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Mapping the Virus and Host Genes Involved in the Resistance Response in Cucumber Mosaic Virus-Infected Arabidopsis thaliana

Mapping the Virus and Host Genes Involved in the Resistance Response in Cucumber Mosaic... Abstract A yellow strain of cucumber mosaic virus (CMV) [CMV(Y)] induces a resistance response characterized by inhibition of virus systemic movement with development of necrotic local lesions in the virus-inoculated leaves of Arabidopsis thaliana ecotype C24. In this report, the avirulence determinant in the virus genome was defined and the resistance gene (RCY1) of C24 was genetically mapped. The response of C24 to CMV containing the chimeric RNA3 between CMV(Y) and a virulent strain of CMV indicated that the coat protein gene of CMV(Y) determined the localization of the virus in the inoculated leaves of C24. The RCY1 locus was mapped between two CAPS markers, DFR and T43968, which were located in the region containing genetically defined disease resistance genes and their homologues. These results indicate that the resistance response to CMV(Y) in C24 is determined by the combination of the coat protein gene and RCY1 on chromosome 5. (Received October 19, 2000; Accepted December 28, 2000). The hypersensitive response (HR), a resistance response that is often accompanied by programmed cell death at the site of infection, is the best characterized host defense response to pathogen infection (Goodman and Novacky 1994). In pathogen-infected plants showing the HR, both pathogenesis-related (PR) proteins and certain other antimicrobial compounds accumulate systemically (Goodman and Novacky 1994). The HR is determined by the molecular interaction between the avirulence gene in the pathogen and the resistance gene in the host plant. Avirulence genes were first investigated genetically by crossing fungal pathogen strains that show differential responses to a resistant plant cultivar (Flor 1971). However, several studies have indicated that certain viral genes may be classified as avirulence genes because of their role in eliciting the virus resistance response within host plants (Keen and Dawson 1992). The first virus-encoded avirulence gene to be characterized was the coat protein gene of some tobacco mosaic virus (TMV) isolates to which N′cultivars of tobacco developed the HR (Saito et al. 1987, Knorr and Dawson 1988). Avirulence and resistance genes that control the resistance response to virus infection also have been extensively studied in N cultivars of tobaccos infected with TMV, in Capsicum plants infected with tobamoviruses, and in Rx and Nx cultivars of potato infected with potato virus X(PVX) (Padgett and Beachy 1993, Berzal-Herranz et al. 1995, De La Cruz et al. 1997, Goulden et al. 1993, Santa Cruz and Baulcombe 1993). The identification of avirulence genes in these viral pathogens indicates that the host plants recognize various kinds of viral gene products, thereby inducing a resistance response to virus infection. From these virus and host combinations, two resistance genes, N and Rx, have been cloned. N is a member of a class of disease resistance genes whose predicted protein products have a putative nucleotide binding site and leucine-rich repeat region (Whitham et al. 1994). The Rx protein is structurally similar to the products of the N gene conferring the HR, although Rx-mediated extreme resistance against PVX in potato did not involve necrotic local lesion formation at the primary infection site (Bendahmane et al. 1999). It is well known that Arabidopsis thaliana is an excellent system for molecular genetic studies of signal transduction during plant/virus interactions (Simon 1994, Kunkel 1996). HR and the HR-like resistance responses to turnip crinkle virus (TCV) have been observed on A. thaliana ecotypes Dijon-0 and Dijon-17 (Simon et al. 1992, Dempsey et al. 1993, Dempsey et al. 1997), and a resistance gene HRT to TCV was recently cloned in Dijon-17 (Cooley et al. 2000). Cucumber mosaic virus (CMV) is well-studied. There are many isolates of CMV that infect various plant species and cause different symptoms (Palukaitis et al. 1992). CMV contains three different positive-strand RNA genomes (RNA1, 2 and 3) plus a subgenomic RNA (RNA4). RNA1 and 2 encode components of viral replicase (Hayes and Buck 1990). RNA3 has two open reading frames encoding the coat protein and the 3a protein, which is involved in cell-to-cell movement (Davis and Symons 1988). The coat protein is translated from subgenomic RNA4, which is transcribed from the minus-strand of RNA3 in virus-infected cells (Schwinghamer and Symons 1977). Recently, in some CMV strains, a 2b protein has been found in CMV-infected plants. This 2b protein, which was translated from subgenomic RNA of RNA2, was associated with host-specific long-distance virus movement (Ding et al. 1995) and with suppressing the gene silencing reaction in host plants (Li et al. 1999). Therefore, RNA1, 2 and 3 are needed to systemically infect the host plant. Previously, we reported that a yellow strain of CMV [CMV(Y)] induces the HR in A. thaliana ecotype C24 but not in ecotype Columbia (Takahashi et al. 1994). The HR in CMV(Y)-inoculated C24 was determined by the interaction between CMV RNA3 and a single dominant resistance gene of C24, RCY1, which imposed the localization of the virus within the inoculated C24 leaves. In this report, to characterize the resistance response to CMV(Y) in C24, the avirulence determinant on CMV(Y) RNA3 was defined, and the resistance gene (RCY1) locus in C24 was genetically mapped. For the propagation of CMV(Y) and an Indonesian isolate of CMV [CMV(B2)] (Tomaru and Hidaka 1960, Suastika et al. 1995), tobacco plants (Nicotiana tabacum cv. Samsun NN) were grown at 26°C in a greenhouse, and the fully expanded tobacco leaves were inoculated with 50 µg ml–1 of CMV. At 7 d after inoculation, virus-inoculated leaves were harvested, and the virus was purified by a procedure described previously (Takahashi and Ehara 1993). Arabidopsis thaliana ecotypes C24 and Columbia (Col) were grown in vermiculite : perlite (1 : 1 mixture) at 24°C under continuous fluorescent light (7,000 lux). One fully expanded leaf of a 14- to 18-day-old (5 to 6 leaf stage) A. thaliana plant was inoculated with 100 µg ml–1 of CMV. The symptom expression on both the virus-inoculated leaf at 3 d after inoculation and on non-inoculated upper leaves at 7 d after inoculation were observed, and then the coat protein within them was immunologically detected by the tissue-printing method (Srinivasan and Tolin 1992). When A. thaliana ecotype C24 was inoculated with CMV(Y), necrotic local lesions (NLLs) began to appear on virus-inoculated C24 leaves at 36 h after inoculation and matured at 3 d after inoculation (Fig. 1A). The coat protein of CMV(Y) was detected on the inoculated leaves (Fig. 1C). However, non-inoculated upper leaves of CMV(Y)-infected C24 plants that developed NLLs on their inoculated leaves had no detectable coat protein at 7 d after inoculation (Fig. 2). When ten C24 plants were inoculated with CMV(B2), the virus spread systemically, causing typical symptoms such as yellowing and stunting (Fig. 1B, 2). On the other hand, when A. thaliana ecotype Columbia (Col) was inoculated with CMV(Y) and CMV(B2), respectively, the NLLs did not develop on either of the virus-inoculated leaves (data not shown). The coat protein was detected on the inoculated and non-inoculated upper leaves of both CMV(Y) and CMV(B2)-infected Col plants (Fig. 2). These results indicated that the systemic spread of the virus was specifically restricted in virus-inoculated leaves of CMV(Y)-infected C24 plants. In our previous report (Takahashi et al. 1994), we used an ordinary strain of CMV[CMV(O)] as the CMV isolate; it systemically infected C24. Although the typical systemic symptoms were observed on non-inoculated upper leaves of CMV(O)-infected C24 at 7 d after inoculation, the NLLs were observed on their inoculated leaves about 12 h later than on CMV(Y)-inoculated C24 leaves in half of CMV(O)-infected C24 (Takahashi et al. 1994). Furthermore, in CMV(O)-inoculated C24 leaves showing such NLLs, the induction of PR-1a gene was detected at the mRNA level (data not shown). These observations indicated that we should consider the possibility that CMV(O) may delay the induction of the HR, thereby infecting C24 systemically. Therefore, in this report, the combination of two CMV strains [CMV(Y) and CMV(B2)] and two A. thaliana ecotypes (C24 and Col) was used for mapping the avirulence determinant on virus genome and the resistance gene of host plant. The host response to a series of pseudorecombinant CMVs, which were constructed by exchanging CMV RNA1+2 and RNA3 between CMV(Y) and CMV(O), indicated that the resistance in CMV(Y)-infected C24 was controlled by RNA3 (Takahashi et al. 1994). Therefore, to define the avirulence determinant on CMV genomes, we focused on RNA3. Chimeric forms of the RNA3-coding region in CMV were constructed at the cDNA level (Fig. 3). pCY3-T7 contains cDNA for CMV(Y) RNA3 under the control of T7 promoter (Nitta et al. 1988, Suzuki et al. 1991). cDNA to CMV(B2) RNA3 was synthesized by RT-PCR with two primers, 5′-CGCGGATCCTAATACGACTCACTATAGTAATCTTACCACTCGTGTGT-3′ containing a BamHI restriction site and the T7 promoter sequence and 5′-CCGGAATTCTGGTCTCCTTTTGGAGGCC-3′ containing a EcoRI restriction site (the viral sequences are underlined) (Baier et al. 1993). The cDNA was cloned into the BamHI and EcoRI sites of pUC118 (TAKARA, Kyoto, Japan). This cDNA sequence has been lodged with DDBJ (accession no. AB042294). Then, because the cDNA to CMV(B2) RNA3 did not have either a SalI-digestion site at 1,298 nucleotide position or a XhoI-digestion site at 1,840 nucleotide positions but pCY3-T7 had both, the nucleotide sequences digested by SalI and XhoI were introduced into 1,298 and 1,840 nucleotide positions in the cDNA to CMV(B2) RNA3, using primers, 5′-CGTAACCGTCGACGTCGTCC-3′ (the SalI restriction site is underlined) and its complimentary sequence for the SalI site, and 5′-CATCCGTCTCGAGCGCATCG-3′ (the XhoI restriction site is underlined) and its complimentary sequence for the XhoI site, by PCR (Higuchi et al. 1998). The introduction of the restriction site of SalI did not cause any changes in the amino acids encoded in those regions, but the XhoI site changed Pro to Leu at the 194 amino acid position in the coat protein. The resulting plasmid was designated as pCB3-T7. pCY3-T7 and pCB3-T7 were used to construct chimeric cDNAs containing the CMV(Y) RNA3 and CMV(B2) RNA3 sequences (Fig. 3). pCY3-BSX and pCB3-YSX were constructed by exchanging the SalI–XhoI fragment (nucleotides 1,298–1,840 of RNA3) between pCY3-T7 and pCB3-T7. Standard molecular techniques were used for vector construction (Sambrook et al. 1989). Construction of these two chimeric cDNAs to CMV RNA3 was confirmed by partial dideoxy-sequencing using primers specific to CMV RNA3. The constructed plasmids containing cDNA to CMV RNA3 were linearized by digestion with EcoRI and in vitro transcribed using T7 RNA polymerase. Each linearized plasmid DNA (2 µg) was added to 50 mM Tris-HCl (pH 7.5), 0.5 mM GTP, 5 mM ATP, 5 mM CTP, 5 mM UTP, 0.1 mM DTT, 5 mM 7mGpppG-cap analog (New England Biolabs, Beverly, MA), 30 units of RNasin (TOYOBO, Osaka, Japan) and 70 units of T7 RNA polymerase (Pharmacia, Piscataway, NJ), and was incubated at 37°C for 1 h. CMV(Y) RNA1 and RNA2 were also transcribed in vitro from NotI-linearized pCY1-T7 and pCY2-T7 (Suzuki et al. 1991). Tobacco plants (N. tabacum cv Xanthi nc) were inoculated with in vitro transcribed CMV RNA1, RNA2 and RNA3. Viruses which contained RNA3 in vitro transcribed from pCY3-T7, pCB3-T7, pCY3-BSX, or pCB3-YSX were designated as CMV(Y), CMV(B2)SX, CMV(Y/BSX) or CMV(B/YSX), respectively. Each virus (100 µg ml–1) was used to inoculate A. thaliana ecotypes C24 and Col. Arabidopsis thaliana ecotype C24 was systemically infected with CMV(B2)SX as well as the parental CMV(B2) (Fig. 4, Table 1), although one amino acid change from Pro to Leu at 194 amino acid position in the coat protein of CMV(B2)SX occurred. To define the avirulence determinant in CMV RNA3, A. thaliana C24 was inoculated with CMV(B/YSX) and CMV(Y/BSX), respectively. In CMV(B/YSX)-inoculated C24 leaves as well as in CMV(Y)-inoculated C24 leaves (Fig. 1A), the NLLs developed at 36 h after inoculation and matured at 3 d after inoculation, and no coat protein was detected on their non-inoculated upper leaves (Fig. 4, Table 1). On the other hand, the coat protein was systemically distributed in CMV(Y/BSX)-infected C24, whose inoculated leaves did not show any symptoms (Fig. 1A, 4, Table 1). The multiplication of the recombinant CMVs in virus-infected C24 was confirmed by sequencing the cDNA to the coat protein gene of the progeny viruses (data not shown). Because, the SalI–XhoI-digested fragment, which includes about 90% of coat protein gene in CMV, had been exchanged at the cDNA level between CMV(Y) and CMV(B2), the virus localization in CMV(B/YSX)-inoculated C24 leaves contrasted with the systemic spread of virus in CMV(Y/BSX)-inoculated C24 leaves indicated that the avirulence determinant in CMV(Y)RNA3 was located in the coat protein gene. The coding region of the coat protein gene had 92.0% and 95.0% identities between CMV(Y) and CMV(B2) at the nucleotide and amino acid sequence levels, respectively. The resistance response to CMV(Y) in C24 seemed to be controlled by the changes of amino acids in the coat protein rather than by an effect of the RNA sequence itself, because, in addition to the capsid function for the assembly of virus particle, several phenotypic properties in CMV-infected plants, such as the induction of chlorosis and stunting response in tobaccos, aphid transmissibility, host specificity, and virus localization with the developing NLLs, are associated with alterations in the viral coat protein itself rather than with the nucleotide sequence of the coat protein (Shintaku et al. 1992, Takahashi and Ehara 1993, Perry et al. 1994, Suzuki et al. 1995, Ryu et al. 1998, Szilassy et al. 1999, Sugiyama et al. 2000, Wikoff et al. 1997). The amino acid sequence alignment of the coat protein of CMV(Y) and CMV(B2)SX indicated six amino acid differences (Fig. 3). We do not know whether the induction of the resistance to CMV(Y) in C24 was controlled by the local secondary structure of the coat protein or by a particular amino acid in the coat protein causing the interaction between the coat protein and unknown host factor(s). To understand this issue, it will be necessary to delimit the amino acid(s) in the coat protein conferring the resistance response. For the genetic mapping analysis of the RCY1 locus, F2 progeny plants of the Col×C24 cross were used. We screened 109 individuals of CMV(Y)-susceptible F2 progenies by detecting the coat protein on non-inoculated upper leaves of CMV(Y)-infected F2 plants at 7d after inoculation. For analysis with the cleaved amplified polymorphic sequence (CAPS) markers, AG (MapPairs from Research Genetics, Huntsville, AL, U.S.A.), PR5K, PHYC, DFR and T43968, the PCR reaction and restriction analysis of the PCR products were performed by the method of Konieczny and Ausubel (1993). The nucleotide sequences for the primers of PR5K and PHYC were derived from the information about new CAPS markers available through the A. thaliana database. Because two of the CAPS markers, DFR and T43968, were not polymorphic between Col and C24, the nucleotide sequences around DFR and T43968 markers in C24 and Col were determined, and then the DFR primers, 5′-AGATCCTGAGGTGAGTTTTTC-3′ and 5′-TGTATATTCTTACCCATCCT-3′, and the T43968 primers, 5′-GGAAACTCTGCAGTCTTGTACA-3′ and 5′-TTGGGAGAACTATCCTCTGCAG-3′, were used for CAPS analysis. The modified DFR and T43968 products were cleaved with HincII and BlnI, respectively, and electrophoresed in 2% agarose gel by the standard protocol (Sambrook et al. 1989). For analysis with the simple sequence length polymorphisms (SSLP) markers, nga280, nga248, nga168, nga162, nga112, nga8, nga 139, nga 106, nga 249 and nga129 (MapPairs from Research Genetics, Huntsville, AL, U.S.A.), the PCR reaction was performed by the method of Bell and Ecker (1994). The obtained PCR products were electrophoresed in 4% agarose gel. The recombination frequencies between RCY1 and the markers in the CMV(Y)-susceptible F2 individuals were calculated by the method of Allard (1956). The map distances in centimorgans between RCY1 locus and the markers were converted from their recombination frequencies by the Kosambi map function (Kosambi 1944). To determine the chromosome location of the RCY1 locus responsible for the resistance to CMV(Y), thirty CMV(Y)-susceptible F2 plants and one to four CAPS or SSLP markers on each chromosome (ch.), nga280 and nga248 on ch.1, nga168 on ch.2, nga162 and nga112 on ch.3, AG and nga8 on ch.4, and nga139, nga106, nga249 and nga129 on ch.5, were initially used. The recombination frequencies between RCY1 locus and the markers on ch. 1, 2, 3, and 4 were more than 40%; however, nga249, nga106, nga139, and nga129 on ch. 5 were 39.7±4.5%, 36.7±4.1%, 30.6±4.1%, and 9.5±2.0% recombination frequencies with the RCY1 locus in 60 chromosomes. The recombination frequency between nga139 and nga129 was 33.0±4.6%. This result suggested that RCY1 locus was located between nga139 and nga129 markers on ch. 5. For further mapping analysis, 109 CMV(Y)-susceptible F2 plants and four CAPS markers between nga139 and nga129, PHYC, PR5K, modified DFR, and modified T43968, were used. Based upon the recombination frequencies between RCY1 locus and each of these four markers, 20.6±3.6% to PHYC, 7.7±1.8% to PR5K, 1.8±0.9% to modified DFR, and 0.5±0.5% to modified T43968 in 218 chromosomes, the RCY1 locus was mapped at approximately 0.5±0.5 cM of modified T43968 and 1.8±0.9 cM of modified DFR (Fig. 5). As the distance between modified T43968 and DFR was found to be 2.0±1.3 cM based upon their recombination frequency using 52 additional resistant and susceptible F3 families, RCY1 was mapped between these two CAPS markers (Fig. 5). Forty-two Arabidopsis expressed sequence tags showing similarities to disease resistance genes (R-ESTs) have been mapped on five chromosomes of A. thaliana (Botella et al. 1997). The R-ESTs encode proteins containing a leucine-rich repeat (LRR) domain. Several of these R-ESTs occur in clusters on the chromosome. RCY1 locus was mapped to one of the R-EST clusters, MRC-J, in which nine defined disease resistance genes (RAC3, RPS4, HRT12, TTR1 and five distinct RPP loci) were genetically mapped. The MRC-J covered about 30 cM of the long arm of ch. 5 on the RI map available through the A. thaliana database, and RCY1 and two CAPS markers, DFR and T43968, were apparently located on the middle of this MRC-J region. The presence of R-EST cluster suggested that the resistance gene in the cluster was generated by gene duplication events (Botella et al. 1997, Meyers et al. 1999). Therefore, the RCY1 locus may be a member of the resistance genes in the R-EST cluster on chromosome 5. The results presented in this report indicate that the resistance response to CMV(Y) in C24 was determined by the combination of the coat protein gene and RCY1 mapped between DFR and T43968 markers on chromosome 5. During the past 7 years, resistance genes that protect the host plants against viruses, bacteria and fungi have been isolated (Hammond-Kosack and Jones 1997). Furthermore, some genes encoding critical components of the signal transduction pathway(s) concerning the induction of disease resistance have been cloned from Arabidopsis, for example NPR1 (Dong 1998). Salicylic acid (SA), jasmonic acid, and ethylene are also important components of the signal transduction pathway leading to disease resistance (Dong 1998). In Arabidopsis, it has been demonstrated that these key signaling-compound-mediated signal transduction pathways were selectively activated by the resistance response to pathogens (Thomma et al. 1998). However, with regard to the HR to viral pathogens in Arabidopsis, only the resistance gene (HRT1) to TCV has been isolated to date, although two genes (RTM1 and RTM2), which restrict the long-distance movement of tobacco etch virus, and a gene (TOM1) encoding a component of the virus RNA replication complex for TMV have been cloned from Arabidopsis (Chisholm et al. 2000, Whitham et al. 2000, Yamanaka et al. 2000). TCV resistance by the HR in Arabidopsis required the SA-dependent signaling pathway (Kachroo et al. 2000). However, nahG C24, expressing SA hydrogenase and thereby failing to accumulate SA, showed resistance to CMV(Y) (Takahashi et al. unpublished results). The characterization of the avirulence determinant on CMV genomes and the resistance gene in C24 provides not only another example of virus resistance on Arabidopsis; it may also lead to greater understanding of SA-independent signal transduction pathway(s) conferring virus disease resistance. Acknowledgements We thank Dr. S. Kanematsu, Tohoku Agriculture Experimental Station, Japan, for technical advice on press-blotting. We also thank the Arabidopsis Biological Resource Center at Ohio State, U.S.A., for providing Arabidopsis seeds. This work was supported in part by a grant-in-aid for scientific research on priority areas (Molecular Mechanisms of Plant—Pathogenic Microbe Interaction—Toward Production of Disease Resistant Plants). 5 Corresponding author: E-mail, takahash@bios.tohoku.ac.jp; Fax, +81-22-717-8659. View largeDownload slide Fig. 1 Symptoms on CMV-infected A. thaliana ecotype C24. Ten C24 plants per virus [CMV(Y), CMV(B2), CMV(Y/BSX) and CMV(B/YSX)], were examined, and representatives are shown here. (A) Symptoms on three independent virus-inoculated leaves at 3 d after inoculation and mock-inoculated leaves (Mock). (B) Systemic symptom expression on CMV(B2)-infected C24 plants at 21 d after inoculation and on the corresponding CMV(Y)-infected C24 plants. (C) Detection of coat protein in CMV-infected A. thaliana ecotype C24 in virus-inoculated leaves at 3 d after inoculation. Leaf protein were press-blotted to 3 MM filter paper, and the coat protein of CMV(Y) and CMV(B2) was immunologically detected. Mock-inoculated C24 (Mock) was used as a control. View largeDownload slide Fig. 1 Symptoms on CMV-infected A. thaliana ecotype C24. Ten C24 plants per virus [CMV(Y), CMV(B2), CMV(Y/BSX) and CMV(B/YSX)], were examined, and representatives are shown here. (A) Symptoms on three independent virus-inoculated leaves at 3 d after inoculation and mock-inoculated leaves (Mock). (B) Systemic symptom expression on CMV(B2)-infected C24 plants at 21 d after inoculation and on the corresponding CMV(Y)-infected C24 plants. (C) Detection of coat protein in CMV-infected A. thaliana ecotype C24 in virus-inoculated leaves at 3 d after inoculation. Leaf protein were press-blotted to 3 MM filter paper, and the coat protein of CMV(Y) and CMV(B2) was immunologically detected. Mock-inoculated C24 (Mock) was used as a control. View largeDownload slide Fig. 2 Detection of the coat protein in CMV-infected A. thaliana ecotypes C24 and Col. Ten C24 and Col plants per virus [CMV(Y) and CMV(B2)] were examined, and representatives are shown here. (A) Entire plants which were press-blotted on the 3 MM filter paper. (B) The coat protein of CMV(Y) and CMV(B2) was detected immunologically in virus-infected whole plants (C24 and Col) at 7 d after inoculation. Mock-inoculated C24 (Mock) was used as a control. View largeDownload slide Fig. 2 Detection of the coat protein in CMV-infected A. thaliana ecotypes C24 and Col. Ten C24 and Col plants per virus [CMV(Y) and CMV(B2)] were examined, and representatives are shown here. (A) Entire plants which were press-blotted on the 3 MM filter paper. (B) The coat protein of CMV(Y) and CMV(B2) was detected immunologically in virus-infected whole plants (C24 and Col) at 7 d after inoculation. Mock-inoculated C24 (Mock) was used as a control. View largeDownload slide Fig. 3 Chimeric CMV RNA3 transcription vector constructs. Rectangular boxes represent open reading frames of the 3a protein gene (3a) and the coat protein gene (CP). cDNA from CMV(Y) is represented as a shaded box; cDNA from CMV(B2) as an unfilled box. Differences in the amino acid sequence, their positions, and the restriction endonuclease sites are indicated. RNA3 is transcribed in vitro from cDNA under the control of T7 promoter (T7). View largeDownload slide Fig. 3 Chimeric CMV RNA3 transcription vector constructs. Rectangular boxes represent open reading frames of the 3a protein gene (3a) and the coat protein gene (CP). cDNA from CMV(Y) is represented as a shaded box; cDNA from CMV(B2) as an unfilled box. Differences in the amino acid sequence, their positions, and the restriction endonuclease sites are indicated. RNA3 is transcribed in vitro from cDNA under the control of T7 promoter (T7). View largeDownload slide Fig. 4 Detection of the coat protein in recombinant CMV-infected A. thaliana ecotype C24. Seven to twelve plants per virus [CMV(Y), CMV(B2)sx, CMV(Y/BSX) and CMV(B/YSX)] were examined (Table 1), and representatives are shown here. The leaf protein was press-blotted to 3 MM filter paper, and the coat protein was detected immunologically. View largeDownload slide Fig. 4 Detection of the coat protein in recombinant CMV-infected A. thaliana ecotype C24. Seven to twelve plants per virus [CMV(Y), CMV(B2)sx, CMV(Y/BSX) and CMV(B/YSX)] were examined (Table 1), and representatives are shown here. The leaf protein was press-blotted to 3 MM filter paper, and the coat protein was detected immunologically. View largeDownload slide Fig. 5 Genetic map position of RCY1 locus on chromosome 5 of Arabidopsis. RCY1 locus was mapped on 109 F2 progeny from a cross between Col×C24. The markers nga 139 and nga129 are SSLP markers. The markers PHYC, PR5K, DFR, and T43968 areCAPSmarkers. The vertical bars indicate positions of loci. Numbers are centimorgans. The MRC-J region containing genetically defined disease resistance genes and their homologous genes is shown by the arrow bar. View largeDownload slide Fig. 5 Genetic map position of RCY1 locus on chromosome 5 of Arabidopsis. RCY1 locus was mapped on 109 F2 progeny from a cross between Col×C24. The markers nga 139 and nga129 are SSLP markers. The markers PHYC, PR5K, DFR, and T43968 areCAPSmarkers. The vertical bars indicate positions of loci. Numbers are centimorgans. The MRC-J region containing genetically defined disease resistance genes and their homologous genes is shown by the arrow bar. Table 1 Response to recombinant CMV in A. thaliana ecotype C24 Virus a  Response in A. thaliana C24  Symptoms on the inoculated leaves  Systemic infection of virus b  CMV(Y)  necrotic local lesions  0 / 12  CMV(B2)sx  no symptoms  7 / 7  CMV(Y/BSX)  no symptoms  10 / 10  CMV(B/YSX)  necrotic local lesions  0 / 12  Virus a  Response in A. thaliana C24  Symptoms on the inoculated leaves  Systemic infection of virus b  CMV(Y)  necrotic local lesions  0 / 12  CMV(B2)sx  no symptoms  7 / 7  CMV(Y/BSX)  no symptoms  10 / 10  CMV(B/YSX)  necrotic local lesions  0 / 12  a CMV(Y), CMV(B2)sx, CMV(Y/BSX) and CMV(B/YSX) contain RNA3 in vitro transcribed from pCY3-T7, pCB3-T7, pCY3-BSX and pCB3-YSX, respectively, in Fig. 3. These recombinant CMVs were used to inoculate A. thaliana ecotype C24. b The number of systemically infected plants is shown over the number of CMV(Y)-inoculated plants. View Large Abbreviations CAPS cleaved amplified polymorphic sequence ch. chromosome Col Columbia CMV cucumber mosaic virus HR hypersensitive response NLLs necrotic local lesions LRR leucine-rich repeat PR pathogenesis-related PVX potato virus X R-ESTs sequence tags showing similarity to disease resistance genes RT reverse transcription SA salicylic acid SSLP simple sequence length polymorphisms TCV turnip crinkle virus TMV tobacco mosaic virus. References Allard, R.W. ( 1956) Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia  24: 235–278. Google Scholar Baier, G., Telford, D., Gulbins, E., Yamada, N., Kawakami, T. and Altman, A. ( 1993) Improved specificity of RT-PCR amplifications using nested cDNA primers. Nucl. Acids Res.  21: 1329–1330. Google Scholar Bell, C.J. and Ecker, J.R. ( 1994) Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics  19: 137–144. Google Scholar Bendahmane, A., Kanyuka, K. and Baulcombe, D.C. ( 1999) The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell  11: 781–791. Google Scholar Berzal-Herranz, A., De La Cruz, A., Tenllado, F., Díaz-Ruíz, J.R., Lóez, L., Sanz, A.I., Vaquero, C., Serra, M.T. and García-Luque, I. ( 1995) The Capsicum L3 gene-mediated resistance against the tobamoviruses is elicited by the coat protein. Virology  209: 498–505. Google Scholar Botella, M.A., Coleman, M.J., Hughes, D.E., Nishimura, M.T., Jones, J.D.G. and Somerville, S.C. ( 1997) Map positions of 47 Arabidopsis sequenceswith sequence similarity to disease resistance genes. Plant J.  12: 1197–1211. Google Scholar Chisholm, S.T., Mahajan, S.K., Whitham, S.A., Yamamoto, M.L. and Carrington, J.C. ( 2000) Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus. Proc. Natl. Acad. Sci. USA  97: 489–494. Google Scholar Cooley, M.B., Pathirana, S., Wu, H-J., Kachroo, P. and Klessig, D.F. ( 2000) Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens. Plant Cell  12: 663–676. Google Scholar Davis, C. and Symons, R.H. ( 1988) Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA3. Virology  165: 216–224. Google Scholar De La Cruz, A., López, L., Tenllado, F., Díaz-Ruíz, J.R., Sanz, A.I., Vaquero, C., Serra, M.T. and García-Luque, I. ( 1997) The coat protein is required for the elicitation of the Capsicum L2 gene-mediated resistance against the tobamoviruses. Mol. Plant-Microbe Interact.  10: 107–113. Google Scholar Dempsey, D.A., Pathirana, M.S., Wobbe, K.K. and Klessig, D.F. ( 1997) Identification of an Arabidopsis locus required for resistance to turnip crinkle virus. Plant J.  11: 301–311. Google Scholar Dempsey, D.A., Wobbe, K.K. and Klessig, D.F. ( 1993) Resistance and susceptible responses of Arabidopsis thaliana to turnip crinkle virus. Phytopathology 83: 1021–1029. Google Scholar Ding, S.W., Li, W.X. and Symons, R.H. ( 1995) A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. EMBO J.  14: 5762–5772. Google Scholar Dong, X. ( 1998) SA, JA, ethylene, and disease resistance in plants. Curr. Opin. Plant Biol.  1: 316–323. Google Scholar Flor, H.H. ( 1971) Current status of the gene-for-gene concept. Annu. Rev. Phytopathol.  9: 275–296. Google Scholar Goodman, R.N. and Novacky, A.J. ( 1994) The Hypersensitive Reaction in Plants to Pathogens. A Resistance Phenomenon.American Phytopathological Society, St. Paul, MN. Google Scholar Goulden, M.G., Köhn, B.A., Santa Cruz, S., Kavanagh, T.A. and Baulcombe, D.C. ( 1993) A feature of the coat protein of potato virus X affects both induced virus resistance in potato and viral fitness. Virology  197: 293–302. Google Scholar Hammond-Kosack, K.E. and Jones, J.D.G. ( 1997) Plant disease resistance genes. Annu. Rev. Plant Physiol. Plant Mol. Biol.  48: 575–607. Google Scholar Hayes, R.J. and Buck, K.W. ( 1990) Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase. Cell  63: 363–368. Google Scholar Higuchi, R., Krummel, B. and Saiki, R.K. ( 1998) A general method ofinvitropreparationand specific mutagenesis of DNA fragments: Study of protein and DNA interactions. Nucl. Acids Res.  16: 7351–7367. Google Scholar Kachroo, P., Yoshioka, K., Shah, J., Dooner, H.K. and Klessig, D.F. ( 2000) Resistance to turnip crinkle virus in Arabidopsis is regulated by two host genes and is salicylic acid dependent but NPR1, ethylene, and jasmonate independent. Plant Cell  12: 677–690. Google Scholar Keen, N.T. and Dawson, W.O. ( 1992) Pathogen avirulence genes and elicitors of plant defense. In Genes Involved in Plant Defense. Edited by Boller, T. and Meins, F. pp. 85–114, Springer-Verlag, Wien. Google Scholar Konieczny, A. and Ausubel, F.M. ( 1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J.  4: 403–410. Google Scholar Kosambi, D.D. ( 1944) The estimation of map distances from recombination values. Ann. Eugen.  12: 172–175. Google Scholar Knorr, D.A. and Dawson, W.O. ( 1988) A point mutation in the tobacco mosaic virus capsid protein gene induces hypersensitivity in Nicotiana sylvestris. Proc. Natl. Acad. Sci. USA  85: 170–174. Google Scholar Kunkel, B.N. ( 1996) A useful weed put to work: Genetic analysis of disease resistance in Arabidopsis thaliana. Trends Genet.  12: 63–69. Google Scholar Li, H.W., Lucy, A.P., Guo, H.-S., Li, W.X., Ji, L.H., Wong, S.M. and Ding, S.W. ( 1999) Strong host resistance targeted against a viral suppressor of the plant gene silencing defense mechanism. EMBO J.  18: 2683–2691. Google Scholar Meyers, B., Dickerman, A.W., Michelmore, R.W., Sivaramakrishnan, S., Sobral, B.W. and Young, N.D. ( 1999) Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. Plant J.  20: 317–332. Google Scholar Nitta, N., Masuta, C., Kuwata, S. and Takanami, Y. ( 1988) Comparative studies on the nucleotide sequence of cucumber mosaic virus RNA3 between Y strain and Q strain. Ann. Phytopathol. Soc. Japan  54: 516–522. Google Scholar Padgett, H.S. and Beachy, R.N. ( 1993) Analysis of a tobacco mosaic virus strain capable of overcoming N gene-mediated resistance. Plant Cell  5: 577–586. Google Scholar Palukaitis, P., Roossinck, M.J., Dietzgen, R.G. and Francki, R.I.B. ( 1992) Cucumber mosaic virus. Adv. Virus Res.  41: 281–348. Google Scholar Perry, K.L., Zhang, L., Shintaku, M.H. and Palukaitis, P. ( 1994) Mapping determinants in cucumber mosaic virus for transmission by Aphis gossypii. Virology  205: 591–595. Google Scholar Ryu, K.H., Kim, C.-H. and Palukaitis, P. ( 1998) The coat protein of cucumber mosaic virus is a host range determinant for infection of maize. Mol. Plant-Microbe Interact.  11: 351–357. Google Scholar Saito, T., Meshi, T., Takamatsu, N. and Okada, Y. ( 1987) Coat protein gene sequence of tobacco mosaic virus encodes a host response determinant. Proc. Natl. Acad. Sci. USA  84: 6074–6077. Google Scholar Sambrook, J., Fritsch, E.F. and Maniatis, T. ( 1989) Molecular Cloning: A Laboratory Manual.2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Google Scholar Santa Cruz, S. and Baulcombe, D. ( 1993) Molecular analysis of potato virus X isolate in relation to the potato hypersensitivity gene Nx. Mol. Plant-Microbe Interact.  6: 707–714. Google Scholar Schwinghamer, M.W. and Symons, R.H. ( 1977) Translation of the four major RNA species of cucumber mosaic virus in plant and animal cell-free systems and in toad oocytes. Virology  79: 88–108. Google Scholar Shintaku, M.H., Zhang, L. and Palukaitis, P. ( 1992) A single amino acid substitution in the coat protein of cucumber mosaic virus induces chlorosis in tobacco. Plant Cell  4: 751–757. Google Scholar Simon, A.E. ( 1994) Interactions between Arabidopsis thaliana and viruses. In Arabidopsis.Edited by Meyerowitz, E.M. and Somerville, C.R. pp. 685–704. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Google Scholar Simon, A.E., Li, X.-H., Lew, J.E., Stange, R., Zhang, C., Polacco, M. and Carpenter, C.D. ( 1992) Susceptibility and resistance of Arabidopsis thaliana to turnip crinkle virus. Mol. Plant-Microbe Interact.  5: 496–503. Google Scholar Srinivasan, I. and Tolin, S.A. ( 1992) Detection of three viruses of clovers by direct tissue immunoblotting. Phytopathology  82: 721. Google Scholar Suastika, G., Tomaru, K., Kurihara, J. and Natsuaki, K.T. ( 1995) Characteristics of two isolates of cucumber mosaic virus obtained from banana plants in Indonesia. Ann. Phytopathol. Soc. Jpn  61: 272. Google Scholar Sugiyama, M., Sato, H., Karasawa, A., Hase, S., Takahashi, H. and Ehara, Y. ( 2000) Characterization of symptom determinants in two mutants of cucumber mosaic virus Y strain, causing distinct mild green mosaic symptoms in tobacco. Physiol. Mol. Plant Pathol.  56: 85–90. Google Scholar Suzuki, M., Kuwata, S., Kataoka, J., Masuta, C., Nitta, N. and Takanami, Y. ( 1991) Functional analysis of deletion mutants of cucumber mosaic virus RNA3 using an in vitro transcription system. Virology  183: 106–113. Google Scholar Suzuki, M., Kuwata, S., Masuta, C. and Takanami, Y. ( 1995) Point mutations in the coat protein of cucumber mosaic virus affect symptom expression and virion accumulation in tobacco. J. Gen. Virol.  76: 1791–1799. Google Scholar Szilassy, D., Salánki, K. and Balázs, E. ( 1999) Stunting induced by cucumber mosaic cucumovirus-infected Nicotiana glutinosa is determined by a single amino acid residue in the coat protein. Mol. Plant-Microbe Interact.  12: 1105–1113. Google Scholar Takahashi, H. and Ehara, Y. ( 1993) Severe chlorotic spot symptoms in cucumber mosaic virus strain Y-infected tobaccos are induced by a combination of the virus coat protein gene and two host recessive genes. Mol. Plant-Microbe Interact.  6: 182–189. Google Scholar Takahashi, H., Goto, N. and Ehara, Y. ( 1994) Hypersensitive response in cucumber mosaic virus-inoculated Arabidopsis thaliana. Plant J.  6: 369–377. Google Scholar Thomma, B.P.H.J., Eggermont, K., Penninckx, I.A.M.A., Mauch-Mani, B., Vogelsang, R., Cammue, B.P.A. and Broekaert, W.F. ( 1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc. Natl. Acad. Sci. USA  95: 15107–15111. Google Scholar Tomaru, K. and Hidaka, J. ( 1960) Strains of cucumber mosaic virus isolated from tobacco plants. III. A yellow strain. Bull. Hatano Tobacco Exp. Stn  46: 143–149. Google Scholar Whitham, S., Dinesh-Kumar, S.P., Chol, D., Hehl, R., Corr, C. and Baker, B. ( 1994) The product of the tobacco mosaic virus resistance gene N: Similarity to toll and the interleukin-1 receptor. Cell  78: 1101–1115. Google Scholar Whitham, S., Anderberg, R.T., Chisholm, S.T. and Carrington, J.C. ( 2000) Arabidopsis RTM2 gene is necessary for specific restriction of tobacco etch virus and encodes an unusual small heat shock-like protein. Plant Cell  12: 569–582. Google Scholar Wikoff, W.R., Tsai, C.J., Wang, G., Baker, T.S. and Johnson, J.E. ( 1997) The structure of cucumber mosaic virus: Cryoelectron microscope, X-ray crystallography, and sequence analysis. Virology  232: 91–97. Google Scholar Yamanaka, T., Ohta, T., Takahashi, M., Meshi, T., Schmidt, R., Dean, C., Naito, S. and Ishikawa, M. ( 2000) TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein. Proc. Natl. Acad. Sci. USA  97: 10107–10112. Google Scholar http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Mapping the Virus and Host Genes Involved in the Resistance Response in Cucumber Mosaic Virus-Infected Arabidopsis thaliana

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Oxford University Press
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0032-0781
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1471-9053
DOI
10.1093/pcp/pce039
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Abstract

Abstract A yellow strain of cucumber mosaic virus (CMV) [CMV(Y)] induces a resistance response characterized by inhibition of virus systemic movement with development of necrotic local lesions in the virus-inoculated leaves of Arabidopsis thaliana ecotype C24. In this report, the avirulence determinant in the virus genome was defined and the resistance gene (RCY1) of C24 was genetically mapped. The response of C24 to CMV containing the chimeric RNA3 between CMV(Y) and a virulent strain of CMV indicated that the coat protein gene of CMV(Y) determined the localization of the virus in the inoculated leaves of C24. The RCY1 locus was mapped between two CAPS markers, DFR and T43968, which were located in the region containing genetically defined disease resistance genes and their homologues. These results indicate that the resistance response to CMV(Y) in C24 is determined by the combination of the coat protein gene and RCY1 on chromosome 5. (Received October 19, 2000; Accepted December 28, 2000). The hypersensitive response (HR), a resistance response that is often accompanied by programmed cell death at the site of infection, is the best characterized host defense response to pathogen infection (Goodman and Novacky 1994). In pathogen-infected plants showing the HR, both pathogenesis-related (PR) proteins and certain other antimicrobial compounds accumulate systemically (Goodman and Novacky 1994). The HR is determined by the molecular interaction between the avirulence gene in the pathogen and the resistance gene in the host plant. Avirulence genes were first investigated genetically by crossing fungal pathogen strains that show differential responses to a resistant plant cultivar (Flor 1971). However, several studies have indicated that certain viral genes may be classified as avirulence genes because of their role in eliciting the virus resistance response within host plants (Keen and Dawson 1992). The first virus-encoded avirulence gene to be characterized was the coat protein gene of some tobacco mosaic virus (TMV) isolates to which N′cultivars of tobacco developed the HR (Saito et al. 1987, Knorr and Dawson 1988). Avirulence and resistance genes that control the resistance response to virus infection also have been extensively studied in N cultivars of tobaccos infected with TMV, in Capsicum plants infected with tobamoviruses, and in Rx and Nx cultivars of potato infected with potato virus X(PVX) (Padgett and Beachy 1993, Berzal-Herranz et al. 1995, De La Cruz et al. 1997, Goulden et al. 1993, Santa Cruz and Baulcombe 1993). The identification of avirulence genes in these viral pathogens indicates that the host plants recognize various kinds of viral gene products, thereby inducing a resistance response to virus infection. From these virus and host combinations, two resistance genes, N and Rx, have been cloned. N is a member of a class of disease resistance genes whose predicted protein products have a putative nucleotide binding site and leucine-rich repeat region (Whitham et al. 1994). The Rx protein is structurally similar to the products of the N gene conferring the HR, although Rx-mediated extreme resistance against PVX in potato did not involve necrotic local lesion formation at the primary infection site (Bendahmane et al. 1999). It is well known that Arabidopsis thaliana is an excellent system for molecular genetic studies of signal transduction during plant/virus interactions (Simon 1994, Kunkel 1996). HR and the HR-like resistance responses to turnip crinkle virus (TCV) have been observed on A. thaliana ecotypes Dijon-0 and Dijon-17 (Simon et al. 1992, Dempsey et al. 1993, Dempsey et al. 1997), and a resistance gene HRT to TCV was recently cloned in Dijon-17 (Cooley et al. 2000). Cucumber mosaic virus (CMV) is well-studied. There are many isolates of CMV that infect various plant species and cause different symptoms (Palukaitis et al. 1992). CMV contains three different positive-strand RNA genomes (RNA1, 2 and 3) plus a subgenomic RNA (RNA4). RNA1 and 2 encode components of viral replicase (Hayes and Buck 1990). RNA3 has two open reading frames encoding the coat protein and the 3a protein, which is involved in cell-to-cell movement (Davis and Symons 1988). The coat protein is translated from subgenomic RNA4, which is transcribed from the minus-strand of RNA3 in virus-infected cells (Schwinghamer and Symons 1977). Recently, in some CMV strains, a 2b protein has been found in CMV-infected plants. This 2b protein, which was translated from subgenomic RNA of RNA2, was associated with host-specific long-distance virus movement (Ding et al. 1995) and with suppressing the gene silencing reaction in host plants (Li et al. 1999). Therefore, RNA1, 2 and 3 are needed to systemically infect the host plant. Previously, we reported that a yellow strain of CMV [CMV(Y)] induces the HR in A. thaliana ecotype C24 but not in ecotype Columbia (Takahashi et al. 1994). The HR in CMV(Y)-inoculated C24 was determined by the interaction between CMV RNA3 and a single dominant resistance gene of C24, RCY1, which imposed the localization of the virus within the inoculated C24 leaves. In this report, to characterize the resistance response to CMV(Y) in C24, the avirulence determinant on CMV(Y) RNA3 was defined, and the resistance gene (RCY1) locus in C24 was genetically mapped. For the propagation of CMV(Y) and an Indonesian isolate of CMV [CMV(B2)] (Tomaru and Hidaka 1960, Suastika et al. 1995), tobacco plants (Nicotiana tabacum cv. Samsun NN) were grown at 26°C in a greenhouse, and the fully expanded tobacco leaves were inoculated with 50 µg ml–1 of CMV. At 7 d after inoculation, virus-inoculated leaves were harvested, and the virus was purified by a procedure described previously (Takahashi and Ehara 1993). Arabidopsis thaliana ecotypes C24 and Columbia (Col) were grown in vermiculite : perlite (1 : 1 mixture) at 24°C under continuous fluorescent light (7,000 lux). One fully expanded leaf of a 14- to 18-day-old (5 to 6 leaf stage) A. thaliana plant was inoculated with 100 µg ml–1 of CMV. The symptom expression on both the virus-inoculated leaf at 3 d after inoculation and on non-inoculated upper leaves at 7 d after inoculation were observed, and then the coat protein within them was immunologically detected by the tissue-printing method (Srinivasan and Tolin 1992). When A. thaliana ecotype C24 was inoculated with CMV(Y), necrotic local lesions (NLLs) began to appear on virus-inoculated C24 leaves at 36 h after inoculation and matured at 3 d after inoculation (Fig. 1A). The coat protein of CMV(Y) was detected on the inoculated leaves (Fig. 1C). However, non-inoculated upper leaves of CMV(Y)-infected C24 plants that developed NLLs on their inoculated leaves had no detectable coat protein at 7 d after inoculation (Fig. 2). When ten C24 plants were inoculated with CMV(B2), the virus spread systemically, causing typical symptoms such as yellowing and stunting (Fig. 1B, 2). On the other hand, when A. thaliana ecotype Columbia (Col) was inoculated with CMV(Y) and CMV(B2), respectively, the NLLs did not develop on either of the virus-inoculated leaves (data not shown). The coat protein was detected on the inoculated and non-inoculated upper leaves of both CMV(Y) and CMV(B2)-infected Col plants (Fig. 2). These results indicated that the systemic spread of the virus was specifically restricted in virus-inoculated leaves of CMV(Y)-infected C24 plants. In our previous report (Takahashi et al. 1994), we used an ordinary strain of CMV[CMV(O)] as the CMV isolate; it systemically infected C24. Although the typical systemic symptoms were observed on non-inoculated upper leaves of CMV(O)-infected C24 at 7 d after inoculation, the NLLs were observed on their inoculated leaves about 12 h later than on CMV(Y)-inoculated C24 leaves in half of CMV(O)-infected C24 (Takahashi et al. 1994). Furthermore, in CMV(O)-inoculated C24 leaves showing such NLLs, the induction of PR-1a gene was detected at the mRNA level (data not shown). These observations indicated that we should consider the possibility that CMV(O) may delay the induction of the HR, thereby infecting C24 systemically. Therefore, in this report, the combination of two CMV strains [CMV(Y) and CMV(B2)] and two A. thaliana ecotypes (C24 and Col) was used for mapping the avirulence determinant on virus genome and the resistance gene of host plant. The host response to a series of pseudorecombinant CMVs, which were constructed by exchanging CMV RNA1+2 and RNA3 between CMV(Y) and CMV(O), indicated that the resistance in CMV(Y)-infected C24 was controlled by RNA3 (Takahashi et al. 1994). Therefore, to define the avirulence determinant on CMV genomes, we focused on RNA3. Chimeric forms of the RNA3-coding region in CMV were constructed at the cDNA level (Fig. 3). pCY3-T7 contains cDNA for CMV(Y) RNA3 under the control of T7 promoter (Nitta et al. 1988, Suzuki et al. 1991). cDNA to CMV(B2) RNA3 was synthesized by RT-PCR with two primers, 5′-CGCGGATCCTAATACGACTCACTATAGTAATCTTACCACTCGTGTGT-3′ containing a BamHI restriction site and the T7 promoter sequence and 5′-CCGGAATTCTGGTCTCCTTTTGGAGGCC-3′ containing a EcoRI restriction site (the viral sequences are underlined) (Baier et al. 1993). The cDNA was cloned into the BamHI and EcoRI sites of pUC118 (TAKARA, Kyoto, Japan). This cDNA sequence has been lodged with DDBJ (accession no. AB042294). Then, because the cDNA to CMV(B2) RNA3 did not have either a SalI-digestion site at 1,298 nucleotide position or a XhoI-digestion site at 1,840 nucleotide positions but pCY3-T7 had both, the nucleotide sequences digested by SalI and XhoI were introduced into 1,298 and 1,840 nucleotide positions in the cDNA to CMV(B2) RNA3, using primers, 5′-CGTAACCGTCGACGTCGTCC-3′ (the SalI restriction site is underlined) and its complimentary sequence for the SalI site, and 5′-CATCCGTCTCGAGCGCATCG-3′ (the XhoI restriction site is underlined) and its complimentary sequence for the XhoI site, by PCR (Higuchi et al. 1998). The introduction of the restriction site of SalI did not cause any changes in the amino acids encoded in those regions, but the XhoI site changed Pro to Leu at the 194 amino acid position in the coat protein. The resulting plasmid was designated as pCB3-T7. pCY3-T7 and pCB3-T7 were used to construct chimeric cDNAs containing the CMV(Y) RNA3 and CMV(B2) RNA3 sequences (Fig. 3). pCY3-BSX and pCB3-YSX were constructed by exchanging the SalI–XhoI fragment (nucleotides 1,298–1,840 of RNA3) between pCY3-T7 and pCB3-T7. Standard molecular techniques were used for vector construction (Sambrook et al. 1989). Construction of these two chimeric cDNAs to CMV RNA3 was confirmed by partial dideoxy-sequencing using primers specific to CMV RNA3. The constructed plasmids containing cDNA to CMV RNA3 were linearized by digestion with EcoRI and in vitro transcribed using T7 RNA polymerase. Each linearized plasmid DNA (2 µg) was added to 50 mM Tris-HCl (pH 7.5), 0.5 mM GTP, 5 mM ATP, 5 mM CTP, 5 mM UTP, 0.1 mM DTT, 5 mM 7mGpppG-cap analog (New England Biolabs, Beverly, MA), 30 units of RNasin (TOYOBO, Osaka, Japan) and 70 units of T7 RNA polymerase (Pharmacia, Piscataway, NJ), and was incubated at 37°C for 1 h. CMV(Y) RNA1 and RNA2 were also transcribed in vitro from NotI-linearized pCY1-T7 and pCY2-T7 (Suzuki et al. 1991). Tobacco plants (N. tabacum cv Xanthi nc) were inoculated with in vitro transcribed CMV RNA1, RNA2 and RNA3. Viruses which contained RNA3 in vitro transcribed from pCY3-T7, pCB3-T7, pCY3-BSX, or pCB3-YSX were designated as CMV(Y), CMV(B2)SX, CMV(Y/BSX) or CMV(B/YSX), respectively. Each virus (100 µg ml–1) was used to inoculate A. thaliana ecotypes C24 and Col. Arabidopsis thaliana ecotype C24 was systemically infected with CMV(B2)SX as well as the parental CMV(B2) (Fig. 4, Table 1), although one amino acid change from Pro to Leu at 194 amino acid position in the coat protein of CMV(B2)SX occurred. To define the avirulence determinant in CMV RNA3, A. thaliana C24 was inoculated with CMV(B/YSX) and CMV(Y/BSX), respectively. In CMV(B/YSX)-inoculated C24 leaves as well as in CMV(Y)-inoculated C24 leaves (Fig. 1A), the NLLs developed at 36 h after inoculation and matured at 3 d after inoculation, and no coat protein was detected on their non-inoculated upper leaves (Fig. 4, Table 1). On the other hand, the coat protein was systemically distributed in CMV(Y/BSX)-infected C24, whose inoculated leaves did not show any symptoms (Fig. 1A, 4, Table 1). The multiplication of the recombinant CMVs in virus-infected C24 was confirmed by sequencing the cDNA to the coat protein gene of the progeny viruses (data not shown). Because, the SalI–XhoI-digested fragment, which includes about 90% of coat protein gene in CMV, had been exchanged at the cDNA level between CMV(Y) and CMV(B2), the virus localization in CMV(B/YSX)-inoculated C24 leaves contrasted with the systemic spread of virus in CMV(Y/BSX)-inoculated C24 leaves indicated that the avirulence determinant in CMV(Y)RNA3 was located in the coat protein gene. The coding region of the coat protein gene had 92.0% and 95.0% identities between CMV(Y) and CMV(B2) at the nucleotide and amino acid sequence levels, respectively. The resistance response to CMV(Y) in C24 seemed to be controlled by the changes of amino acids in the coat protein rather than by an effect of the RNA sequence itself, because, in addition to the capsid function for the assembly of virus particle, several phenotypic properties in CMV-infected plants, such as the induction of chlorosis and stunting response in tobaccos, aphid transmissibility, host specificity, and virus localization with the developing NLLs, are associated with alterations in the viral coat protein itself rather than with the nucleotide sequence of the coat protein (Shintaku et al. 1992, Takahashi and Ehara 1993, Perry et al. 1994, Suzuki et al. 1995, Ryu et al. 1998, Szilassy et al. 1999, Sugiyama et al. 2000, Wikoff et al. 1997). The amino acid sequence alignment of the coat protein of CMV(Y) and CMV(B2)SX indicated six amino acid differences (Fig. 3). We do not know whether the induction of the resistance to CMV(Y) in C24 was controlled by the local secondary structure of the coat protein or by a particular amino acid in the coat protein causing the interaction between the coat protein and unknown host factor(s). To understand this issue, it will be necessary to delimit the amino acid(s) in the coat protein conferring the resistance response. For the genetic mapping analysis of the RCY1 locus, F2 progeny plants of the Col×C24 cross were used. We screened 109 individuals of CMV(Y)-susceptible F2 progenies by detecting the coat protein on non-inoculated upper leaves of CMV(Y)-infected F2 plants at 7d after inoculation. For analysis with the cleaved amplified polymorphic sequence (CAPS) markers, AG (MapPairs from Research Genetics, Huntsville, AL, U.S.A.), PR5K, PHYC, DFR and T43968, the PCR reaction and restriction analysis of the PCR products were performed by the method of Konieczny and Ausubel (1993). The nucleotide sequences for the primers of PR5K and PHYC were derived from the information about new CAPS markers available through the A. thaliana database. Because two of the CAPS markers, DFR and T43968, were not polymorphic between Col and C24, the nucleotide sequences around DFR and T43968 markers in C24 and Col were determined, and then the DFR primers, 5′-AGATCCTGAGGTGAGTTTTTC-3′ and 5′-TGTATATTCTTACCCATCCT-3′, and the T43968 primers, 5′-GGAAACTCTGCAGTCTTGTACA-3′ and 5′-TTGGGAGAACTATCCTCTGCAG-3′, were used for CAPS analysis. The modified DFR and T43968 products were cleaved with HincII and BlnI, respectively, and electrophoresed in 2% agarose gel by the standard protocol (Sambrook et al. 1989). For analysis with the simple sequence length polymorphisms (SSLP) markers, nga280, nga248, nga168, nga162, nga112, nga8, nga 139, nga 106, nga 249 and nga129 (MapPairs from Research Genetics, Huntsville, AL, U.S.A.), the PCR reaction was performed by the method of Bell and Ecker (1994). The obtained PCR products were electrophoresed in 4% agarose gel. The recombination frequencies between RCY1 and the markers in the CMV(Y)-susceptible F2 individuals were calculated by the method of Allard (1956). The map distances in centimorgans between RCY1 locus and the markers were converted from their recombination frequencies by the Kosambi map function (Kosambi 1944). To determine the chromosome location of the RCY1 locus responsible for the resistance to CMV(Y), thirty CMV(Y)-susceptible F2 plants and one to four CAPS or SSLP markers on each chromosome (ch.), nga280 and nga248 on ch.1, nga168 on ch.2, nga162 and nga112 on ch.3, AG and nga8 on ch.4, and nga139, nga106, nga249 and nga129 on ch.5, were initially used. The recombination frequencies between RCY1 locus and the markers on ch. 1, 2, 3, and 4 were more than 40%; however, nga249, nga106, nga139, and nga129 on ch. 5 were 39.7±4.5%, 36.7±4.1%, 30.6±4.1%, and 9.5±2.0% recombination frequencies with the RCY1 locus in 60 chromosomes. The recombination frequency between nga139 and nga129 was 33.0±4.6%. This result suggested that RCY1 locus was located between nga139 and nga129 markers on ch. 5. For further mapping analysis, 109 CMV(Y)-susceptible F2 plants and four CAPS markers between nga139 and nga129, PHYC, PR5K, modified DFR, and modified T43968, were used. Based upon the recombination frequencies between RCY1 locus and each of these four markers, 20.6±3.6% to PHYC, 7.7±1.8% to PR5K, 1.8±0.9% to modified DFR, and 0.5±0.5% to modified T43968 in 218 chromosomes, the RCY1 locus was mapped at approximately 0.5±0.5 cM of modified T43968 and 1.8±0.9 cM of modified DFR (Fig. 5). As the distance between modified T43968 and DFR was found to be 2.0±1.3 cM based upon their recombination frequency using 52 additional resistant and susceptible F3 families, RCY1 was mapped between these two CAPS markers (Fig. 5). Forty-two Arabidopsis expressed sequence tags showing similarities to disease resistance genes (R-ESTs) have been mapped on five chromosomes of A. thaliana (Botella et al. 1997). The R-ESTs encode proteins containing a leucine-rich repeat (LRR) domain. Several of these R-ESTs occur in clusters on the chromosome. RCY1 locus was mapped to one of the R-EST clusters, MRC-J, in which nine defined disease resistance genes (RAC3, RPS4, HRT12, TTR1 and five distinct RPP loci) were genetically mapped. The MRC-J covered about 30 cM of the long arm of ch. 5 on the RI map available through the A. thaliana database, and RCY1 and two CAPS markers, DFR and T43968, were apparently located on the middle of this MRC-J region. The presence of R-EST cluster suggested that the resistance gene in the cluster was generated by gene duplication events (Botella et al. 1997, Meyers et al. 1999). Therefore, the RCY1 locus may be a member of the resistance genes in the R-EST cluster on chromosome 5. The results presented in this report indicate that the resistance response to CMV(Y) in C24 was determined by the combination of the coat protein gene and RCY1 mapped between DFR and T43968 markers on chromosome 5. During the past 7 years, resistance genes that protect the host plants against viruses, bacteria and fungi have been isolated (Hammond-Kosack and Jones 1997). Furthermore, some genes encoding critical components of the signal transduction pathway(s) concerning the induction of disease resistance have been cloned from Arabidopsis, for example NPR1 (Dong 1998). Salicylic acid (SA), jasmonic acid, and ethylene are also important components of the signal transduction pathway leading to disease resistance (Dong 1998). In Arabidopsis, it has been demonstrated that these key signaling-compound-mediated signal transduction pathways were selectively activated by the resistance response to pathogens (Thomma et al. 1998). However, with regard to the HR to viral pathogens in Arabidopsis, only the resistance gene (HRT1) to TCV has been isolated to date, although two genes (RTM1 and RTM2), which restrict the long-distance movement of tobacco etch virus, and a gene (TOM1) encoding a component of the virus RNA replication complex for TMV have been cloned from Arabidopsis (Chisholm et al. 2000, Whitham et al. 2000, Yamanaka et al. 2000). TCV resistance by the HR in Arabidopsis required the SA-dependent signaling pathway (Kachroo et al. 2000). However, nahG C24, expressing SA hydrogenase and thereby failing to accumulate SA, showed resistance to CMV(Y) (Takahashi et al. unpublished results). The characterization of the avirulence determinant on CMV genomes and the resistance gene in C24 provides not only another example of virus resistance on Arabidopsis; it may also lead to greater understanding of SA-independent signal transduction pathway(s) conferring virus disease resistance. Acknowledgements We thank Dr. S. Kanematsu, Tohoku Agriculture Experimental Station, Japan, for technical advice on press-blotting. We also thank the Arabidopsis Biological Resource Center at Ohio State, U.S.A., for providing Arabidopsis seeds. This work was supported in part by a grant-in-aid for scientific research on priority areas (Molecular Mechanisms of Plant—Pathogenic Microbe Interaction—Toward Production of Disease Resistant Plants). 5 Corresponding author: E-mail, takahash@bios.tohoku.ac.jp; Fax, +81-22-717-8659. View largeDownload slide Fig. 1 Symptoms on CMV-infected A. thaliana ecotype C24. Ten C24 plants per virus [CMV(Y), CMV(B2), CMV(Y/BSX) and CMV(B/YSX)], were examined, and representatives are shown here. (A) Symptoms on three independent virus-inoculated leaves at 3 d after inoculation and mock-inoculated leaves (Mock). (B) Systemic symptom expression on CMV(B2)-infected C24 plants at 21 d after inoculation and on the corresponding CMV(Y)-infected C24 plants. (C) Detection of coat protein in CMV-infected A. thaliana ecotype C24 in virus-inoculated leaves at 3 d after inoculation. Leaf protein were press-blotted to 3 MM filter paper, and the coat protein of CMV(Y) and CMV(B2) was immunologically detected. Mock-inoculated C24 (Mock) was used as a control. View largeDownload slide Fig. 1 Symptoms on CMV-infected A. thaliana ecotype C24. Ten C24 plants per virus [CMV(Y), CMV(B2), CMV(Y/BSX) and CMV(B/YSX)], were examined, and representatives are shown here. (A) Symptoms on three independent virus-inoculated leaves at 3 d after inoculation and mock-inoculated leaves (Mock). (B) Systemic symptom expression on CMV(B2)-infected C24 plants at 21 d after inoculation and on the corresponding CMV(Y)-infected C24 plants. (C) Detection of coat protein in CMV-infected A. thaliana ecotype C24 in virus-inoculated leaves at 3 d after inoculation. Leaf protein were press-blotted to 3 MM filter paper, and the coat protein of CMV(Y) and CMV(B2) was immunologically detected. Mock-inoculated C24 (Mock) was used as a control. View largeDownload slide Fig. 2 Detection of the coat protein in CMV-infected A. thaliana ecotypes C24 and Col. Ten C24 and Col plants per virus [CMV(Y) and CMV(B2)] were examined, and representatives are shown here. (A) Entire plants which were press-blotted on the 3 MM filter paper. (B) The coat protein of CMV(Y) and CMV(B2) was detected immunologically in virus-infected whole plants (C24 and Col) at 7 d after inoculation. Mock-inoculated C24 (Mock) was used as a control. View largeDownload slide Fig. 2 Detection of the coat protein in CMV-infected A. thaliana ecotypes C24 and Col. Ten C24 and Col plants per virus [CMV(Y) and CMV(B2)] were examined, and representatives are shown here. (A) Entire plants which were press-blotted on the 3 MM filter paper. (B) The coat protein of CMV(Y) and CMV(B2) was detected immunologically in virus-infected whole plants (C24 and Col) at 7 d after inoculation. Mock-inoculated C24 (Mock) was used as a control. View largeDownload slide Fig. 3 Chimeric CMV RNA3 transcription vector constructs. Rectangular boxes represent open reading frames of the 3a protein gene (3a) and the coat protein gene (CP). cDNA from CMV(Y) is represented as a shaded box; cDNA from CMV(B2) as an unfilled box. Differences in the amino acid sequence, their positions, and the restriction endonuclease sites are indicated. RNA3 is transcribed in vitro from cDNA under the control of T7 promoter (T7). View largeDownload slide Fig. 3 Chimeric CMV RNA3 transcription vector constructs. Rectangular boxes represent open reading frames of the 3a protein gene (3a) and the coat protein gene (CP). cDNA from CMV(Y) is represented as a shaded box; cDNA from CMV(B2) as an unfilled box. Differences in the amino acid sequence, their positions, and the restriction endonuclease sites are indicated. RNA3 is transcribed in vitro from cDNA under the control of T7 promoter (T7). View largeDownload slide Fig. 4 Detection of the coat protein in recombinant CMV-infected A. thaliana ecotype C24. Seven to twelve plants per virus [CMV(Y), CMV(B2)sx, CMV(Y/BSX) and CMV(B/YSX)] were examined (Table 1), and representatives are shown here. The leaf protein was press-blotted to 3 MM filter paper, and the coat protein was detected immunologically. View largeDownload slide Fig. 4 Detection of the coat protein in recombinant CMV-infected A. thaliana ecotype C24. Seven to twelve plants per virus [CMV(Y), CMV(B2)sx, CMV(Y/BSX) and CMV(B/YSX)] were examined (Table 1), and representatives are shown here. The leaf protein was press-blotted to 3 MM filter paper, and the coat protein was detected immunologically. View largeDownload slide Fig. 5 Genetic map position of RCY1 locus on chromosome 5 of Arabidopsis. RCY1 locus was mapped on 109 F2 progeny from a cross between Col×C24. The markers nga 139 and nga129 are SSLP markers. The markers PHYC, PR5K, DFR, and T43968 areCAPSmarkers. The vertical bars indicate positions of loci. Numbers are centimorgans. The MRC-J region containing genetically defined disease resistance genes and their homologous genes is shown by the arrow bar. View largeDownload slide Fig. 5 Genetic map position of RCY1 locus on chromosome 5 of Arabidopsis. RCY1 locus was mapped on 109 F2 progeny from a cross between Col×C24. The markers nga 139 and nga129 are SSLP markers. The markers PHYC, PR5K, DFR, and T43968 areCAPSmarkers. The vertical bars indicate positions of loci. Numbers are centimorgans. The MRC-J region containing genetically defined disease resistance genes and their homologous genes is shown by the arrow bar. Table 1 Response to recombinant CMV in A. thaliana ecotype C24 Virus a  Response in A. thaliana C24  Symptoms on the inoculated leaves  Systemic infection of virus b  CMV(Y)  necrotic local lesions  0 / 12  CMV(B2)sx  no symptoms  7 / 7  CMV(Y/BSX)  no symptoms  10 / 10  CMV(B/YSX)  necrotic local lesions  0 / 12  Virus a  Response in A. thaliana C24  Symptoms on the inoculated leaves  Systemic infection of virus b  CMV(Y)  necrotic local lesions  0 / 12  CMV(B2)sx  no symptoms  7 / 7  CMV(Y/BSX)  no symptoms  10 / 10  CMV(B/YSX)  necrotic local lesions  0 / 12  a CMV(Y), CMV(B2)sx, CMV(Y/BSX) and CMV(B/YSX) contain RNA3 in vitro transcribed from pCY3-T7, pCB3-T7, pCY3-BSX and pCB3-YSX, respectively, in Fig. 3. These recombinant CMVs were used to inoculate A. thaliana ecotype C24. b The number of systemically infected plants is shown over the number of CMV(Y)-inoculated plants. View Large Abbreviations CAPS cleaved amplified polymorphic sequence ch. chromosome Col Columbia CMV cucumber mosaic virus HR hypersensitive response NLLs necrotic local lesions LRR leucine-rich repeat PR pathogenesis-related PVX potato virus X R-ESTs sequence tags showing similarity to disease resistance genes RT reverse transcription SA salicylic acid SSLP simple sequence length polymorphisms TCV turnip crinkle virus TMV tobacco mosaic virus. References Allard, R.W. ( 1956) Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia  24: 235–278. Google Scholar Baier, G., Telford, D., Gulbins, E., Yamada, N., Kawakami, T. and Altman, A. ( 1993) Improved specificity of RT-PCR amplifications using nested cDNA primers. Nucl. Acids Res.  21: 1329–1330. Google Scholar Bell, C.J. and Ecker, J.R. ( 1994) Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics  19: 137–144. Google Scholar Bendahmane, A., Kanyuka, K. and Baulcombe, D.C. ( 1999) The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell  11: 781–791. Google Scholar Berzal-Herranz, A., De La Cruz, A., Tenllado, F., Díaz-Ruíz, J.R., Lóez, L., Sanz, A.I., Vaquero, C., Serra, M.T. and García-Luque, I. ( 1995) The Capsicum L3 gene-mediated resistance against the tobamoviruses is elicited by the coat protein. Virology  209: 498–505. Google Scholar Botella, M.A., Coleman, M.J., Hughes, D.E., Nishimura, M.T., Jones, J.D.G. and Somerville, S.C. ( 1997) Map positions of 47 Arabidopsis sequenceswith sequence similarity to disease resistance genes. Plant J.  12: 1197–1211. Google Scholar Chisholm, S.T., Mahajan, S.K., Whitham, S.A., Yamamoto, M.L. and Carrington, J.C. ( 2000) Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus. Proc. Natl. Acad. Sci. USA  97: 489–494. Google Scholar Cooley, M.B., Pathirana, S., Wu, H-J., Kachroo, P. and Klessig, D.F. ( 2000) Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens. Plant Cell  12: 663–676. Google Scholar Davis, C. and Symons, R.H. ( 1988) Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA3. Virology  165: 216–224. Google Scholar De La Cruz, A., López, L., Tenllado, F., Díaz-Ruíz, J.R., Sanz, A.I., Vaquero, C., Serra, M.T. and García-Luque, I. ( 1997) The coat protein is required for the elicitation of the Capsicum L2 gene-mediated resistance against the tobamoviruses. Mol. Plant-Microbe Interact.  10: 107–113. Google Scholar Dempsey, D.A., Pathirana, M.S., Wobbe, K.K. and Klessig, D.F. ( 1997) Identification of an Arabidopsis locus required for resistance to turnip crinkle virus. Plant J.  11: 301–311. Google Scholar Dempsey, D.A., Wobbe, K.K. and Klessig, D.F. ( 1993) Resistance and susceptible responses of Arabidopsis thaliana to turnip crinkle virus. Phytopathology 83: 1021–1029. Google Scholar Ding, S.W., Li, W.X. and Symons, R.H. ( 1995) A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. EMBO J.  14: 5762–5772. Google Scholar Dong, X. ( 1998) SA, JA, ethylene, and disease resistance in plants. Curr. Opin. Plant Biol.  1: 316–323. Google Scholar Flor, H.H. ( 1971) Current status of the gene-for-gene concept. Annu. Rev. Phytopathol.  9: 275–296. Google Scholar Goodman, R.N. and Novacky, A.J. ( 1994) The Hypersensitive Reaction in Plants to Pathogens. A Resistance Phenomenon.American Phytopathological Society, St. Paul, MN. Google Scholar Goulden, M.G., Köhn, B.A., Santa Cruz, S., Kavanagh, T.A. and Baulcombe, D.C. ( 1993) A feature of the coat protein of potato virus X affects both induced virus resistance in potato and viral fitness. Virology  197: 293–302. Google Scholar Hammond-Kosack, K.E. and Jones, J.D.G. ( 1997) Plant disease resistance genes. Annu. Rev. Plant Physiol. Plant Mol. Biol.  48: 575–607. Google Scholar Hayes, R.J. and Buck, K.W. ( 1990) Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase. Cell  63: 363–368. Google Scholar Higuchi, R., Krummel, B. and Saiki, R.K. ( 1998) A general method ofinvitropreparationand specific mutagenesis of DNA fragments: Study of protein and DNA interactions. Nucl. Acids Res.  16: 7351–7367. Google Scholar Kachroo, P., Yoshioka, K., Shah, J., Dooner, H.K. and Klessig, D.F. ( 2000) Resistance to turnip crinkle virus in Arabidopsis is regulated by two host genes and is salicylic acid dependent but NPR1, ethylene, and jasmonate independent. Plant Cell  12: 677–690. Google Scholar Keen, N.T. and Dawson, W.O. ( 1992) Pathogen avirulence genes and elicitors of plant defense. In Genes Involved in Plant Defense. Edited by Boller, T. and Meins, F. pp. 85–114, Springer-Verlag, Wien. Google Scholar Konieczny, A. and Ausubel, F.M. ( 1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J.  4: 403–410. Google Scholar Kosambi, D.D. ( 1944) The estimation of map distances from recombination values. Ann. Eugen.  12: 172–175. Google Scholar Knorr, D.A. and Dawson, W.O. ( 1988) A point mutation in the tobacco mosaic virus capsid protein gene induces hypersensitivity in Nicotiana sylvestris. Proc. Natl. Acad. Sci. USA  85: 170–174. Google Scholar Kunkel, B.N. ( 1996) A useful weed put to work: Genetic analysis of disease resistance in Arabidopsis thaliana. Trends Genet.  12: 63–69. Google Scholar Li, H.W., Lucy, A.P., Guo, H.-S., Li, W.X., Ji, L.H., Wong, S.M. and Ding, S.W. ( 1999) Strong host resistance targeted against a viral suppressor of the plant gene silencing defense mechanism. EMBO J.  18: 2683–2691. Google Scholar Meyers, B., Dickerman, A.W., Michelmore, R.W., Sivaramakrishnan, S., Sobral, B.W. and Young, N.D. ( 1999) Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. Plant J.  20: 317–332. Google Scholar Nitta, N., Masuta, C., Kuwata, S. and Takanami, Y. ( 1988) Comparative studies on the nucleotide sequence of cucumber mosaic virus RNA3 between Y strain and Q strain. Ann. Phytopathol. Soc. Japan  54: 516–522. Google Scholar Padgett, H.S. and Beachy, R.N. ( 1993) Analysis of a tobacco mosaic virus strain capable of overcoming N gene-mediated resistance. Plant Cell  5: 577–586. Google Scholar Palukaitis, P., Roossinck, M.J., Dietzgen, R.G. and Francki, R.I.B. ( 1992) Cucumber mosaic virus. Adv. Virus Res.  41: 281–348. Google Scholar Perry, K.L., Zhang, L., Shintaku, M.H. and Palukaitis, P. ( 1994) Mapping determinants in cucumber mosaic virus for transmission by Aphis gossypii. Virology  205: 591–595. Google Scholar Ryu, K.H., Kim, C.-H. and Palukaitis, P. ( 1998) The coat protein of cucumber mosaic virus is a host range determinant for infection of maize. Mol. Plant-Microbe Interact.  11: 351–357. Google Scholar Saito, T., Meshi, T., Takamatsu, N. and Okada, Y. ( 1987) Coat protein gene sequence of tobacco mosaic virus encodes a host response determinant. Proc. Natl. Acad. Sci. USA  84: 6074–6077. Google Scholar Sambrook, J., Fritsch, E.F. and Maniatis, T. ( 1989) Molecular Cloning: A Laboratory Manual.2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Google Scholar Santa Cruz, S. and Baulcombe, D. ( 1993) Molecular analysis of potato virus X isolate in relation to the potato hypersensitivity gene Nx. Mol. Plant-Microbe Interact.  6: 707–714. Google Scholar Schwinghamer, M.W. and Symons, R.H. ( 1977) Translation of the four major RNA species of cucumber mosaic virus in plant and animal cell-free systems and in toad oocytes. Virology  79: 88–108. Google Scholar Shintaku, M.H., Zhang, L. and Palukaitis, P. ( 1992) A single amino acid substitution in the coat protein of cucumber mosaic virus induces chlorosis in tobacco. Plant Cell  4: 751–757. Google Scholar Simon, A.E. ( 1994) Interactions between Arabidopsis thaliana and viruses. In Arabidopsis.Edited by Meyerowitz, E.M. and Somerville, C.R. pp. 685–704. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Google Scholar Simon, A.E., Li, X.-H., Lew, J.E., Stange, R., Zhang, C., Polacco, M. and Carpenter, C.D. ( 1992) Susceptibility and resistance of Arabidopsis thaliana to turnip crinkle virus. Mol. Plant-Microbe Interact.  5: 496–503. Google Scholar Srinivasan, I. and Tolin, S.A. ( 1992) Detection of three viruses of clovers by direct tissue immunoblotting. Phytopathology  82: 721. Google Scholar Suastika, G., Tomaru, K., Kurihara, J. and Natsuaki, K.T. ( 1995) Characteristics of two isolates of cucumber mosaic virus obtained from banana plants in Indonesia. Ann. Phytopathol. Soc. Jpn  61: 272. Google Scholar Sugiyama, M., Sato, H., Karasawa, A., Hase, S., Takahashi, H. and Ehara, Y. ( 2000) Characterization of symptom determinants in two mutants of cucumber mosaic virus Y strain, causing distinct mild green mosaic symptoms in tobacco. Physiol. Mol. Plant Pathol.  56: 85–90. Google Scholar Suzuki, M., Kuwata, S., Kataoka, J., Masuta, C., Nitta, N. and Takanami, Y. ( 1991) Functional analysis of deletion mutants of cucumber mosaic virus RNA3 using an in vitro transcription system. Virology  183: 106–113. Google Scholar Suzuki, M., Kuwata, S., Masuta, C. and Takanami, Y. ( 1995) Point mutations in the coat protein of cucumber mosaic virus affect symptom expression and virion accumulation in tobacco. J. Gen. Virol.  76: 1791–1799. Google Scholar Szilassy, D., Salánki, K. and Balázs, E. ( 1999) Stunting induced by cucumber mosaic cucumovirus-infected Nicotiana glutinosa is determined by a single amino acid residue in the coat protein. Mol. Plant-Microbe Interact.  12: 1105–1113. Google Scholar Takahashi, H. and Ehara, Y. ( 1993) Severe chlorotic spot symptoms in cucumber mosaic virus strain Y-infected tobaccos are induced by a combination of the virus coat protein gene and two host recessive genes. Mol. Plant-Microbe Interact.  6: 182–189. Google Scholar Takahashi, H., Goto, N. and Ehara, Y. ( 1994) Hypersensitive response in cucumber mosaic virus-inoculated Arabidopsis thaliana. Plant J.  6: 369–377. Google Scholar Thomma, B.P.H.J., Eggermont, K., Penninckx, I.A.M.A., Mauch-Mani, B., Vogelsang, R., Cammue, B.P.A. and Broekaert, W.F. ( 1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc. Natl. Acad. Sci. USA  95: 15107–15111. Google Scholar Tomaru, K. and Hidaka, J. ( 1960) Strains of cucumber mosaic virus isolated from tobacco plants. III. A yellow strain. Bull. Hatano Tobacco Exp. Stn  46: 143–149. Google Scholar Whitham, S., Dinesh-Kumar, S.P., Chol, D., Hehl, R., Corr, C. and Baker, B. ( 1994) The product of the tobacco mosaic virus resistance gene N: Similarity to toll and the interleukin-1 receptor. Cell  78: 1101–1115. Google Scholar Whitham, S., Anderberg, R.T., Chisholm, S.T. and Carrington, J.C. ( 2000) Arabidopsis RTM2 gene is necessary for specific restriction of tobacco etch virus and encodes an unusual small heat shock-like protein. Plant Cell  12: 569–582. Google Scholar Wikoff, W.R., Tsai, C.J., Wang, G., Baker, T.S. and Johnson, J.E. ( 1997) The structure of cucumber mosaic virus: Cryoelectron microscope, X-ray crystallography, and sequence analysis. Virology  232: 91–97. Google Scholar Yamanaka, T., Ohta, T., Takahashi, M., Meshi, T., Schmidt, R., Dean, C., Naito, S. and Ishikawa, M. ( 2000) TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein. Proc. Natl. Acad. Sci. USA  97: 10107–10112. Google Scholar

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Plant and Cell PhysiologyOxford University Press

Published: Mar 15, 2001

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