Induction of tobacco chlorosis by certain cucumber mosaic virus satellite RNAs is specific to subgroup II helper strains

VIROLOGY 176,292-295

(1990)

Induction of Tobacco Chlorosis by Certain Cucumber Mosaic Virus Satellite RNAs Is Specific to Subgroup II Helper Strains DAVID E. SLEATAND PETER PALUKAITIS’ Department

of Plant Pathology, Received November

Cornell University,

Ithaca, New York 14853-5098

3, 1989; accepted January 26, 1990

Two satellite (sat) RNAs of cucumber mosaic virus (CMV), B2- and WLS-sat RNAs, which induce systemic chlorosis on tobacco, were inoculated onto tobacco with a number of CMV strains. Systemic chlorosis was observed only when these satellite RNAs were associated with subgroup II CMV strains. Infection of tobacco with various pseudorecombinants of subgroup I and II CMV strains, together with WL3- or BP-sat RNA, suggests that chlorosis is associated with RNA 2 of subgroup II CMV strains. Chlorosis was not induced when B2- or WL3-sat RNAs were inoculated onto tobacco with tomato aspermy virus. In contrast, the induction of chlorosis on tomato by Bl-sat RNA did not show any clear dependence on the subgroup of its CMV helper strain although chlorosis did tend to be more severe in association o 199oAcademic press. ~nc with subgroup II CMV strains.

was confirmed by dideoxynucleotide chain-termination sequencing (12) before reinoculation. Satellite RNAs were inoculated onto tobacco or tomato plants at a concentration of 5-10 pg/ml with 300-500 pg/ml of each of the different helper virus strains tested, in 50 mh/l sodium phosphate, pH 7.0. Tobacco and tomato plants were kept in the dark for 24 and 48 hr, respectively, then dusted with Carborundum before inoculation. Plants were tested for the presence of satellite RNA 1O-l 4 days after inoculation using a satellite RNA-specific cDNA probe. Plants were kept in growth chambers at 25” with a 16-hr photoperiod and symptoms were observed for 14-2 1 days after inoculation. CMV strains have been classified into two subgroups based on the ability of the total RNA of any given strain to hybridize to cDNAs of either Fny-CMV, a subgroup I strain, or WL-CMV,* a subgroup II strain (13). This classification system is supported by the observation of two distinct CMV serotypes (14, 15), by competitive rehybridization of double-stranded CMV RNAs (16), and by nucleotide sequence comparisons (17-21). However, while subgroup I strains tend to induce more severe symptoms on some hosts than subgroup II strains (73; P. Palukaitis, unpublished data), no other distinct pathological differences between the subgroups have been observed. The CMV strains tested have been described previously (13) with some exceptions. SB-CMV, a subgroup II isolate (M. Roossinck and P. Palukaitis, unpub-

Satellite RNAs of cucumber mosaic virus (CMV) (I) can influence the pathology of their associated helper viruses in a number of ways on different host plant species. Different satellite RNAs with the same strain of helper virus can attenuate symptoms (2-4), induce lethal necrosis (5, 6), induce chlorosis (3, 4, 7, 8), induce stunting, or cause combinations of these effects (9). Other satellite RNAs may have no effect on CMV disease expression (4, 9). These small (approximately 330-370 nucleotides), single-stranded RNA molecules consequently have been the focus of considerable attention as model systems of relatively small genome size that can induce severe pathogenic effects. We have examined the ability of CMV satellite RNAs, in association with a number of different CMV helper strains, to induce chlorosis on tobacco and tomato plants. Three satellite RNAs were used: (i) B2-sat RNA, which induces chlorosis on tobacco (4); (ii) Bl -sat RNA, which induces chlorosis on tomato (4); and (iii) WL3sat RNA, which was originally isolated from tobacco infected with WL-CMV (8) and which induces chlorosis on tobacco while differing from B2-sat RNA at 6 nucleotide positions (our unpublished data). Each satellite RNA was propagated in association with the LS strain of CMV. Virus was purified from tobacco (70) and extracted with phenol/chloroform to isolate the RNA (7 1). Satellite RNAs were then separated from the viral genomic RNAs by electrophoresis on 6% polyacrylamide gels and purified by the excision and elution of the appropriate gel-band after staining with toluidine blue (3). Each satellite RNA sequence

’ Subgroup II CMV strains. or individual RNAs derived from subgroup II strains within pseudorecombinant strains, hereafter are indicated in boldface.

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SHORT COMMUNICATIONS TABLE 1 SYMPTOMS INDUCED ON TOBACCO BY WL3- AND B2-sat RNAs WITH SUBGROUP I AND II CMV STRAINS

Virus strain

Virus/B2-sat

Virus alone

Subgroup I Fny-CMV Pf-CMV Uh-CMV M-CMV

Mosaic; rugosity; dark-green (As for Fny-CMV) (As for Fny-CMV) Severe chlorosis

Subgroup II LS-CMV SB-CMV U-CMV WL-CMV Q-CMV 2A-CMV

Very mild mosaic; or no symptoms Very mild mosaic Very mild mosaic Very mild mosaic Symptomless Very mild mosaic

islands

lished data), came from squash plants in New York. UCMV, 2A-CMV, and the various pseudorecombinants were obtained from Dr. R. Francki (University of Adelaide). WL3- and B2-sat RNAs were inoculated onto tobacco with a number of CMV strains of both subgroups. The results of these infectivity assays are presented in Table 1. WL3- and B2-sat RNAs induced severe interveinal chlorosis on tobacco only in the presence of subgroup II CMV strains (i.e., LS-, SB-, U-, WL-, Q-, and 2A-CMV) (Table 1). By contrast, B2- and WL3sat RNAs tended to attenuate symptoms induced by subgroup I CMV strains. This was particularly evident with M-CMV, a subgroup I strain that causes severe yellow/white chlorosis on tobacco in the absence of a satellite RNA. In the presence of either of the two satellite RNAs tested, M-CMV symptoms were modified to a severe chlorosis of the veins with little interveinal chlorosis. The ability of CMV strains to induce chlorosis on tobacco with either WL3- or B2-sat RNA therefore appears to represent a clear difference in pathogenicity between the two CMV subgroups. WL3- and B2-sat RNAs were also inoculated onto tobacco with a number of pseudorecombinant CMV strains to determine which RNA(s) of the subgroup II strains would allow an interaction with either satellite RNA leading to chlorosis. The results of these analyses are presented in Table 2. Although not all combinations of pseudorecombinants between thevarious CMV strains were available, a number of conclusions can still be drawn from these results: (i) The presence of RNA 1 of subgroup II strains within a pseudorecombinant was not specifically required for chlorosis; e.g., K,U&-CMV induced chlorosis as severe as that caused by U-CMV. This observation was confirmed by the fact that the complementary

Mild mosaic Mild mosaic Mild mosaic Vein-yellowing; chlorosis Severe Severe Severe Severe Severe Severe

chlorosis chlorosis chlorosis chlorosis chlorosis chlorosis

RNA

mild interveinal

VirusNVL3sat

Mild mosaic Mild mosaic Mild mosaic Vein-yellowing; chlorosis Severe Severe Severe Severe Severe Severe

RNA

mild interveinal

chlorosis chlorosis chlorosis chlorosis chlorosis chlorosis

pseudorecombinant, U,K,K,-CMV, did not induce chlorosis with either satellite RNA. (ii) The presence of RNA 3 from a subgroup II strain was not solely sufficient to induce chlorosis, since the infection of tobacco with the pseudorecombinants T,T& and K,K&CMV with either WL3- or B2-sat RNA did not result in a chlorotic response. (iii) Pseudorecombinants Q,Q2G3and Q1Q2M8-CMV, which contain RNAs 1 and 2 from a subgroup II strain, induced chlorosis with WL3- and B2-sat RNAs. These results imply that the ability of the subgroup II CMV strains to support satellite RNA-dependent chlorosis is associated with RNA 2 alone, since RNA 1 from a subgroup II CMV strain within a pseudorecombinant was not specifically required for chlorosis induction (see above). No clear pathogenic differences between the CMV subgroups were observed with the induction of chlorosis by Bl -sat RNA on tomato (Table 3) although chlorosis did tend to be more pronounced with B 1-sat RNA and the subgroup II CMV strains. Bl-sat RNA tended to attenuate the fernleaf and stunting caused by subgroup I CMV strains, and, in some cases, caused some degree of veinal chlorosis or vein clearing. In contrast, Bl-sat RNA tended to exacerbate symptoms caused by subgroup II strains, in addition to causing severe chlorosis. This was particularly marked with LS-, SB-, and K,U,U,-CMV, which cause relatively mild symptoms in the absence of satellite RNA. In the presence of Bl -sat RNA, these CMV strains induced very severe chlorosis, fernleaf, severe stunting, and pronounced leaf distortion. It is possible that the other symptoms induced on these plants are simply a consequence of the severe chlorosis. The effects of WL3-sat RNA on symptoms induced by another cucumovirus, tomato aspermy virus (TAV),

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SHORT COMMUNICATIONS TABLE 2 SYMPTOMStNoucEo ON TOBACCO BYWL3- AND B2-sat RNAs WITH PSEUDORECOMBINANTS OF CMV STRAINSOF BOTH SUBGROUPS CMV stratn U,U,U,-CMV” U,K,K,-CMV K,U&CMV K,K&CMV Q,Q,Q,-CMV Q,Q*G&MV M,M,M,-CMV Q,QPM&MV T,TPQ&MV

’ RNAs derived from subgroup strains of the pseudorecombinants their own satellite RNA.

Virus/B2-sat

Virus alone

Severe chlorosis Mild mosaic Severe chlorosts Mild vein-clearing; Severe chlorosis Severe chlorosls Severe mosaic Severe chlorosis Mosaic

Very mild mosaic Severe mosaic Mosaic Mosaic Symptomless Mild mosaic Severe chlorosis Mosaic Mosaic

RNA

mosaic

Virus/WLB-sat Severe chlorosls Mild mosaic Severe chlorosis Mild vein-clearing; Severe chlorosis Severe chlorosis Severe mosaic Severe chlorosis Mosaic

RNA

mosaic

II CMV strains are indicated by bold type. U,lJ&CMV is equivalent to U-CMV. A number of the parental were not tested with WL3- and B2-sat RNAs because they either were not available or already contained

on tobacco were also examined. Chlorosis was not induced and the TAV symptoms, which are severe mosaic, stunting, and leaf-distortion, were attenuated (data not shown), an effect which was previously observed with D- and K-sat RNAs (22). In this respect at least, TAV gave the same results as the subgroup I CMV strains. Y-sat RNA induces chlorosis in tobacco in association with the subgroup I strain, Y-CMV (7). Therefore, the mechanism by which this satellite RNA induces chlorosis may be different from that of WL3- and BZsat RNA. This difference may reflect the sequence differences between Y-sat RNA and the satellite RNAs examined here; however, WL3- and B2-sat RNAs should be tested with a satellite RNA-free isolate of YCMV before such conclusions can be drawn. Moreover, if such a difference in subgroup specificity between Y-sat RNA and WL3- and B2-sat RNAs should

be confirmed, sequence comparisons may prove valuable in the characterization of a sequence within satellite RNAs that dictates the strain-specificity of chlorosis. The interaction between a satellite RNA and its helper CMV strain that results in chlorosis can therefore be addressed in two ways: (i) by determination of the sequences within the satellite RNA that determine strain-specificity of chlorosis, and (ii) by determination of the sequences within the subgroup II CMV strains that are required for such an interaction. The latter question might best be examined by pseudorecombination and recombination of biologically active clones of RNA 2 from CMV strains of both subgroups. Such a strategy does assume that a recombinant subgroup I/ II RNA 2 would be biologically viable, which may not be the case given the differences between the CMV subgroups at the nucleic acid and amino acid se-

TABLE 3 EFFECTOF Bl-sat RNA ON SYMPTOM INDUCTIONON TOMATO BY DIFFERENTCMV STRAINSAND PSEUDORECOMBINANTS Virus strain

Virus alone

Virus/B1 -sat RNA

Subgroup I Fny-CMV Pf-CMV Uh-CMV

Severe stunting; fernleaf; mosaic Severe stunting; fernleaf; leaf-distortion (As for Fny-CMV)

Slight mosaic; mild chlorosis; slight stunting; fernleaf Slight mosaic only Slight stunting; veinal chlorosls; fernleaf

Subgroup II LS-CMV SB-CMV WL-CMV

Slight stunting; mild fernleaf Stunting; fernleaf Stunting; severe fernleaf

Severe chlorosis; stunting; leaf-distortion Severe stunting; severe chlorosis; leaf-distortion Stunting; mild fernleaf; mild veinal chlorosis

Pseudorecombinants U,K,K,-CMVa K,U&CMV

Severe stunting; fernleaf Stunting; fernleaf

Yellow chlorosis only Very stunted; very severe chlorosis;

’ RNAs derived from subgroup II CMV strains are indicated by boldface.

severe leaf-distortion

SHORT COMMUNICATIONS

quence levels (17). However, determination of the sequences within both the viral and the satellite RNAs that together result in a chlorotic phenotype may prove an important step in determining how satellite RNAs modify CMV symptoms, and, more generally, how many plant viruses induce disease states. ACKNOWLEDGMENTS This work was supported by a grant from the Cornell Biotechnology Program which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, the U.S. Army Research Office, and the National Science Foundation, and in part by Grant DE-FG02-86ER13505 from the Department of Energy. The authors thank Susan Potts for her excellent technical assistance.

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