Cucumber mosaic virus (CMV) is a well-characterized tripartite virus comprising three positive single-stranded RNA genomes: RNA1, RNA2, and RNA3. The RNA1 and RNA2 genomes encode the components of the viral replicase (1a and 2a proteins), and RNA3 contains two open reading frames (ORFs) encoding the cell-to-cell movement protein (3a protein) and the coat protein (CP) [5]. The CP is translated from RNA4, which is a subgenomic RNA containing the ORF encoding the CP that is transcribed from the minus-strand of RNA3 in virus-infected cells. The RNA2 genome contains a small second ORF encoding the 2b protein, which is also translated from a subgenomic RNA of RNA2 in virus-infected cells and acts as viral suppressor of RNA silencing. Various strains and isolates of CMV exist that induce symptoms of different severity or different host responses [2, 5]. Arabidopsis thaliana ecotype C24 carrying the RESISTANCE TO CMV(Y) 1 (RCY1) gene exhibits resistance to a yellow strain of CMV [CMV(Y)] but ecotype Col-0 is susceptible to this strain of CMV [10]. Meanwhile, the CMV B2 strain [CMV(B2)] exhibits virulence to both Arabidopsis ecotypes C24 and Col-0 [14]. Analysis of CMV resistance using a series of CMV strains reassortant between CMV(Y) and CMV(B2) indicates that the CP gene in CMV(Y) is the corresponding avirulence gene to the Arabidopsis RCY1 gene [14]. RCY1 is encoded by the RCY1/RPP8/HRT locus, which represents a singleton R gene encoding a coiled-coil (CC)-nucleotide binding (NB)-leucine rich repeat (LRR) protein [7, 11] that has likely become evolutionally diversified among three ecotypes to confer resistance to three different pathogens, including CMV in A. thaliana ecotype C24, turnip crinkle virus (TCV) in Arabidopsis ecotype Di-17, and Hyaloperonospora arabidopsidis in Arabidopsis ecotype Ler [1, 4, 11].
In the present study, we first confirmed that the CMV(Y) CP functions as the avirulence molecule independent of the other CMV(Y) RNA-coding proteins (1a, 2a 2b and 3a proteins). Next, to further dissect the avirulence function of the CP, a series of binary vectors constructed by partially exchanging CP-coding sequences between CMV(Y) and CMV(B2) or by inducing nucleotide substitutions in the CP-coding sequences were transiently expressed in Nicotiana benthamiana #4-3 leaves transformed with modified RCY1 cDNA (P/P/C-HA) containing the CC-NB domain of RPP8 and the LRR domain of RCY1 with an HA-epitope tag sequence. Subsequent analyses of hypersensitive resistance-cell death (HCD), CP accumulation, and defense gene expression in the leaves transiently expressing each CP, revealed that a single amino acid in the CP might determine avirulence function in RCY1-conferred resistance to CMV(Y).
The cDNAs for CMV(Y) RNA1, RNA2, RNA3, CP-coding RNA, and 2b-coding RNA sequences were synthesized by RT-PCR using a set of primers shown in Table S1 with pCY1-T7, pCY2-T7, or pCY3-T7 as a template DNA [15]. These cDNAs were then cloned between the CaMV 35S promoter and HSP terminator in the binary vector pRI201-AN (Takara-Bio, Shiga, Japan) according to a standard protocol [6]. The resultant vector constructs were named 35S::CY1, 35S::CY2, 35S::CY3, and 35S::Y2b-HA (Figs. 1A and S1A). A 35S::GFP construct was also generated to use as a control for gene and protein expression (Figs. 1A and S1A).
The cDNAs of the CMV(Y) CP and CMV(B2) CPs (Fig. 2A), the 10 cDNAs encoding CPs chimeric between CMV(Y) and CMV(B2) (Fig. 2A), the three cDNAs encoding a single amino acid substitution at positions 17, 28, or 31 in the protein sequence of CMV(B2) CP, and the three cDNAs encoding double amino acid substitutions at positions 17 and 28, 28 and 31, or 17 and 31 in the protein sequence of the CMV(B2) CP (Fig. 3A) were all chemically synthesized using nucleoside phosphoramidites according to the nucleotide sequences of CMV(Y) RNA3 (GenBank/EMBL/DDBJ accession number M57602) and CMV(B2) CP (AB069971). Each synthesized cDNA was cloned between the CaMV 35S promoter and HSP terminator of binary vector pRI201-AN. The resultant vector constructs were named YCP, B2CP, CP#1, CP#2, CP#3, CP#4, CP#5, CP#6, CP#7, CP#8, CP#9, and CP#10 (Fig. 2A); B2CP.L17P, B2CP.S28A, B2CP.N31T, B2CP.L17P/S28A, B2CP. S28A/N31T, and B2CP.L17P /N31T (Fig. 3A).
Agrobacterium tumefaciens LBA4404 (TAKARA-Bio, Shiga, Japan) was transformed with each of the binary vectors by electroporation according to a standard protocol [6] and was then infiltrated into the leaves of wild-type or P/P/C-HA-transformed N. benthamiana according to a procedure described previously [13]. At 48 h after infiltration of Agrobacterium into leaves, the transient expression of each cDNA (i.e., agroinfiltration), indicated by the accumulation of P/P/C-HA protein and HA-tagged 2b protein was detected by immunoblotting with anti-HA monoclonal antibody (clone 3F10, dilution 1:10,000; Roche, IN, USA) according to a previously described method [8]. The transcripts of transiently expressed cDNAs introduced into leaves of wild-type N. benthamiana by infiltration of Agrobacterium were detected by RT-PCR using sets of primers as shown in Table S2. Total RNA for RT-PCR was isolated from the agroinfiltrated sites according to a standard protocol [6]. The HCD on CMV(Y)-inoculated leaves was visualized at 2 days after agroinfiltration by staining with Trypan blue according to a standard protocol [16]. The CMV CP was immunologically detected using immunoblotting [6]. The expression of pathogenesis-related protein 1 (PR-1) gene was detected by quantitative PCR (qPCR) using the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA). The procedure for qPCR was conducted following a previously described method [12, 13].
The constructs 35S::CY1, 35S::CY2, 35S::CY3, 35S::YCP, 35S::Y2b-HA, or 35S::GFP as a control (Figs. 1A and S1A) were each transiently expressed in wild-type N. benthamiana. Transcripts (35S::CY1, 35S::CY2, 35S::CY3, or 35S::YCP) in the agroinfiltrated leaves were then detected by amplification of the cDNA fragment corresponding to each construct by RT-PCR (Fig. S1B). The transient expression of 35S::Y2b-HA in the agroinfiltrated leaves of wild-type N. benthamiana was immunologically detected by the accumulation of 2b-HA protein (Fig. S1C).
After transient expression of each vector in wild-type N. benthamiana was confirmed, the constructs 35S::CY1, 35S::CY2, 35S::CY3, 35S::YCP, 35S::Y2b-HA, or 35S::GFP as a control (Fig. 2A), were then each transiently expressed in P/P/C-HA-transformed N. benthamiana #4-3 (Fig. 2B). HCD developed only at the site transiently expressing 35S::YCP (Fig. 2B), whereas HCD did not develop at the other sites transiently expressing the cDNA constructs encoding the other CMV proteins (Fig. 2B). These results suggested that the CMV(Y) CP functions as the avirulence molecule independent of the other CMV(Y) RNA-coding proteins (the 1a, 2a, 2b, and 3a proteins). Moreover, at the 35S::YCP-agroinfiltrated site, the CP was detected but P/P/C-HA protein was not, while the accumulation of P/P/C-HA protein was confirmed at each agroinfiltrated site of other vectors (Fig. 1C and D). Thus, the development of HCD during the accumulation of the CMV(Y) CP might be related to the degradation of RCY1.This correlation of HCD and the degradation of the RCY1 is completely consistent with our previous report [13].
To further characterize the critical region in the CMV(Y) CP that could be responsible for the avirulence function against RCY1, Agrobacterium carrying each of a series of chimeric cDNA constructs: CP#1–10, 35S::YCP, or 35S::B2CP (Fig. 2A), were agroinfiltrated into P/P/C-HA-transformed N. benthamiana #4-3. HCD developed at the sites agroinfiltrated with the vectors 35S::YCP, CP#1, CP#2, CP#3, CP#4, or CP#5, which each encode the 5′-terminal region from nucleotide positions 1–95 of the CMV(Y) CP (Fig. 2A and B). However, transient expression of 35S::B2CP, CP#6, CP#7, CP#8, CP#9, or CP#10, which each encode the 5′-terminal region from nucleotide positions 1–95 of CMV(B2) CP, did not induce HCD (Fig. 2A and B). The CP of CMV(B2) and chimeric proteins equally accumulated in similar amounts at all agroinfiltrated sites (Fig. 2C). Moreover, pathogenesis-related gene 1 (PR-1) gene expression increased at the sites agroinfiltrated with 35S::YCPor CP#1–5, but not at the sites agroinfiltrated with 35S::B2CP or CP#6–10 (Fig. S2), which suggests that defense responses might have been induced along with the development of HCD. These results suggest that the 5′-terminal region containing nucleotide positions 1–95 of the CMV CP-coding sequence might determine the avirulence function against RCY1.
We generated three non-synonymous nucleotide substitutions between CMV(Y) and CMV(B2) in their 5′-terminal CP-coding regions from nucleotide positions 1–95 (Fig. 2A). In P/P/C-HA-transformed N. benthamiana #4-3 leaves agroinfiltrated with B2CP.L17P, B2CP.S28A, B2CP.N31T, B2CP.L17P/S28A, B2CP.L17P/N31T, or B2CP.S28A/N31T (Fig. 3A), transient expression of the constructs B2CP.N31T, B2CP.L17P/N31T, or B2CP.S28A/N31T containing 31(N-T) in their N-terminal regions induced HCD (Fig. 3B), although the CMV CP could be immunologically detected in sites agroinfiltrated with any of the constructs (Fig. 3C). These results indicated that a single amino acid at N-terminal 31 position of the CP might be associated with the avirulence function of the CMV CP for the induction of RCY1-conferred HCD.
The results obtained in this study indicate that the CMV(Y) CP functions as an avirulence determinant against RCY1, independent of other CMV proteins (1a, 2a, 2b and 3a proteins), although it is not still clear whether CMV(Y) CP directly or indirectly interacts with RCY1 for resistance to CMV(Y) in Arabidopsis ecotype C24. However, a single amino acid at position 31 of the CP likely determines this avirulence function. According to analysis of the putative conformation of the CMV CP subunit, the amino acid at position 31 of N-terminal region of the CP is located in an α-helical motif that might interact with the CP subunits [9]. Substitution of amino acid N to T at position 31 might affect this proposed α-helical structure and thereby cause loss of the avirulence function of the CP.
The gene HR TO TCV INFECTION (HRT), which is allelic to RCY1 [1, 11] and was identified in A. thaliana ecotype Di-17 [1], confers resistance to turnip crinkle virus (TCV) strain M [TCV(M)]. Interestingly, the CP of TCV(M) also acts as an avirulence determinant against HRT [17], and the N-terminal 52-amino acid R domain of the TCV CP is critical for inducing HRT-dependent HCD in N. benthamiana [3]. Because two amino acid substitutions at amino acid positions 4 and 5 (D4N and P5S), or a single amino acid substitution R6A in the R domain can change the degree of the induction of HRT-mediated HCD [3. 17], such conformational changes in the R domain seem to be associated with HRT-conferred resistance to TCV(M). However, direct interaction of the HRT and TCV(M) CP [18] as well as the relationship between RCY1 and CMV(Y) CP have not yet been confirmed. Analysis of the avirulence function of the CMV(Y) CP revealed that although CMV and TCV are classified into different viral taxonomic families, their avirulence functions might be governed by the conformation of the N-terminal regions of the CPs of CMV(Y) and TCV(M) that might interact with RCY1 and HRT, respectively. The similarities and differences among RCY1-conferred resistance to CMV(Y) and HRT-conferred resistance to TCV(M) should provide new insights that will allow us to elucidate the mechanisms of NB-LRR-class R-protein-mediated immune system responses against virus infection in plants.