnote: Trees were inoculated on 6/17/87. Assay was by ELISAof cambium scrapings from an approximately 1 cm diameter

disk cut from below the inoculation patch. The bark diskwas cut just above the graft union in the 1987. For the

1988 assay the bark disk was cut from the region of the

graft union and not below the site of the 1987 sample.Assignment of results to the category of "not infected" wason the basis of ELISA values comparable to those obtainedfrom uninoculated trees.

(2) Transformation of walnut tissue with STobRV RNA seQuences. Initial

constructions for transforming walnut embryos have either a "monomeric"

(plasmid pW1) or "trimeric" (plasmid pW2) satellite RNA sequence, as

illustrated in OBJECTIVE. They also contain a chimeric kanamycinresistance gene to allow transformed walnut embryos to be selected.

Somatic embryos were obtained from the repetitively embryogenic line SU-2,derived from an open pollinated seed of the English walnut cultivarSunland. About 250 somatic embryos were exposed for 10 min to 2.5 x 108

colony forming units/ml of A~robacterium tumefaciens bearing either pW1 orpW2, as described (McGranahan et al., 1988). Embryos were plated on solidbasal DKW medium (Driver and Kuniyuki, 1984) containing100 uMacetosyringone, a compound that induces the DNA-transfering capability ofthe bacterium. After 24 hr (A~robacterium inoculation period), embryoswere transferred to solid basal DKW medium containing 500 mg/l cefotaximeto inhibit further growth of bacteria.

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Assay of 9/14/87 Assay of 9/30/88

Tree Not NotInoculum No. Infected Infected Infected Infected

CLRV +STobRV RNA 1 0.14 0.07

2 0.17 0.053 0.15 1.344 0.21 0.065 1.51 1.12

CLRV 1 1.78 0.862 1.41 0.913 1.23 1.23

, 4 > 2 1.705 1.83 1.46

Half of the embryos were exposed to 100 mg/l kanamycin after two weeks,the other half after four weeks. Initial selection for transformation is

based on the ability to continue to produce new embryos. After three

months 13 pW1-derived embryo lines showed continued embryogenesis. Two of

these (WP-96 and WP-180) were clearly less sensitive than the others. Allof these are being tested for their ability to generate satellite RNA

sequences by "northern blot" analysis and for the arrangement of satellitesequences in the nuclear DNA of the cells.

Of the 13 pW1-derived embyro lines, WP-118 and WP-180 were found to besatellite sequence-expressing. Thus we have our first indication that the

STobRV RNA sequences are not incompatible with walnut cells. WP-118 andWP-180 are being cultured for production of green shoots for further

testing.

The trimeric pW2-derived embryogenic cultures are under selection and will

be analyzed as described above for the pWl-derived cultures.

A new set of plasmids has been constructed. These plasmids are designedto reduce the time and effort, and increase the accuracy of the selection

process. They will simultaneously transfer three genes to the walnut

embryo, not only the chimeric kanamycin-resistance gene and the monomericor trimeric satellite RNA constructions, but also a beta-glucuronidase

gene (GUS). The GUS gene product will convert a chromogenic, glucuronic

acid-containing substrate into an intensely blue-colored product. We

already have tested the GUS marker as an independent construction and haveshown that it is actively expressed in transformed walnut embryos.

The new, "triple-threat" plasmids proved to be difficult to construct.

There are many possible arrangements of kanamycin, satellite RNA and GUS

sequences and of the plant promoter and 3' sequences necessary for theirfunction, and the several arrangements initially tested proved to be

unstable. We anticipate that GUS activity derived from the new plasmidswill allow us to identify much more readily the transformed embryogenic

cultures. Initial transformation experiments are in progress.

(3) Improvin~ assays. The experiments described in part (1) of thissection gave us two experimental points per tree. We wished to increasethe number of assays per tree. We have grafted rectangular patches ofBlack or Paradox (~ regia x ~ hindsii) into trunks of English trees.The trees were inoculated no only at the usual site above the graft union

but also at sites above each patch derived from Black or Paradox.Spacings between the site of inoculation and the patch or graft union were

5 cm or 10 cm, the former distance designed in decrease the time to WBLdevelopment. This approach triples the number of inoculation sites that

can be accommodated conveniently on a tree. Unfortunately, the CLRV used

in these experiments appeared to be less potent than expected, and too few

of the sites have become infected to draw any conclusion at this time.

However, we did learn that it is critical to use large (several cm longand wide) bark patches to foster consistent taking of the graft.

An important component of this research is the assessment of regenerated

walnut trees for resistance to CLRV. The usual inoculation procedure

requires that the cambium of saplings be inoculated. There was a question

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as to what state of development a regenerated walnut tree must be takenbefore its CLRV-resistance can be tested. We suspected that walnut

shoots, freshly regenerated from embryos and unhardened, may be

susceptible, perhaps more susceptible than seedlings. Such shoots, leaf-inoculated with purified CLRV, showed no symptoms. However, ELISA

analysis for CLRV antigen even 2 months after inoculation revealed thatthey were uniformly infected and producing a high titer of CLRV. Thisresult shows that the ELISA signals were from replicated CLRV rather thanfrom the inoculum.

CONCLUSIONS

We have confirmed the ability of STobRV RNA to interfere with the usual

increase of CLRV in inoculated cambium of English walnut. In addition, we

have shown for the first time that symptoms of WBL did not develop inthose trees in which STobRV RNA had restricted the CLRV increase. In an

experiment critical to the future of this research, transgenic walnut

tissue expressed STobRV RNA sequences. We found that walnut shoots newlyregenerated from non-transformed embryos are suitable test hosts for CLRV.

Therefore, we have established the necessary background and methods togenerate and test STobRV RNA-expressing walnut shoots for their expectedresistance to CLRV.

REFERENCES

Buzayan, J.M., Gerlach, W.L., and Bruening, G. 1986. Satellite tobaccoringspot virus RNA: A subset of the RNA sequence is sufficient forautolytic processing. Proc. Nat. Acad. Sci. USA 83:8859-8862.

Driver, J.A., and Kuniyuki, A.H. 1984. In vitro propagation of Paradoxwalnut rootstock. Hortscience 19:507-509.

McGranahan, G.H., Leslie, C.A., Uratsu, S.L., Martin, L.A., and Dandekar,A.M. 1988. Agrobacterium mediated transformation of walnut somaticembryos and regeneration of transgenic plants. Bio/Technology 6:800-804.

Mircetich, S.M., DeZoeten, G.A., and Lauritis, J.A. 1980a.

natural spread of blackline disease of English walnut trees.Phytopathol. Acad. Sci. Hung. 15:147-151.

Etiology andActa

Mircetich, S. M., Sanborn, R. R., and Ramos, D. E. 1980b. Naturalspread, graft transmission, and possible etiology of walnut blacklinedisease. Phytopathology 70:962-968.

Mircetich, S. M., and Rowhani, A. 1984. The relationship of cherryleafroll virus and blackline disease of English walnut trees.Phytopathology 74:423-428.

Ponz, F., Rowhani, A., Mircetich, S. M., and Bruening, G. 1987. Cherryleafroll virus infections are affected by a satellite RNA that the virusdoes not support. Virology 160:183-190.

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