Pathogenic interactions between variants of cauliflower mosaic virus and Arabidopsis thaliana

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1 Journal of Experimental Botany, Vol. 49, No. 321, pp , April 1998 Pathogenic interactions between variants of cauliflower mosaic virus and Arabidopsis thaliana Edi Cecchini1, Nadia S. Al-Kaff2, Andrew Bannister2, Maria E. Giannakou1, Don G. McCallum2, Andrew J. Maule2, Joel J. Milner1 and Simon N. Covey2,3 1 Plant Molecular Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Glasgow University, Glasgow G12 8QQ, UK 2 Department of Virus Research, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK Received 29 September 1997; Accepted 18 November 1997 Abstract Pathogenic interactions between genetic variants of cauliflower mosaic virus (CaMV) and Arabidopsis thaliana were characterized to identify combinations potentially useful in molecular genetic analysis. Infections of a glabrous mutant (gl1) ofarabidopsis ecotype Columbia (Col-0 gl1) by 30 CaMV isolates were assessed by recording symptom character. Thirteen isolates failed to cause symptoms; the remainder induced symptoms that varied between mild and very severe. Some CaMV isolates produced symptoms in Arabidopsis that differed significantly in severity or character from those produced in a standard host Brassica rapa (turnip). A greater variety of symptom types was observed in a single Arabidopsis ecotype infected with a range of CaMV isolates than was found in a range of Arabidopsis ecotypes infected with a single, typical CaMV isolate (Cabb B-JI). One isolate, Bari-1, that was asymptomatic but accumulated virus in Arabidopsis ecotype Col-0 gl1, caused mild symp- toms in ecotype Ler gl1. A hybrid virus constructed from CaMV isolates Cabb B-JI and Bari-1 produced symptoms in Arabidopsis variants that were more severe than in either parental isolate. From a screen of EMS-mutagenized Arabidopsis, one mutant (Col-0 dv1) with a pale-green, dark-vein phenotype which had an altered symptom response to CaMV, was isolated. From this, a phenotypically near-normal revertant (Col-0 dv1r) spontaneously arose, but which showed altered responses to CaMV. Infection of Col-0 dv1r by CaMV isolate Bari-1 elicited symptoms unlike the parent Arabidopsis ecotype (Col-0 gl1). Also, Col-0 dv1 and Col-0 dv1r expressed an uncharacteristic necrotic reaction to CaMV. Key words: Caulimovirus, Cruciferae, plant mutants, symptom expression. Introduction Development of viral disease in plants is a consequence of the expression of specific viral and host genes during a compatible interaction. Differences in disease character elicited by particular host virus combinations can reflect genetic variation in either the host or the pathogen. Therefore, the contribution of the viral and host genome to determining disease components can be analysed indi- vidually or in combination by exploiting genetic variants of both partners. Because the viral genome is more accessible than that of the host, more progress has been made in mapping specific viral determinants of symptom expression and this is particularly exemplified in the plant DNA virus, cauliflower mosaic virus (CaMV ) (Covey, 1991). It has been shown that distinct pathogenic determinants are associated with particular regions of the viral genome. This was determined by constructing hybrid viruses from naturally-occurring isolates that elicit different types of symptoms in plants of the Cruciferae and Solanaceae (Daubert, 1988; Stratford and Covey, 1989; Qiu and Schoelz, 1992). From this, it was concluded that the CaMV gene VI, which encodes a protein that accumulates in subcellular inclusion bodies (Covey and Hull, 1981), and functions as a translational transactivator ( Rothnie et al., 1994), plays a major role in viral pathogenicity. In fact, the gene VI protein, when expressed from a trans- gene, can still elicit symptomatic effects in non-infected Nicotiana plants (Baughman et al., 1988; Takahashi et al., 3 To whom correspondence should be addressed. Fax: Covey@BBSRC.AC.UK Oxford University Press 1998

2 732 Cecchini et al. 1989) as well as the CaMV host plant Arabidopsis (Zijlstra of apical dominance etc., were assessed qualitatively. Light et al., 1996; Cecchini et al., 1997). intensity and the light quality of fluorescent tubes used in growth chambers to propagate Arabidopsis were found to Use of Arabidopsis as an experimental subject should influence symptom expression. Some comparisons were made also facilitate identification of host genes involved in viral in different growth chambers in which symptom expression was pathogenesis. CaMV readily infects Arabidopsis (Balàzs slightly different. These differences were accounted for by and Lebeurier, 1981; Melcher, 1989) and there have been comparing infections relative to the symptoms produced in some investigations of the infection process in wild-type plants by the standard CaMV isolate, Cabb B-JI, used in this study. and mutant plants (Melcher, 1989; Leisner and Howell, 1992; Leisner et al., 1992). In a previous study of the Preparation of antiserum and assay of virus pathogenic interactions between variants of CaMV and Rabbit antiserum was raised to virions of the CaMV isolate Brassica hosts, significant differences in symptom patho- Bari-1 according to Ismail et al. (1987). Samples for serological logy were reported (Al-Kaff and Covey, 1994). In the assay were prepared by a method similar to that described by present study, the combination of naturally-occurring Cecchini et al. (1997) with minor modifications. CaMV coat variants and in vitro-generated mutants of both CaMV protein was assayed by indirect ELISA essentially as described by Ismail et al. (1987) except that TMB Microwell peroxidase and Arabidopsis have been exploited to identify pathosubstrate ( Kirkegaard and Perry Laboratories) was used as genic combinations that should provide the material to developer. investigate the molecular genetic processes involved in viral pathogenesis. Results Materials and methods Symptom expression of CaMV variants in Arabidopsis Plants and viruses The response of a single Arabidopsis variant, a glabrous CaMV isolates used in this study were obtained as described in mutant of ecotype Columbia (Col-0 gl1), to infection by Al-Kaff and Covey (1994, 1995) and comprised BSE, NZ-1, a broad range of naturally-occurring CaMV isolates, was NZ-2, Bari-3, Nomé, W, Cost-2, Bari-2, Bari-2b, Braunsweig, first compared. Glabrous plants were found to be slightly Phatak, Cabb B-JI, Aust, 11/3 7, XJ, CM4 184, CM1841, easier to infect than hairy plants. The infections were also Cabb-B2, Belt-7, Hung-1, Belt, Nch-1, Yug-3, 11/3 7, Brass-6, Cabb-B, California, Campbell, Strasbourg, Bari-1, and Bari-4. compared to the response of the standard experimental The hybrid virus Baji-31 comprised primarily the Cabb B-JI CaMV host, turnip, used in this study. Initially, some genome with a substituted portion from gene VI of the Bari-1 variation in the susceptibility of Arabidopsis plants to a isolate between nucleotides 6103 to 7027 (McCallum, 1994). given CaMV isolate was found, but this improved with Arabidopsis variants were obtained from a local collection and experience of manual inoculation to obtain consistent comprised ecotypes Al-0, Be-0, Bla-10, Bur-0, C-24, Columbia (Col-0 gl1), Ct-1, Ge-0, JI-1, Kos-7, Li-6, Le-0, Landsberg infection with sap or purified virus. Infections of erecta (Ler gl1), Ms-0, Nd-0, No-0, Dy-0, Po-1, Se-0, Sl-0, Arabidopsis with cloned viral DNA were also achieved. Sy-0, Tu-1, and Zu-1. Seed of Arabidopsis Col-0 gl1 that had In general, the time-course of symptom development been mutagenized by EMS treatment (M4 generation) was in Arabidopsis was similar to that in turnip (Al-Kaff and obtained from Lehle Seeds ( USA). Virus was propagated in Covey, 1994). Inoculation of the second or subsequent turnip (Brassica rapa-rapifera, cv. Just Right ) in a glasshouse as described previously (Al-Kaff and Covey, 1994). leaves of Arabidopsis seedlings with CaMV isolate Cabb B-JI often produced what appeared to be a single local Growth and infection of plants lesion at about 7 days post inoculation (dpi). With time, Plants were sown and propagated either as described before this lesion increased in size eventually covering a large (Bannister et al., 1993), or seed was sown directly into compost proportion of the leaf surface (data not shown). This and plants were propagated under warm white fluorescent tubes contrasts with larger host plants such as turnip in which in a growth chamber at C in an 8 or 10 h photoperiod, to maintain the plants in a rosette and delay flowering. Plants many lesions typically develop on the inoculated leaf. were inoculated either with sap from infected turnip plants Systemic symptoms in Cabb B-JI-infected Arabidopsis (Al-Kaff and Covey, 1994), or with virus purified as described appeared dpi, initially as vein clearing, developing by Gardner and Shepherd (1980). Arabidopsis seedlings were into vein banding and chlorosis with leaf stunting and inoculated on leaves 2, 3 or 4. This was achieved by pipetting curling by dpi (Fig. 1). 1 ml of inoculum containing a trace of celite abrasive and gently rubbing with a Pasteur pipette tip flattened into a spatula A different pattern of responses was observed, to a shape. When screening larger numbers of plants, it was easier variety of CaMV isolates, in Arabidopsis Col-0 gl1 com- to inoculate larger leaves, but this increased the proportion of pared with turnip ( Table 1). Infections of Arabidopsis escapes. Symptoms were scored comparatively, in 5 to 20 plants with 10 different CaMV isolates led to plant death by 30 per experiment, and assessed relative to those caused by CaMV dpi. Only one isolate, Aust, frequently causes early death isolate Cabb B-JI. Assessments of overall severity of systemic symptoms, and of vein chlorosis and plant stunting, were made in turnip. In contrast, 13 CaMV isolates caused no on a scale of 0 5 (indicated as + in Tables). Other symptom symptoms in Arabidopsis whilst all isolates tested elicited characters, for example, leaf coloration patterns, necrosis, loss symptoms in turnip ( Table 1). Several isolates that were

3 CaMV and Arabidopsis interactions 733 Fig. 1. Symptom expression by CaMV isolates in Arabidopsis ecotype Col-0 gl1 systemically-infected at about 25 dpi. (A) Healthy plants; plants infected with CaMV isolates (B) Aust, (C) CM4 184, (D) Phatak, (E) Cabb B2, (G) Cabb B-JI, (H) Braunsweig, (F) turnip plant infected with Aust. Plants are shown at between 1 and 3 their normal sizes except for the plant in (F) which is about one-sixth life-size. Table 1. Symptoms produced by infection of Arabidopsis and severe in turnip were asymptomatic in Arabidopsis whereas turnip with CaMV isolates isolate Braunsweig was very severe in Arabidopsis, but mild in turnip. CM4 184 and CM1841 were the only two CaMV isolate Arabidopsis Col-0 gl1 Turnip CaMV isolates that showed a similar degree of severity BSE +++++* ++++ in both host species. Responses of Arabidopsis to three NZ * ++++ CaMV isolates showed greater plant-to-plant variability NZ * ++++ than the other isolates. Also tested, was a recombinant Bari * ++++ Nomé +++++* ++++ CaMV construct, Baji-31, in which most of the genome W +++++* ++++ was derived from Cabb B-JI, but with the central portion Cost * ++++ of gene VI, from the mild CaMV isolate Bari-1 (Stratford Bari * ++++ Bari-2b +++++* ++++ et al., 1988). Infection of turnip with Baji-31 produced Braunsweig +++++* + an infection with a degree of severity intermediate between Phatak +++++# ++++ that produced by the parental isolates, but in Arabidopsis Cabb B-JI Aust +++# +++++** the infection was more severe than that of either parent. 11/ # ++++ This was especially interesting as Bari-1 was asympto- XJ matic in Arabidopsis ecotype Col-0 gl1 ( Table 1). CM CM In addition to differences in the overall severity of Cabb-B infections produced by CaMV isolates, differences in Belt specific symptom characters produced in Arabidopsis were Hung Belt ++++ noted. Typical examples of these differences are shown in Nch Fig. 1. Compared with an uninfected Arabidopsis plant Yug /3 + ( Fig. 1A), isolate Aust caused relatively mild vein clearing Brass and leaf curling in Arabidopsis ( Fig. 1B) which contrasts Cabb-B +++ with its lethal effects in turnip. CM4 184 produced leaf California ++ Campbell ++ mosaics with dark green islands in Arabidopsis ( Fig. 1C); Strasbourg ++ Phatak caused development of miniature leaves ( Fig. 1D); Bari-1 + isolate Cabb B2 produced a fine mottling and chlorosis Bari-4 + Baji * +++ of the minor veins (Fig. 1E); Cabb B-JI caused leaf stunting and vein banding in Arabidopsis (Fig. 1G) +++++, Very severe; * most plants dead at 30 dpi; ** usually although later in infection, it produced stunting and more lead to plant death; ++++, severe; +++, less severe; ++, mild; +, very mild;, no symptoms. #, symptom variability observed. Baji-31 generalized chlorosis as in turnip ( Fig. 1F); Braunsweig is a hybrid of isolates Cabb B-JI and Bari-1. produced a highly stunted and distorted rosette ( Fig. 1H ).

4 734 Cecchini et al. CaMV symptom expression in different Arabidopsis arbitrary units). Preliminary ELISA results also indicated ecotypes only small differences between virus titres in Arabidopsis Ler gl1 infected with CaMV isolates Cabb B-JI, Bari-1 The influence of the host genotype on symptom expression and Baji-31 (data not shown). during CaMV infection in Arabidopsis was investigated next. CaMV isolate Cabb B-JI was selected as the infectious agent since this is our laboratory type isolate and Arabidopsis mutant screen has a moderately severe effect in Arabidopsis. Cabb B-JI This study of naturally-occurring Arabidopsis ecotypes infections were compared in a total of 23 Arabidopsis suggested a relatively limited range of differences in ecotypes ( listed in Materials and methods) and, surpris- response to CaMV infection. Therefore, an EMS-mutagenized ingly, no significant differences were observed between population of Col-0 gl1 Arabidopsis plants was them. In general, the variation in symptom expression screened for altered symptom expression. Approximately between individuals of a given ecotype was similar to that mutant plants were assessed following manual between ecotypes. In a second series of infections, the inoculation with Cabb B-JI. From these, several plants effect of selected CaMV isolates on Arabidopsis ecotypes were identified with apparently different symptomatic Col-0 gl1 and Ler gl1 were compared and a slight, but properties, but only one such mutant produced viable consistently more severe, response in Ler gl1 was observed seed. This mutant, provisionally designated dv1 (Col-0 ( Fig. 2). Most significant of these interactions was the dv1), had an uninfected phenotype of pale green leaves finding that CaMV isolate Bari-1 was asymptomatic in with serrated margins, dark veins, reduced apical domin- Col-0 gl1, but produced mild, but distinct vein clearing ance, late flowering and low fertility, in addition to symptoms in Ler gl1. The recombinant CaMV, Baji-31, retaining the glabrous phenotype of the parent ( Fig. 3A). always caused more severe symptoms than either of the Col-0 dv1 exhibited leaf stunting and distortion ( Fig. 3B) parental isolates but the increased severity in Ler gl1 about 10 d later than wild-type plants following infection compared with Col-0 gl1 was still clearly apparent with Cabb B-JI. Vein clearing symptoms, typical of wildtype (Fig. 2). infections, were not observed in infected Col-0 dv1 Serological estimations of virus titre by ELISA were ( Fig. 3C) presumably because of the masking effect of undertaken to determine whether asymptomatic plants the abnormal phenotype. Crossing of Col-0 dv1 with Ler were infected. Relative virus levels were measured in leaf gl1 and analysis of the F and F progeny indicated that 1 2 samples harvested 21 dpi following inoculation with the uninfected phenotype of the Arabidopsis mutant CaMV isolate Bari-1. A value of 8.3±1.0 was recorded segregated in a manner consistent with its being controlled in asymptomatic Arabidopsis Col-0 gl1 plants, and by a single recessive locus (data not shown). 11.1±1.7 in Ler gl1 plants showing mild vein clearing During propagation of Col-0 dv1, a spontaneous phenotypic symptoms. Values were obtained from eight measurements revertant arose (Col-0 dv1r) that resembled the on each of two separately-infected plants and are original Col-0 gl1 parent in leaf coloration and morphology, expressed relative to levels of Bari-1 virus in turnip (100 but produced a somewhat larger rosette. Compared Fig. 2. Response of Arabidopsis ecotypes Col-0 gl1 (top row) and Ler gl1 (bottom row) to CaMV isolates (A, F ) Bari-1, (B, G) Cabb B-JI, (C, H) Baji-31, (D, I ) 11/3 7, (E, J) Aust.

5 CaMV and Arabidopsis interactions 735 Fig. 3. Arabidopsis mutant Col-0 dv1 (A) uninfected plant, (B) infected with CaMV isolate Cabb B-JI. (C) Col-0 gl1 infected with Cabb B-JI. with the parent Col-0 gl1, both Col-0 dv1 and Col-0 dv1r showed distinct differences in their symptom responses following infection with six variants of CaMV ( Fig. 4; Table 2). In particular, isolate Bari-1, which was asymptomatic in Col-0 gl1, induced necrosis early in infection, and mild vein clearing and leaf necrosis later in infection of Col-0 dv1r ( Table 2; compare Fig. 4A, B with Fig. 2A). Levels of Bari-1 coat protein in Col-0 dv1r, assayed serologically, were about 60% of the levels in the Col-0 gl1 parent. The revertant Col-0 dv1r also developed localized leaf necrosis with three out of six CaMV variants compared, particularly late in infection ( Table 2). This type of leaf necrosis was rarely seen in any Arabidopsis ecotype or in Fig. 4. Symptoms expressed in the Arabidopsis revertant mutant Col-0 dv1r infected with (A) Bari-1 showing vein clearing, (B) Bari-1 showing necrosis of the emerging leaves, (C) Baji-31 with vein-clearing and stunting, (D) Baji-31 showing loss of apical dominance, (E) Cabb B-JI with vein clearing and stunting, (F) 11/3 7 showing necrosis, (G) Aust with necrosis, (H) Aust causing loss of apical dominance. Table 2. Response of Arabidopsis mutants to CaMV variants Plant Col-0 gl1 Col-0 dv1 Col-0 dv1r Stage E L E L E L Isolate Bari-1,,,,, N V++, S,N Cabb B-JI V+++, S V++++, S++++ V, S V, S++++ V++, S V++++, S++ Baji-31 V+++, S V+++++, S+++++ V, S V, S+++++ V+++, S V+++++, S+++++, AD 11/3 7 V+++, S V+++++, S+++++ V, S+++ V, S+++++, N V, S, N V, S, N Phatak V++, S V+++, S++ V, S V, S+++ V++++, S V+++++, S+++, N Aust V++++, S V++++, S++ V, S V, S+++ V, S, N V, S, N, AD V, Vein clearing/banding; S, stunting; N, necrosis; AD, loss of apical dominance. Symbols of symptom severity are as in Table 1.

6 736 Cecchini et al. turnip upon infection with any of these six CaMV vari- developmental factors and the Arabidopsis CaMV system ants, or indeed with the other 28 isolates listed in Table 1. offers potential in probing these phenomena. Necrosis was, however, observed in late infections by Although CaMV isolate Bari-1 did not elicit symptoms CaMV isolate 11/3 7 in Col-0 dv1. Another notable in Arabidopsis ecotype Col-0 gl1 under the growth condidifference between Col-0 dv1r and both Col-0 dv1 and tions employed here, it did produce mild vein clearing in Col-gl1 was that Col-0 dv1r showed reduced apical the Ler gl1 ecotype. Differences in symptom expression in dominance in response to two of the more severe CaMV Arabidopsis ecotypes that are apparently independent of variants ( Table 2, Fig. 4H). virus accumulation have also been observed in infections by the RNA virus tobacco ringspot nepovirus (Lee et al., 1996). Taken together, these observations suggest that Discussion specific host loci influence symptom expression independently of virus accumulation. In the case of CaMV, recombinant inbred lines between Col and Ler (Lister and Dean, In these studies, it was found that there was a greater degree of genetic variation in apparent susceptibility and 1993) should provide a means to investigate such loci. symptom character defined by the viral genome than by One interesting mutant (Col-0 dv1) was isolated from the plant genome when comparing wild-type variants. In a screen of EMS-mutagenized seed, and, from this mutant, a previous study involving three CaMV isolates and three a revertant arose. Both variants showed altered responses Arabidopsis ecotypes (Leisner and Howell, 1992), some to CaMV infection. The original mutation was presumvariation in symptom expression was recorded although ably pleiotropic, and genetic analysis indicated that the the most significant differences were found with a CaMV visible phenotype resulted from a recessive mutation at a single locus. The revertant, Col-0 dv1r, which was phenoisolate ( W260) and an Arabidopsis ecotype ( En-2) that typically near normal but retained an altered response to were not included in this study. It was also found that CaMV, is currently being characterized further. Of parthe response of Arabidopsis to particular CaMV variants ticular interest was the symptomatic response of Col-0 was sometimes quite different from that exhibited in other dv1r to isolate Bari-1 compared with its asymptomatic CaMV hosts, B. rapa and B. oleraceae (Al-Kaff and Col-0 gl1 parent. Whatever the basis for such a change, Covey, 1994). Of particular interest was the finding that results herein indicate that the mutation does not appear Arabidopsis ecotype Col-0 gl1 was apparently asymptoto give rise to an increased capacity by the plant to matic to a number of CaMV isolates. In the case of support replication of Bari-1. Bari-1, despite the lack of symptoms, the virus titre in Unlike either its parent or other Arabidopsis ecotypes, these plants was not significantly different from the titre Col-0 dv1r showed a necrotic response to four out of the in the symptomatic ecotype Ler gl1. Indeed, preliminary six CaMV isolates which were tested. Necrosis is an serological data also suggested that virus levels in infected atypical feature of CaMV infections in general, and it plants may also not differ greatly between mild and severe might indicate a predisposition to mounting a hypersensitvirus isolates. ive response. Necrosis is often associated with host resist- So-called developmental resistance to CaMV in early- ance to virus infection as has been found in the response flowering Arabidopsis variants, where an early source to of Arabidopsis to the RNA virus turnip crinkle virus sink transition, or other host factors, might prevent long (Dempsey et al., 1993, 1997). It is yet to be determined distance movement of virus from the site of inoculation whether Arabidopsis necrosis due to CaMV infection is ( Leisner et al., 1992; Callaway et al., 1996), has been linked to resistance. Resistance of crucifers to CaMV has suggested as a possible explanation for the apparent lack recently been shown to involve a pathogen-elicited gene of susceptibility of some Arabidopsis ecotypes. Differences silencing response (Covey et al., 1997). It should be were sometimes observed in Arabidopsis susceptibility and possible to exploit Arabidopsis variants to unravel this symptom expression in different growth environments, and other host virus pathogenic interactions. with lighting being a particularly important variable which could influence plant development and systemic virus movement indirectly. It is possible, therefore, that Acknowledgements the failure of some Arabidopsis ecotypes to become This work was supported by a grant from the BBSRC No. infected, or the development of asymptomatic infections, P04725, and to the John Innes Centre. We thank Dr ID may be due to inappropriate environmental conditions Hamilton for production of the antiserum. Experiments were performed under MAFF license No. PHF 1491/982/34. rather than immunity. However, it remains to be determined which of the virus host combinations in this study produced an asymptomatic but productive infection like References Bari-1 and Col-0 gl1, and which did not. There is still Al-Kaff NS, Covey SN Variation in biological properties much to learn about the relationships between plant of cauliflower mosaic virus clones. Journal of General Virology susceptibility, symptom expression and environmental/ 75,

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