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Isolation and validation of a candidate Rsv3 gene from a soybean genotype that confers strain-specific resistance to soybean mosaic virus
Virology 513 (2018) 153–159
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Isolation and validation of a candidate Rsv3 gene from a soybean genotype that confers strain-specific resistance to soybean mosaic virus Phu-Tri Trana,b, Kristin Widyasaria, Jang-Kyun Seoc, Kook-Hyung Kima,b,d,
MARK
⁎
a
Department of Agricultural Biotechnology and College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea c Department of International Agricultural Technology and Institutes of Green Bio Science and Technology, Seoul National University, Pyeongchang 25354, Republic of Korea d Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Keywords: Glycine max (L.) Merr. Soybean mosaic virus Resistance gene Rsv3
Soybean mosaic virus (SMV), a member of the genus Potyvirus, significantly reduces soybean production worldwide. Rsv3, which confers strain-specific resistance to SMV, was previously mapped between the markers A519F/R and M3Satt in chromosome 14 of the soybean [Glycine max (L.) Merr.] genotype L29. Analysis of the soybean genome database revealed that five different NBS-LRR sequences exist between the flanking markers. Among these candidate Rsv3 genes, the full-length cDNA of the Glyma.14g204700 was successfully cloned from L29. Over-expression of Glyma.14g204700 in leaves inoculated with SMV inhibited viral infection in a soybean genotype lacking Rsv3. In addition, the transient silencing of the candidate gene caused a high accumulation of an avirulent strain in L29 carrying Rsv3. Our results therefore provide additional line of evidence to support that Glyma.14g204700 is likely Rsv3 gene that confers strain-specific resistance to SMV.
1. Introduction Soybean [Glycine max (L.) Merr.] is the world's largest source of animal protein feed and the second largest source of vegetable oil for human consumption. According to The Food and Agriculture Organization Corporate Statistical Database, the global production of soybean during the period 2005–2007 was about 217.6 million metric tons (Masuda and Goldsmith, 2009). Soybean production, however, is greatly reduced by plant pathogens; the production loss in 2006 was estimated to be about 59.9 million metric tons in the top eight soybeanproducing countries (Wrather et al., 2010). Soybean viruses are emerging threats to soybean production (Hill and Whitham, 2014), and soybean mosaic virus (SMV) is the most damaging (Ramteke et al., 2015). SMV is a member of the genus Potyvirus in the family Potyviridae and consists of an approximately 9.6kilobase (kb) single-stranded positive-sense RNA. It was long believed that the viral genome encodes a long polyprotein that is processed into 10 mature proteins; however, Chung et al. (2008) discovered another short PIPO fusion protein as a P3-PIPO resulting from ribosomal frameshifting or transcriptional slippage at a highly conserved G1–2A6–7 sequence within the coding region of P3 protein. This seed-born and aphid-transmitted virus can induce diverse symptoms, including mosaic, mottling, chlorosis, and wrinkling (Babu et al., 2008). ⁎
To protect against virus infection, plants have evolved various types of resistance genes. Plant disease resistance genes can be classified into two groups: recessive and dominant genes. Recessive resistance is passive and results from a lack of a specific host component (or mutations in that component) required for virus replication (Diaz‐Pendon et al., 2004). Dominant resistance involves an active mechanism in which R proteins recognize specific avirulent (Avr) factors from viruses and trigger resistance responses (Chisholm et al., 2006). According to the gene-for-gene concept, a plant that produces an R gene product is specifically resistant to a pathogen that produces a corresponding avirulent (Avr) gene product (Flor, 1971). Most R genes encode proteins containing a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) region (Ellis et al., 2000). The NBS domain includes a functional nucleotide-binding site that binds to and hydrolyzes ATP (Tameling et al., 2002) and is involved in signaling of resistant responses (Rairdan and Moffett, 2006). The LRR domain consists of conserved repeats of an 11 or 12 residue stretch, LxxLxLxxN/LxL or LxxLxLxxN/LxxL, in which “L” is Leu/Ile/Val/or Phe, “N” is Asn/Thr/Ser/or Cys, and “x” denotes any amino acid; the LRR domain is believed to be responsible for Avr recognition (Ellis et al., 2007; McHale et al., 2006). Based on the structure of the N-terminal domain, these NBS-LRR proteins are classified into two groups: the TIR-NBS-LRR proteins that contain an Nterminal domain with Toll/Interleukin-1 receptor (TIR) homology,
Corresponding author at: Department of Agricultural Biotechnology and College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea. E-mail address: [email protected] (K.-H. Kim).
http://dx.doi.org/10.1016/j.virol.2017.10.014 Received 4 September 2017; Received in revised form 17 October 2017; Accepted 19 October 2017 0042-6822/ © 2017 Elsevier Inc. All rights reserved.
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Fig. 1. The representative structures of the Glyma.14g204700 coding sequence and putative protein. A, chromosomal position and exon distribution of Glyma.14g204700 on soybean genome; numbers indicate nucleotide position on chromosome 14. B, representative diagram indicated the Glyma.14g204700 putative protein containing a possible coiled-coil domain (CC), a nucleotide-binding domain (NB-ARC), and a leucine-rich repeat domain (LRR); the number indicates the amino acid position from the N terminal.
2009a, 2009b, 2009c). In the present study, we isolated the cDNA sequence of the Rsv3 candidate Glyma.14g204700 from soybean cultivar L29. Co-inoculation of soybean genotype Lee74, which lacks Rsv3, with a gene delivery vector expressing Glyma.14g204700 and an SMV infectious cDNA clone resulted in inhibition of the local infection and a reduction in the systemic accumulation of SMV strain G5H (SMV-G5H, avirulent in Rsv3-mediated resistance) but not of SMV strain G7H (SMV-G7H, virulent in Rsv3-mediated resistance). In addition, loss-offunction effects of Glyma.14g204700 on SMV infection in genotype L29, which has Rsv3, were examined using a bean pod mottle virus (BPMV)based virus-induced gene silencing (VIGS) tool (Zhang et al., 2010). The results showed that transient silencing of this candidate gene caused an increase in the accumulation of SMV-G5H in L29; the viral accumulation then decreased during the recovery of the Glyma.14g204700 in the transiently silenced plant. Our results therefore suggest that the cloned candidate gene Glyma.14g204700 is likely the resistance gene Rsv3, which confers strain-specific resistance to SMV.
while the CC-NBS-LRR proteins that are characterized by an N-terminal coiled-coil (CC) motif (Dangl and Jones, 2001). SMV was classified into several distinct strains based on disease reactions on different soybean genotypes (Cho and Goodman, 1979). Four dominant loci containing Rsv1, Rsv3, Rsv4, or Rsv5 have been genetically mapped in soybean. The Rsv1 locus, which confers extreme resistance (ER) to SMV strain G1 through G6 but not to strain G7, is in chromosome 13 of soybean genotype PI 96983 (Chen et al., 1991; Hajimorad and Hill, 2001; Yu et al., 1994); Rsv1 resistance was predicted to result from at least one of three NBS-LRR sequences (3gG2, 5gG3, and 6gG9) (Zhang et al., 2012). The Rsv4 locus, which mediates resistance to all seven SMV strains (Chen et al., 1993; Ma et al., 1995), was mapped between the markers Rat2 and S6a in chromosome 2 of soybean genotype VP-5152; no NBS-LRR type R sequence was found in this region, suggesting that the Rsv4 gene belongs to a new class of resistance genes (Ilut et al., 2016). Recently, the Rsv5 was reassigned from the Rsv1-y that is from soybean cultivar York and highly linked to the Rsv1 locus on chromosome 13 with a genetic distance 2.2 cM (Klepadlo et al., 2017). The Rsv3 gene that confers ER against G5 to G7 but not G1 to G4 was previously identified between the markers A519F/R and M3Satt in chromosome 14 of soybean genotype L29 (Jeong et al., 2002). Sequence analysis of the 154-kbp region between these two markers revealed five candidate Rsv3 genes that contain NBSLRR domains (Suh et al., 2011). According to the recent annotation of the soybean genome database derived from the soybean cultivar William 82 (Glyma. Wm82.a2.v1, phytozome.net), the five candidate Rsv3 genes were named Glyma.14g204500, Glyma.14g204600, Glyma.14g204700, Glyma.14g205000, and Glyma.14g205300. A comparative sequence analysis of various susceptible and resistant soybean cultivars showed that Glyma.14g204700 (Glyma.14g38533 in previous annotation) has highest transcript abundance and highest number of genotype-phenotype correlations; most of the polymorphisms of this gene were identified in LRR domain (Redekar et al., 2016). This suggested that the Glyma.14g204700 is most likely responsible for the Rsv3 resistance. In Korea, the repeated cultivation of soybean cultivars with resistance against the most prevalent strain, SMV-G5H, caused the emergence of a new SMV strain, G7H. In the late 1990s, G7H caused systemic mosaic symptoms or a lethal systemic hypersensitive response (HR) in certain soybean cultivars that are resistant to G5H (Kim, 2000; Kim et al., 2003). We previously constructed infectious cDNA clones of SMV strains G5H (SMV-G5H) and G7H (SMV-G7H) under the control of the 35 S promoter of cauliflower mosaic virus (CaMV), and demonstrated that the cylindrical inclusion (CI) gene is the elicitor of Rsv3mediated ER and is also a pathogenic determinant that caused the emergence of the resistance-breaking SMV-G7H strain (Seo et al.,
2. Results 2.1. Sequence analysis of Glyma.14g204700, an Rsv3 candidate gene from soybean genotype L29 Because L29 carrying Rsv3 is resistant to SMV-G5H, we sought to isolate the cDNA sequences of Rsv3 candidates from the genotype's total RNA. We obtained a full-length, double-stranded cDNA clone of the candidate Glyma.14g204700. Based upon the gene annotation from soybean genome database Glyma. W82.a2.v1, a 15593 bp genomic sequence of Glyma.14g204700 was found which includes 8 exons and 7 introns (Fig. 1A). The cloned coding gene, which contains 3888 base pairs (bp), encodes a putative NBS-LRR protein that contains 1295 amino acid (aa) residues (Fig. 1A and S1). As shown in Fig. 1B and S1, the positions of domains CC, NB-ARC, and LRR were predicted by bioinformatics tools. Firstly, a coiled-coil prediction method (Combet et al., 2000) indicated that the region ranging from the 39th to the 66th aa was likely to have (with a probability of P > 0.99) a coiled-coil domain containing heptad repeats, hxxhcxc, of hydrophobic (h) and charged (c) amino-acid residues. Secondly, an NCBI-based domain and motif search (Marchler-Bauer et al., 2016) revealed an NB-ARC domain in the region ranging from the 171st to the 424th aa, which includes the conserved motifs of P-loop (with motif GxxxxGKS/T, in which x indicates any residue), kinase 2 (with motif hhhhDD/E, in which h is mostly a hydrophobic residue), and kinase 3a (motif hhhhToR, in which o is an alcoholic residue). Finally, twenty-three LRR motifs (named LRR1 to LRR23) were detected from the 590th to the 1157th aa by using an LRR searching tool (Bej et al., 2014); four LRRs (number 3, 6, 154
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had been inoculated with either the empty vector BPMV or with BPMV::Gm14g204700 (Fig. 4A). The expression level of Glyma.14g204700 in the upper non-inoculated leaves of the plants inoculated with BPMV::Gm14g204700 remained about 20% of that in the plants inoculated with the empty vector, and BPMV infection itself had very little effect on the expression of Glyma.14g204700 (Fig. 4B). Three weeks after inoculation, the expression level of Glyma.14g204700 had almost completely recovered, indicating that the BPMV-based silencing of Glyma.14g204700 was only temporary (Fig. S4A). When the target gene was down-regulated to the lowest level (2 weeks after VIGS application based on the above findings), we used sap containing SMV-G5H::GFP to inoculate the leaves previously infected by BPMV::Gm14g204700; we then monitored virus accumulation for 1 week. The similar sap inoculation was also carried out in the soybean genotype lacking Rsv3 (Lee74). One day after the SMV challenging, the virus level in inoculated leaves of the L29 plants infected with BPMV::Gm14g204700 was not significantly different from the one in Lee74. From 1–2 dpi, the local accumulations of SMV-G5H::GFP were significantly higher in the silenced L29 plants than in the vector-treated plants. However, in contrast to the logarithmic replication of the virus in the inoculated leaves of Lee74, the local accumulation of SMVG5H::GFP in the silenced L29 plants gradually declined until 7 days post inoculation (Fig. 4C). During this period, the expression level of Glyma.14g204700 increased, i.e., the silencing of Glyma.14g204700 was only temporary (Fig. 4D). In the L29 carrying Rsv3, a sap inoculation with the virulent SMVG7H::GFP into the leaves of the L29 previously infected with BPMV::Gm14g204700 resulted in systemic GFP expression which indicated that the BPMV alone did not negatively interfere to the SMV infection (Fig. S4B). In the soybean genotype lacking Rsv3 (Lee74), the sap inoculation resulted in a high accumulation of SMV-G5H::GFP, indicating that the sap of SMV-G5H::GFP was fully infectious (Fig. S4, C and D, left panels). However, neither local nor systemic expression of the GFP delivered by SMV-G5H::GFP was detected, although Glyma.14g204700 was transiently silenced, in the L29 (Fig. S4, panel C and D). This indicated that the transient silencing of Glyma.14g204700 using the BPMV-based VIGS system does not completely suppress Rsv3mediated resistance.
16, and 18) have one aa mismatch with the consensus motif. These analyses suggested that Glyma.14g204700 belongs to the CC-NBS-LRR family. To compare the amino acid sequences of Glyma.14g204700 in a susceptible soybean genotype (William 82) vs. a resistant soybean genotype (L29), we aligned the two aa sequences using Mega6 software. The two putative proteins share 90% sequence identity. Compared to the corresponding sequence from the genotype William 82, the candidate gene from L29 has 3 aa deletions, 2 aa insertions, and 100 aa substitutions (Fig. S1). Only 7 of the 100 substitutions are outside the LRR domain; the others are inside this region, with 54 substitutions located in 22 LRR motifs. These comparisons suggest that the putative proteins Glyma.14g204700 from resistant and susceptible genotypes significantly differ in the LRR domain and that such differences might explain differences in Avr recognition. 2.2. Glyma.14g204700 mediates strain-specific resistance against SMV To evaluate the effect of the candidate Rsv3 on infectivity of SMV, we first cloned Glyma.14g204700 in a pPZP-based binary vector. DNA plasmids of the expression clone and the SMV infectious cDNA clones (Seo et al., 2009a) expressing green fluorescent protein (GFP) were then used to co-inoculate the primary leaves of the susceptible soybean cultivar. Two weeks after inoculation, the viral infection was assessed based on expression of GFP. The results showed that the expression of GFP and replication of SMV-G5H were inhibited in the inoculated leaves which were confirmed by microscopic observation and qRT-PCR, respectively (Fig. 2, panels A and C). A similar result was obtained when Lee74 trifoliate leaves were inoculated with sap containing SMVG5H 2 days after they had been inoculated with the candidate gene (Fig. S3). An attenuation of GFP expression and virus replication in the upper leaves reflected the lower amount of virus in the inoculated leaves (Fig. 2, panels B and C). When the virulent strain G7H was used instead of G5H in the co-inoculation experiment, the expression of GFP was not inhibited or decreased in either the inoculated or upper noninoculated leaves (Fig. 3). This indicated that the transient expression of Glyma.14g204700 did not inhibit infection of the SMV virulent strain G7H.
3. Discussion 2.3. Transient silencing of Glyma.14g204700 temporarily decreases Rsv3-mediated resistance against SMV
The comparative analyses of the polymorphisms between the sequences of the Rsv3 candidate from resistant and susceptible soybean genotypes suggested that Glyma.14g204700 is the most likely candidate (Redekar et al., 2016). However, no full-length cDNA sequence of any of the five candidates had been cloned and validated before the current study. In the current research, we isolated a cDNA sequence of the Glyma.14g204700 gene from the soybean genotype L29. We could not clone cDNAs for the other four NBS-LRR candidate genes perhaps because the expression levels of these four genes are low in response to SMV-G5H infection (Fig. S6). Our findings are consistent with the high abundance of Glyma.14g204700 in the transcriptome of L29, as reported by Redekar et al. (2016). In China, researchers have mapped another locus system (named Rsc) of SMV resistance genes from soybean cultivars Kefeng No. 1 (Rsc7 and Rsc8), Qihuang No. 1 (Rsc3 and Rsc14) and Dabaima (Rsc4) on chromosome 2, 13 and 14, respectively (Ma et al., 2011; Wang et al., 2011a, b; Yan et al., 2015; Zheng et al., 2014). The relationships between these resistance genes and the Rsv loci system is not yet understood. However, a recent analysis of locus Rsc4 in cultivar Dabaima revealed three resistance candidates whose sequences are identical to Glyma.14g204500, Glyma.14g204600, and Glyma.14g204700 (Li et al., 2016). The variations in expression profiles and amino acids of the three candidates from resistant and susceptible cultivars were also consistent with the results in this study. It is still unclear whether the same genes are involved in the Rsc4- and Rsv3mediated resistances; this can be determined when the corresponding
Virus-induced gene silencing (VIGS) is widely used to study the functions of genes in legumes (Pflieger et al., 2013). To investigate the genes responsible for Rsv1 resistance in soybeans, Zhang and colleagues used a bean pod mottle virus (BPMV)-based VIGS system (Zhang et al., 2012). Following a similar strategy, we used the BPMV vector to silence the expression of Glyma.14g204700 in the soybean cultivar L29, which carries Rsv3. To check the applicability of this system under our experimental conditions, we cloned a short fragment of the gene encoding the soybean enzyme phytoene desaturase (GmPDS) into the BPMV vector; the BPMV::GmPDS clone was then used to inoculate the primary leaves of L29. Two weeks after inoculation, a photo-bleaching phenotype was evident on the upper non-inoculated leaves of the inoculated plant (Fig. 4A); the BPMV vector alone also induced yellow mosaic symptoms and reduced the GmPDS expression level by 55% compared to that in the healthy plants. The silencing efficiency of GmPDS was more than 99.9% in the plants infected by the BPMV::GmPDS clone (Fig. 4B). This result indicated that the BPMV-based VIGS vector system works efficiently under our experimental conditions. To down-regulate the expression of Glyma.14g204700 in the resistant genotype, we inoculated L29 plants with the BPMV VIGS vector expressing a short, antisense RNA fragment of the target gene (BPMV::Gm14g204700). Two weeks later, mottle and mosaic symptoms were evident on the upper non-inoculated leaves of the L29 plants that 155
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Fig. 2. Inhibition of SMV-G5H::GFP accumulation by Glyma.14g204700 in soybean cultivar Lee74 was observed 2 weeks after the primary leaves were inoculated with SMV-G5H::GFP. A, the images captured under UV-light (scale bar = 1 cm) and fluorescent microscopy (scale bar = 200 µm). B, the systemic expression of GFP (delivered by SMV-G5H::GFP) in the upper leaves. C, Quantitation of SMV-G5H::GFP in the soybean leaves by qRT-PCR; error bars are standard deviations of three biological replications; asterisks indicate significant t-test results, * P < 0.05. D. Confirmation of GFP expression on soybean leaves using immunodetection by anti-GFP antibody; the controls for protein loading and efficient blotting are indicated as the Rubisco band (about 55 kDa) in the SDS-PGE gel and as the protein marker in the blotted membrane, respectively; bands of GFP with corrected size are indicated. The positions of the leaves are indicated as primary and trifoliate (from 1st to 4th). The soybean primary leaves were co-inoculated with one of the following DNA plasmids: (1) the empty vector pPZP and the Glyma.14g204700 expression clone; (2) the empty vector and SMV-G5H::GFP; and (3) the Glyma.14g204700 expression clone and SMV-G5H::GFP.
effect was only temporary. G5H failed to systemically infect the L29 plants inoculated with BPMV::Gm14g204700 perhaps because the temporary silencing occurred only in the inoculated leaves. Therefore, a permanent deletion of the candidate gene from resistant plants by further mutagenesis will be necessary to confirm the systemic breaking of Rsv3-mediated resistance. As noted, the SMV CI protein was previously identified as an avirulence determinant of Rsv3 resistance (Seo et al., 2009c). Recently, a transcriptome analysis based on resistance and susceptible responses of the genotype L29 against SMV suggested that the abscisic acid pathway mediates the signaling of Rsv3 resistance (Seo et al., 2014). However, the interactions between the R-Avr proteins and the regulatory components of Rsv3 resistance are not yet understood. The molecular characterization an Rsv3 resistance gene, Glyma.14g204700, and the corresponding SMV avirulence factor will enable us to further characterize the other partners in the interaction between the Rsv3 resistance gene and the SMV CI in planta and to rapidly evaluate the possible function(s) of the gene.
genes are isolated and characterized. Functional validations of R genes or resistance-related genes using transient expression systems have been previously reported (Cui et al., 2017; Schweizer et al., 1999). In our study, the function of Glyma.14g204700 in soybean resistance against SMV was evaluated in vivo through the transient co-expression of the candidate gene and viral infectious clones. The strain-specific inhibition of SMV accumulation by Glyma.14g204700 was consistent with the natural resistance mediated by Rsv3. However, SMV infection was not completely inhibited probably because the transient expression was only local and temporary. The response against SMV-G5H::GFP in this study was not as strong as the ER of the Rsv3-carrying soybeans which completely prevents replication of the SMV-G5H (Seo et al., 2014). Thus, stable transgenic plants in which the candidate gene is systemically over-expressed should be developed. In a BPMV-based VIGS assay to verify the Rsv1 candidates, the replication of an SMV avirulent strain in inoculated leaves was used as an indicator of Rsv1 resistance breaking (Zhang et al., 2012). In our study, the role of Glyma.14g204700 in resistance response against SMV-G5H was also evaluated with a BPMV-based VIGS assay. The resulting silencing of Glyma.14g204700 increased SMV-G5H accumulation, but the 156
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Fig. 3. Effect of Glyma.14g204700 on SMV-G7H::GFP accumulation in soybean cultivar Lee74 2 weeks after the primary leaves were inoculated with SMV-G7H::GFP. A, Images of the soybean primary leaves under UV light (scale bars = 1 cm) and fluorescent microscopy (scale bars = 200 µm). B, Systemic expression of GFP (delivered by SMV-G7H::GFP) in the upper leaves. C, Quantitation of SMV-G7H::GFP in the soybean leaves by qRT-PCR; error bars are standard deviations of three biological replications. D, Confirmation of GFP expression in the third trifoliate leaves based on immunodetection with anti-GFP antibody; the Rubisco band (about 55 kDa) in SDS-PAGE gel and the protein marker in the blotted membrane was used as the loading and blotting control, respectively; bands of GFP with corrected size are indicated. The positions of the leaves are indicated as primary and trifoliate (from 1st to 4th). The soybean primary leaves were co-inoculated with one of the following plasmids: (1) the empty vector pPZP and the Glyma.14g204700 expression clone; (2) the empty vector and SMVG7H::GFP; or (3) the Glyma.14g204700 expression clone and SMV-G7H::GFP.
signal was found (data not shown). With the use of flanking primers, the full-length cDNA was obtained by overlapping PCR and was then sub-cloned into a TA vector (pGEM-T Easy, Promega) (Fig. S7, panels A and B). The cloned sequence was confirmed by sequencing with genespecific primers. The sequence alignment with the sequences of five Rsv3 candidates from the Phytozome database indicated that the cloned sequence shared highest sequence similarity with Glyma.14g204700 (Fig. S7C). The clone with consensus sequence and without any mutation was then cloned into the pPZP-based expression vector in which the transient expression is controlled by the 35 S promoter and Tnos terminator (Fig. S7D). Primers used for detection, amplification, and sequencing of the candidates are listed in Table S1. The nucleotide sequence of the Glyma.14g204700 was deposited in GenBank, with accession number MF687340.
4. Materials and methods 4.1. Isolation of the candidate Rsv3 from soybean L29 To isolate Rsv3 candidates, we inoculated the first trifoliate of soybean L29 with the SMV avirulent strain G5H. Eight hours after inoculation, the total RNAs were extracted from the inoculated leaves using Triazol reagent (RNAiso Plus, Takara). The cDNA was obtained by reverse transcription using the oligodT (20 mer, Bioneer) and a reverse transcriptase (Goscripts, Promega). Because the sequences at the outermost ends of some candidates are identical, we designed specific internal primers to obtain the full-length cDNA clone via overlapping PCR (Fig. S5A). Two halves of the doublestranded cDNA Glyma.14g204700 were amplified with internally specific primers (Fig. S5B) and outermost primers (Fig. S6, panels A and B). Partial regions homologous to Glyma.14g204500 and Glyma.14g204600 were detected and confirmed in the genomic DNA but not in the cDNA of L29 (Fig. S6C). The two candidates Glyma.14g205000 and Glyma.14g205300 were not detected in either the genomic DNA or cDNA by partial sequence amplifications (Fig. S6D); another primer set was designed and used to detect these two candidates, but no positive
4.2. SMV resistance assay To evaluate the effect of the cloned candidate on SMV resistance, the DNA plasmid of the empty vector pPZP, the expression clone of Glyma.14g204700, and infectious clones of SMV::GFP were prepared with a maxi-prep DNA plasmid purification kit (Nucleobond® Xtra 157
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Fig. 4. Effect of transient silencing of Glyma.14g204700 in soybean cultivar L29 on SMVG5H accumulation. A, Symptoms of the third trifoliate leaf of the soybean plants 2 weeks after the plants were inoculated with phosphate buffer, BPMV empty vector, or BPMV clones specific to GmPDS and Glyma.14g204700 (scale bar = 1 cm). B, Relative expression level of GmPDS and Glyma.14g204700 2 weeks after plants were inoculated with phosphate buffer (healthy), BPMV only (vector), and BPMV clones specific to the targets (VIGS). C, Time course accumulation of SMV-G5H in inoculated plants that were silenced (VIGS) or not silenced (vector). D, Relative expression level of Glyma.14g204700 within 1 week after silenced or non-silenced (vector) plants were inoculated with SMV. In panels B, C, and D, error bars are standard deviations of three biological replications. Asterisks indicate significant t-test results, * P < 0.05, ** P < 0.01.
used as a negative control for the non-silenced plants. The plasmid DNAs were prepared with the maxi-prep plasmid DNA purification kit. For delivery of the VIGS clones, the plasmid DNAs (0.5 µg/µl) corresponding to RNA1 and RNA2 of the BPMV were mixed at 1:1 ratios in 50 mM phosphate buffer (pH 7.4). The mixtures then were then used to inoculate the primary leaves of 10-day-old plants of soybean cultivar L29 (with 10 µg of total DNA plasmids per leaf, two leaves per plant, and three plants per experiment). Two weeks after the inoculation, the silencing of the target genes was assessed by quantifying the expression levels of the target gene in plants treated with VIGS clones and with the empty vector.
Maxi, MN). A 1:1 ratio mixture (0.5 µg/µl) of the candidate and infectious clone was prepared in 50 mM phosphate buffer (pH 7.4). The empty vector was used in the control instead of the candidate. These mixtures were then used to inoculate the primary leaves of 2-week-old soybean cultivar Lee74 (with 10 µg of total DNA plasmids per leaf, two leaves per plant, and three plants per experiment); Lee74 is susceptible to SMV G5H. Lee74. Detailed experimental steps were shown in Fig. S2A. After inoculation, the GFP images of the inoculated and upper non-inoculated leaves were captured under UV light with a digital camera (D700, Nikon) with a green filter. The microscopic images of the SMV::GFP in the inoculated leaves were recorded with a fluorescent microscope (Axio Imager A1, Carl Zeiss). To check the effect of the candidate on SMV infectivity, the trifoliate leaves of soybean Lee74 were first mechanically inoculated with the DNA plasmid of expression clones with 60 µg per trifoliate. Two days later, the same leaves were inoculated with “viral sap”, which was prepared by grinding 100 mg of soybean tissue infected by SMVG5H::GFP in 1 ml of 50 mM phosphate buffer (pH 7.4); each leaf was inoculated with 20 µl of the sap. Four days after inoculation with the virus, the expression of GFP was assessed as described above.
4.4. Immuno-blotting To evaluate the expression level of GFP, about 100 mg of tissue was collected and ground in liquid nitrogen. Total protein was extracted with a plant total protein extraction kit (Sigma). Protein concentration was measured by the Bradford assay using Bradford reagent (Bio-Rad). A 40-μg quantity of total protein for each sample was separated by 12% SDS-PAGE and electro-blotted onto PVDF membranes (GE Healthcare). Protein blots were probed with rabbit anti-GFP antibody (Santa Cruz, 1:20,000 dilution) followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad, 1:20,000 dilution). The probed blots were visualized using Clarity western ECL substrate (Bio-Rad) in a Fusion Fx imaging instrument (Vilber Lourmat).
4.3. Virus-induced gene silencing To down-regulate the expression of the candidate in soybean genotype L29, which contains Rsv3 and is resistant to G5H, we used a previously described BPMV-based system (Zhang et al., 2010). The experimental procedure was symbolically represented in the Fig. S2B. A 347-bp DNA fragment amplified from Glyma.14g204700 was inserted in reverse orientation into the BPMV RNA2 vector via BamHI-XhoI restriction enzyme sites. To test the applicability of the BPMV vector in this study, we used a similar insertion of a 327-bp DNA fragment amplified from the GmPDS gene (NM_001249840). Primers for amplification of the target fragments are listed in Table S1. The empty vector was
4.5. Real-time RT-PCR For quantification of target gene expression and SMV accumulation, total RNAs were extracted from leaves with Triazol reagent and were treated with DNase I (RQ1, Promega), and cDNA was synthesized using oligodT and reverse transcriptase. Real-time PCR was then carried out with biological triplicates and technically replicates in a real-time PCR 158
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Effect of diseases on soybean yield in the top eight producing countries in 2006. Plant Health Prog. 10, 1094. Yan, H., Wang, H., Cheng, H., Hu, Z., Chu, S., Zhang, G., Yu, D., 2015. Detection and fine‐mapping of SC7 resistance genes via linkage and association analysis in soybean. J. Integr. Plant Biol. 57, 722–729. Yu, Y., Saghai Maroof, M., Buss, G., Maughan, P., Tolin, S., 1994. RFLP and microsatellite mapping of a gene for soybean mosaic virus resistance. Phytopathology 84, 60–64. Zhang, C., Bradshaw, J.D., Whitham, S.A., Hill, J.H., 2010. The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing. Plant Physiol. 153, 52–65. Zhang, C., Grosic, S., Whitham, S.A., Hill, J.H., 2012. The requirement of multiple defense genes in soybean Rsv1–mediated extreme resistance to soybean mosaic virus. Mol. Plant-Microbe Interact. 25, 1307–1313. 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system (CFX96, Bio-rad) using Sybr green master mix (IQ SYBR green supermix, Bio-rad) as described by the manufacturer. The soybean gene encoding CDPK-related protein kinase (AW396185) was used as reference for data normalization as described previously (Libault et al., 2008). Primers used for the qRT-PCRs are listed in Table S1. The expression levels of the target sequence were normalized based on Ct values of the target or the reference and were statistically calculated with Microsoft Excel 2010 software (Schmittgen, 2006). Differences between the tested group and the control group were accessed by an unpaired two-tailed t-test. Acknowledgements This research was supported in part by grants from the NextGeneration BioGreen 21 Program (no. PJ01101401) and the Rural Development Administration and the Vegetable Breeding Research Center (no. 710001-3) through the Agriculture, the Food and Rural Affairs Research Center Support Program from the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. PT and KW were supported by research fellowships from the Brain Korea 21 Plus Project. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2017.10.014. References Babu, M., Gagarinova, A.G., Brandle, J.E., Wang, A., 2008. Association of the transcriptional response of soybean plants with soybean mosaic virus systemic infection. J. Gen. Virol. 89, 1069–1080. Bej, A., Sahoo, B.R., Swain, B., Basu, M., Jayasankar, P., Samanta, M., 2014. LRRsearch: an asynchronous server-based application for the prediction of leucine-rich repeat motifs and an integrative database of NOD-like receptors. Comput. Biol. Med. 53, 164–170. Chen, P., Buss, G., Roane, C., Tolin, S., 1991. Allelism among genes for resistance to soybean mosaic virus in strain-differential soybean cultivars. Crop Sci. 31, 305–309. Chen, P., Buss, G., Tolin, S., 1993. Resistance to soybean mosaic virus conferred by two independent dominant genes in PI 486355. J. Hered. 84, 25–28. Chisholm, S.T., Coaker, G., Day, B., Staskawicz, B.J., 2006. Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124, 803–814. Cho, E.-K., Goodman, R.M., 1979. Strains of soybean mosaic virus: classification based on virulence in resistant soybean cultivars. Phytopathology 69, 467–470. Chung, B.Y.-W., Miller, W.A., Atkins, J.F., Firth, A.E., 2008. An overlapping essential gene in the Potyviridae. Proc. Natl. Acad. Sci. USA 105, 5897–5902. Combet, C., Blanchet, C., Geourjon, C., Deleage, G., 2000. NPS@: network protein sequence analysis. Trends Biochem. Sci. 25, 147–150. Cui, M.-Y., Wei, W., Gao, K., Xie, Y.-G., Guo, Y., Feng, J.-Y., 2017. A rapid and efficient Agrobacterium-mediated transient gene expression system for strawberry leaves and the study of disease resistance proteins. Plant Cell Tiss. Org. 130, 1–14. Dangl, J.L., Jones, J.D., 2001. Plant pathogens and integrated defence responses to infection. Nature 411, 826–833. Diaz‐Pendon, J.A., Truniger, V., Nieto, C., Garcia‐Mas, J., Bendahmane, A., Aranda, M.A., 2004. Advances in understanding recessive resistance to plant viruses. Mol. Plant Pathol. 5, 223–233. Ellis, J., Dodds, P., Pryor, T., 2000. Structure, function and evolution of plant disease resistance genes. Curr. Opin. Plant Biol. 3, 278–284. Ellis, J.G., Dodds, P.N., Lawrence, G.J., 2007. Flax rust resistance gene specificity is based on direct resistance-avirulence protein interactions. Annu. Rev. Phytopathol. 45, 289–306. Flor, H.H., 1971. Current status of the gene-for-gene concept. Annu. Rev. Phytopathol. 9, 275–296. Hajimorad, M., Hill, J., 2001. Rsv1-mediated resistance against soybean mosaic virus-N is hypersensitive response-independent at inoculation site, but has the potential to initiate a hypersensitive response-like mechanism. Mol. Plant-Microbe Interact. 14, 587–598. Hill, J.H., Whitham, S.A., 2014. Control of virus diseases in soybeans. Adv. Virus Res. 90, 355–390. Ilut, D.C., Lipka, A.E., Jeong, N., Bae, D.N., Kim, D.H., Kim, J.H., Redekar, N., Yang, K., Park, W., Kang, S.-T., 2016. Identification of haplotypes at the Rsv4 genomic region in soybean associated with durable resistance to soybean mosaic virus. Theor. Appl. Genet. 129, 453–468. Jeong, S., Kristipati, S., Hayes, A., Maughan, P., Noffsinger, S., Gunduz, I., Buss, G., Maroof, M., 2002. Genetic and sequence analysis of markers tightly linked to the resistance gene, Rsv3. Crop Sci. 42, 265–270.
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Isolation and validation of a candidate Rsv3 gene from a soybean genotype that confers strain-specific resistance to soybean mosaic virus Phu-Tri Trana,b, Kristin Widyasaria, Jang-Kyun Seoc, Kook-Hyung Kima,b,d,
MARK
⁎
a
Department of Agricultural Biotechnology and College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea c Department of International Agricultural Technology and Institutes of Green Bio Science and Technology, Seoul National University, Pyeongchang 25354, Republic of Korea d Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Keywords: Glycine max (L.) Merr. Soybean mosaic virus Resistance gene Rsv3
Soybean mosaic virus (SMV), a member of the genus Potyvirus, significantly reduces soybean production worldwide. Rsv3, which confers strain-specific resistance to SMV, was previously mapped between the markers A519F/R and M3Satt in chromosome 14 of the soybean [Glycine max (L.) Merr.] genotype L29. Analysis of the soybean genome database revealed that five different NBS-LRR sequences exist between the flanking markers. Among these candidate Rsv3 genes, the full-length cDNA of the Glyma.14g204700 was successfully cloned from L29. Over-expression of Glyma.14g204700 in leaves inoculated with SMV inhibited viral infection in a soybean genotype lacking Rsv3. In addition, the transient silencing of the candidate gene caused a high accumulation of an avirulent strain in L29 carrying Rsv3. Our results therefore provide additional line of evidence to support that Glyma.14g204700 is likely Rsv3 gene that confers strain-specific resistance to SMV.
1. Introduction Soybean [Glycine max (L.) Merr.] is the world's largest source of animal protein feed and the second largest source of vegetable oil for human consumption. According to The Food and Agriculture Organization Corporate Statistical Database, the global production of soybean during the period 2005–2007 was about 217.6 million metric tons (Masuda and Goldsmith, 2009). Soybean production, however, is greatly reduced by plant pathogens; the production loss in 2006 was estimated to be about 59.9 million metric tons in the top eight soybeanproducing countries (Wrather et al., 2010). Soybean viruses are emerging threats to soybean production (Hill and Whitham, 2014), and soybean mosaic virus (SMV) is the most damaging (Ramteke et al., 2015). SMV is a member of the genus Potyvirus in the family Potyviridae and consists of an approximately 9.6kilobase (kb) single-stranded positive-sense RNA. It was long believed that the viral genome encodes a long polyprotein that is processed into 10 mature proteins; however, Chung et al. (2008) discovered another short PIPO fusion protein as a P3-PIPO resulting from ribosomal frameshifting or transcriptional slippage at a highly conserved G1–2A6–7 sequence within the coding region of P3 protein. This seed-born and aphid-transmitted virus can induce diverse symptoms, including mosaic, mottling, chlorosis, and wrinkling (Babu et al., 2008). ⁎
To protect against virus infection, plants have evolved various types of resistance genes. Plant disease resistance genes can be classified into two groups: recessive and dominant genes. Recessive resistance is passive and results from a lack of a specific host component (or mutations in that component) required for virus replication (Diaz‐Pendon et al., 2004). Dominant resistance involves an active mechanism in which R proteins recognize specific avirulent (Avr) factors from viruses and trigger resistance responses (Chisholm et al., 2006). According to the gene-for-gene concept, a plant that produces an R gene product is specifically resistant to a pathogen that produces a corresponding avirulent (Avr) gene product (Flor, 1971). Most R genes encode proteins containing a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) region (Ellis et al., 2000). The NBS domain includes a functional nucleotide-binding site that binds to and hydrolyzes ATP (Tameling et al., 2002) and is involved in signaling of resistant responses (Rairdan and Moffett, 2006). The LRR domain consists of conserved repeats of an 11 or 12 residue stretch, LxxLxLxxN/LxL or LxxLxLxxN/LxxL, in which “L” is Leu/Ile/Val/or Phe, “N” is Asn/Thr/Ser/or Cys, and “x” denotes any amino acid; the LRR domain is believed to be responsible for Avr recognition (Ellis et al., 2007; McHale et al., 2006). Based on the structure of the N-terminal domain, these NBS-LRR proteins are classified into two groups: the TIR-NBS-LRR proteins that contain an Nterminal domain with Toll/Interleukin-1 receptor (TIR) homology,
Corresponding author at: Department of Agricultural Biotechnology and College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea. E-mail address: [email protected] (K.-H. Kim).
http://dx.doi.org/10.1016/j.virol.2017.10.014 Received 4 September 2017; Received in revised form 17 October 2017; Accepted 19 October 2017 0042-6822/ © 2017 Elsevier Inc. All rights reserved.
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Fig. 1. The representative structures of the Glyma.14g204700 coding sequence and putative protein. A, chromosomal position and exon distribution of Glyma.14g204700 on soybean genome; numbers indicate nucleotide position on chromosome 14. B, representative diagram indicated the Glyma.14g204700 putative protein containing a possible coiled-coil domain (CC), a nucleotide-binding domain (NB-ARC), and a leucine-rich repeat domain (LRR); the number indicates the amino acid position from the N terminal.
2009a, 2009b, 2009c). In the present study, we isolated the cDNA sequence of the Rsv3 candidate Glyma.14g204700 from soybean cultivar L29. Co-inoculation of soybean genotype Lee74, which lacks Rsv3, with a gene delivery vector expressing Glyma.14g204700 and an SMV infectious cDNA clone resulted in inhibition of the local infection and a reduction in the systemic accumulation of SMV strain G5H (SMV-G5H, avirulent in Rsv3-mediated resistance) but not of SMV strain G7H (SMV-G7H, virulent in Rsv3-mediated resistance). In addition, loss-offunction effects of Glyma.14g204700 on SMV infection in genotype L29, which has Rsv3, were examined using a bean pod mottle virus (BPMV)based virus-induced gene silencing (VIGS) tool (Zhang et al., 2010). The results showed that transient silencing of this candidate gene caused an increase in the accumulation of SMV-G5H in L29; the viral accumulation then decreased during the recovery of the Glyma.14g204700 in the transiently silenced plant. Our results therefore suggest that the cloned candidate gene Glyma.14g204700 is likely the resistance gene Rsv3, which confers strain-specific resistance to SMV.
while the CC-NBS-LRR proteins that are characterized by an N-terminal coiled-coil (CC) motif (Dangl and Jones, 2001). SMV was classified into several distinct strains based on disease reactions on different soybean genotypes (Cho and Goodman, 1979). Four dominant loci containing Rsv1, Rsv3, Rsv4, or Rsv5 have been genetically mapped in soybean. The Rsv1 locus, which confers extreme resistance (ER) to SMV strain G1 through G6 but not to strain G7, is in chromosome 13 of soybean genotype PI 96983 (Chen et al., 1991; Hajimorad and Hill, 2001; Yu et al., 1994); Rsv1 resistance was predicted to result from at least one of three NBS-LRR sequences (3gG2, 5gG3, and 6gG9) (Zhang et al., 2012). The Rsv4 locus, which mediates resistance to all seven SMV strains (Chen et al., 1993; Ma et al., 1995), was mapped between the markers Rat2 and S6a in chromosome 2 of soybean genotype VP-5152; no NBS-LRR type R sequence was found in this region, suggesting that the Rsv4 gene belongs to a new class of resistance genes (Ilut et al., 2016). Recently, the Rsv5 was reassigned from the Rsv1-y that is from soybean cultivar York and highly linked to the Rsv1 locus on chromosome 13 with a genetic distance 2.2 cM (Klepadlo et al., 2017). The Rsv3 gene that confers ER against G5 to G7 but not G1 to G4 was previously identified between the markers A519F/R and M3Satt in chromosome 14 of soybean genotype L29 (Jeong et al., 2002). Sequence analysis of the 154-kbp region between these two markers revealed five candidate Rsv3 genes that contain NBSLRR domains (Suh et al., 2011). According to the recent annotation of the soybean genome database derived from the soybean cultivar William 82 (Glyma. Wm82.a2.v1, phytozome.net), the five candidate Rsv3 genes were named Glyma.14g204500, Glyma.14g204600, Glyma.14g204700, Glyma.14g205000, and Glyma.14g205300. A comparative sequence analysis of various susceptible and resistant soybean cultivars showed that Glyma.14g204700 (Glyma.14g38533 in previous annotation) has highest transcript abundance and highest number of genotype-phenotype correlations; most of the polymorphisms of this gene were identified in LRR domain (Redekar et al., 2016). This suggested that the Glyma.14g204700 is most likely responsible for the Rsv3 resistance. In Korea, the repeated cultivation of soybean cultivars with resistance against the most prevalent strain, SMV-G5H, caused the emergence of a new SMV strain, G7H. In the late 1990s, G7H caused systemic mosaic symptoms or a lethal systemic hypersensitive response (HR) in certain soybean cultivars that are resistant to G5H (Kim, 2000; Kim et al., 2003). We previously constructed infectious cDNA clones of SMV strains G5H (SMV-G5H) and G7H (SMV-G7H) under the control of the 35 S promoter of cauliflower mosaic virus (CaMV), and demonstrated that the cylindrical inclusion (CI) gene is the elicitor of Rsv3mediated ER and is also a pathogenic determinant that caused the emergence of the resistance-breaking SMV-G7H strain (Seo et al.,
2. Results 2.1. Sequence analysis of Glyma.14g204700, an Rsv3 candidate gene from soybean genotype L29 Because L29 carrying Rsv3 is resistant to SMV-G5H, we sought to isolate the cDNA sequences of Rsv3 candidates from the genotype's total RNA. We obtained a full-length, double-stranded cDNA clone of the candidate Glyma.14g204700. Based upon the gene annotation from soybean genome database Glyma. W82.a2.v1, a 15593 bp genomic sequence of Glyma.14g204700 was found which includes 8 exons and 7 introns (Fig. 1A). The cloned coding gene, which contains 3888 base pairs (bp), encodes a putative NBS-LRR protein that contains 1295 amino acid (aa) residues (Fig. 1A and S1). As shown in Fig. 1B and S1, the positions of domains CC, NB-ARC, and LRR were predicted by bioinformatics tools. Firstly, a coiled-coil prediction method (Combet et al., 2000) indicated that the region ranging from the 39th to the 66th aa was likely to have (with a probability of P > 0.99) a coiled-coil domain containing heptad repeats, hxxhcxc, of hydrophobic (h) and charged (c) amino-acid residues. Secondly, an NCBI-based domain and motif search (Marchler-Bauer et al., 2016) revealed an NB-ARC domain in the region ranging from the 171st to the 424th aa, which includes the conserved motifs of P-loop (with motif GxxxxGKS/T, in which x indicates any residue), kinase 2 (with motif hhhhDD/E, in which h is mostly a hydrophobic residue), and kinase 3a (motif hhhhToR, in which o is an alcoholic residue). Finally, twenty-three LRR motifs (named LRR1 to LRR23) were detected from the 590th to the 1157th aa by using an LRR searching tool (Bej et al., 2014); four LRRs (number 3, 6, 154
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had been inoculated with either the empty vector BPMV or with BPMV::Gm14g204700 (Fig. 4A). The expression level of Glyma.14g204700 in the upper non-inoculated leaves of the plants inoculated with BPMV::Gm14g204700 remained about 20% of that in the plants inoculated with the empty vector, and BPMV infection itself had very little effect on the expression of Glyma.14g204700 (Fig. 4B). Three weeks after inoculation, the expression level of Glyma.14g204700 had almost completely recovered, indicating that the BPMV-based silencing of Glyma.14g204700 was only temporary (Fig. S4A). When the target gene was down-regulated to the lowest level (2 weeks after VIGS application based on the above findings), we used sap containing SMV-G5H::GFP to inoculate the leaves previously infected by BPMV::Gm14g204700; we then monitored virus accumulation for 1 week. The similar sap inoculation was also carried out in the soybean genotype lacking Rsv3 (Lee74). One day after the SMV challenging, the virus level in inoculated leaves of the L29 plants infected with BPMV::Gm14g204700 was not significantly different from the one in Lee74. From 1–2 dpi, the local accumulations of SMV-G5H::GFP were significantly higher in the silenced L29 plants than in the vector-treated plants. However, in contrast to the logarithmic replication of the virus in the inoculated leaves of Lee74, the local accumulation of SMVG5H::GFP in the silenced L29 plants gradually declined until 7 days post inoculation (Fig. 4C). During this period, the expression level of Glyma.14g204700 increased, i.e., the silencing of Glyma.14g204700 was only temporary (Fig. 4D). In the L29 carrying Rsv3, a sap inoculation with the virulent SMVG7H::GFP into the leaves of the L29 previously infected with BPMV::Gm14g204700 resulted in systemic GFP expression which indicated that the BPMV alone did not negatively interfere to the SMV infection (Fig. S4B). In the soybean genotype lacking Rsv3 (Lee74), the sap inoculation resulted in a high accumulation of SMV-G5H::GFP, indicating that the sap of SMV-G5H::GFP was fully infectious (Fig. S4, C and D, left panels). However, neither local nor systemic expression of the GFP delivered by SMV-G5H::GFP was detected, although Glyma.14g204700 was transiently silenced, in the L29 (Fig. S4, panel C and D). This indicated that the transient silencing of Glyma.14g204700 using the BPMV-based VIGS system does not completely suppress Rsv3mediated resistance.
16, and 18) have one aa mismatch with the consensus motif. These analyses suggested that Glyma.14g204700 belongs to the CC-NBS-LRR family. To compare the amino acid sequences of Glyma.14g204700 in a susceptible soybean genotype (William 82) vs. a resistant soybean genotype (L29), we aligned the two aa sequences using Mega6 software. The two putative proteins share 90% sequence identity. Compared to the corresponding sequence from the genotype William 82, the candidate gene from L29 has 3 aa deletions, 2 aa insertions, and 100 aa substitutions (Fig. S1). Only 7 of the 100 substitutions are outside the LRR domain; the others are inside this region, with 54 substitutions located in 22 LRR motifs. These comparisons suggest that the putative proteins Glyma.14g204700 from resistant and susceptible genotypes significantly differ in the LRR domain and that such differences might explain differences in Avr recognition. 2.2. Glyma.14g204700 mediates strain-specific resistance against SMV To evaluate the effect of the candidate Rsv3 on infectivity of SMV, we first cloned Glyma.14g204700 in a pPZP-based binary vector. DNA plasmids of the expression clone and the SMV infectious cDNA clones (Seo et al., 2009a) expressing green fluorescent protein (GFP) were then used to co-inoculate the primary leaves of the susceptible soybean cultivar. Two weeks after inoculation, the viral infection was assessed based on expression of GFP. The results showed that the expression of GFP and replication of SMV-G5H were inhibited in the inoculated leaves which were confirmed by microscopic observation and qRT-PCR, respectively (Fig. 2, panels A and C). A similar result was obtained when Lee74 trifoliate leaves were inoculated with sap containing SMVG5H 2 days after they had been inoculated with the candidate gene (Fig. S3). An attenuation of GFP expression and virus replication in the upper leaves reflected the lower amount of virus in the inoculated leaves (Fig. 2, panels B and C). When the virulent strain G7H was used instead of G5H in the co-inoculation experiment, the expression of GFP was not inhibited or decreased in either the inoculated or upper noninoculated leaves (Fig. 3). This indicated that the transient expression of Glyma.14g204700 did not inhibit infection of the SMV virulent strain G7H.
3. Discussion 2.3. Transient silencing of Glyma.14g204700 temporarily decreases Rsv3-mediated resistance against SMV
The comparative analyses of the polymorphisms between the sequences of the Rsv3 candidate from resistant and susceptible soybean genotypes suggested that Glyma.14g204700 is the most likely candidate (Redekar et al., 2016). However, no full-length cDNA sequence of any of the five candidates had been cloned and validated before the current study. In the current research, we isolated a cDNA sequence of the Glyma.14g204700 gene from the soybean genotype L29. We could not clone cDNAs for the other four NBS-LRR candidate genes perhaps because the expression levels of these four genes are low in response to SMV-G5H infection (Fig. S6). Our findings are consistent with the high abundance of Glyma.14g204700 in the transcriptome of L29, as reported by Redekar et al. (2016). In China, researchers have mapped another locus system (named Rsc) of SMV resistance genes from soybean cultivars Kefeng No. 1 (Rsc7 and Rsc8), Qihuang No. 1 (Rsc3 and Rsc14) and Dabaima (Rsc4) on chromosome 2, 13 and 14, respectively (Ma et al., 2011; Wang et al., 2011a, b; Yan et al., 2015; Zheng et al., 2014). The relationships between these resistance genes and the Rsv loci system is not yet understood. However, a recent analysis of locus Rsc4 in cultivar Dabaima revealed three resistance candidates whose sequences are identical to Glyma.14g204500, Glyma.14g204600, and Glyma.14g204700 (Li et al., 2016). The variations in expression profiles and amino acids of the three candidates from resistant and susceptible cultivars were also consistent with the results in this study. It is still unclear whether the same genes are involved in the Rsc4- and Rsv3mediated resistances; this can be determined when the corresponding
Virus-induced gene silencing (VIGS) is widely used to study the functions of genes in legumes (Pflieger et al., 2013). To investigate the genes responsible for Rsv1 resistance in soybeans, Zhang and colleagues used a bean pod mottle virus (BPMV)-based VIGS system (Zhang et al., 2012). Following a similar strategy, we used the BPMV vector to silence the expression of Glyma.14g204700 in the soybean cultivar L29, which carries Rsv3. To check the applicability of this system under our experimental conditions, we cloned a short fragment of the gene encoding the soybean enzyme phytoene desaturase (GmPDS) into the BPMV vector; the BPMV::GmPDS clone was then used to inoculate the primary leaves of L29. Two weeks after inoculation, a photo-bleaching phenotype was evident on the upper non-inoculated leaves of the inoculated plant (Fig. 4A); the BPMV vector alone also induced yellow mosaic symptoms and reduced the GmPDS expression level by 55% compared to that in the healthy plants. The silencing efficiency of GmPDS was more than 99.9% in the plants infected by the BPMV::GmPDS clone (Fig. 4B). This result indicated that the BPMV-based VIGS vector system works efficiently under our experimental conditions. To down-regulate the expression of Glyma.14g204700 in the resistant genotype, we inoculated L29 plants with the BPMV VIGS vector expressing a short, antisense RNA fragment of the target gene (BPMV::Gm14g204700). Two weeks later, mottle and mosaic symptoms were evident on the upper non-inoculated leaves of the L29 plants that 155
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Fig. 2. Inhibition of SMV-G5H::GFP accumulation by Glyma.14g204700 in soybean cultivar Lee74 was observed 2 weeks after the primary leaves were inoculated with SMV-G5H::GFP. A, the images captured under UV-light (scale bar = 1 cm) and fluorescent microscopy (scale bar = 200 µm). B, the systemic expression of GFP (delivered by SMV-G5H::GFP) in the upper leaves. C, Quantitation of SMV-G5H::GFP in the soybean leaves by qRT-PCR; error bars are standard deviations of three biological replications; asterisks indicate significant t-test results, * P < 0.05. D. Confirmation of GFP expression on soybean leaves using immunodetection by anti-GFP antibody; the controls for protein loading and efficient blotting are indicated as the Rubisco band (about 55 kDa) in the SDS-PGE gel and as the protein marker in the blotted membrane, respectively; bands of GFP with corrected size are indicated. The positions of the leaves are indicated as primary and trifoliate (from 1st to 4th). The soybean primary leaves were co-inoculated with one of the following DNA plasmids: (1) the empty vector pPZP and the Glyma.14g204700 expression clone; (2) the empty vector and SMV-G5H::GFP; and (3) the Glyma.14g204700 expression clone and SMV-G5H::GFP.
effect was only temporary. G5H failed to systemically infect the L29 plants inoculated with BPMV::Gm14g204700 perhaps because the temporary silencing occurred only in the inoculated leaves. Therefore, a permanent deletion of the candidate gene from resistant plants by further mutagenesis will be necessary to confirm the systemic breaking of Rsv3-mediated resistance. As noted, the SMV CI protein was previously identified as an avirulence determinant of Rsv3 resistance (Seo et al., 2009c). Recently, a transcriptome analysis based on resistance and susceptible responses of the genotype L29 against SMV suggested that the abscisic acid pathway mediates the signaling of Rsv3 resistance (Seo et al., 2014). However, the interactions between the R-Avr proteins and the regulatory components of Rsv3 resistance are not yet understood. The molecular characterization an Rsv3 resistance gene, Glyma.14g204700, and the corresponding SMV avirulence factor will enable us to further characterize the other partners in the interaction between the Rsv3 resistance gene and the SMV CI in planta and to rapidly evaluate the possible function(s) of the gene.
genes are isolated and characterized. Functional validations of R genes or resistance-related genes using transient expression systems have been previously reported (Cui et al., 2017; Schweizer et al., 1999). In our study, the function of Glyma.14g204700 in soybean resistance against SMV was evaluated in vivo through the transient co-expression of the candidate gene and viral infectious clones. The strain-specific inhibition of SMV accumulation by Glyma.14g204700 was consistent with the natural resistance mediated by Rsv3. However, SMV infection was not completely inhibited probably because the transient expression was only local and temporary. The response against SMV-G5H::GFP in this study was not as strong as the ER of the Rsv3-carrying soybeans which completely prevents replication of the SMV-G5H (Seo et al., 2014). Thus, stable transgenic plants in which the candidate gene is systemically over-expressed should be developed. In a BPMV-based VIGS assay to verify the Rsv1 candidates, the replication of an SMV avirulent strain in inoculated leaves was used as an indicator of Rsv1 resistance breaking (Zhang et al., 2012). In our study, the role of Glyma.14g204700 in resistance response against SMV-G5H was also evaluated with a BPMV-based VIGS assay. The resulting silencing of Glyma.14g204700 increased SMV-G5H accumulation, but the 156
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Fig. 3. Effect of Glyma.14g204700 on SMV-G7H::GFP accumulation in soybean cultivar Lee74 2 weeks after the primary leaves were inoculated with SMV-G7H::GFP. A, Images of the soybean primary leaves under UV light (scale bars = 1 cm) and fluorescent microscopy (scale bars = 200 µm). B, Systemic expression of GFP (delivered by SMV-G7H::GFP) in the upper leaves. C, Quantitation of SMV-G7H::GFP in the soybean leaves by qRT-PCR; error bars are standard deviations of three biological replications. D, Confirmation of GFP expression in the third trifoliate leaves based on immunodetection with anti-GFP antibody; the Rubisco band (about 55 kDa) in SDS-PAGE gel and the protein marker in the blotted membrane was used as the loading and blotting control, respectively; bands of GFP with corrected size are indicated. The positions of the leaves are indicated as primary and trifoliate (from 1st to 4th). The soybean primary leaves were co-inoculated with one of the following plasmids: (1) the empty vector pPZP and the Glyma.14g204700 expression clone; (2) the empty vector and SMVG7H::GFP; or (3) the Glyma.14g204700 expression clone and SMV-G7H::GFP.
signal was found (data not shown). With the use of flanking primers, the full-length cDNA was obtained by overlapping PCR and was then sub-cloned into a TA vector (pGEM-T Easy, Promega) (Fig. S7, panels A and B). The cloned sequence was confirmed by sequencing with genespecific primers. The sequence alignment with the sequences of five Rsv3 candidates from the Phytozome database indicated that the cloned sequence shared highest sequence similarity with Glyma.14g204700 (Fig. S7C). The clone with consensus sequence and without any mutation was then cloned into the pPZP-based expression vector in which the transient expression is controlled by the 35 S promoter and Tnos terminator (Fig. S7D). Primers used for detection, amplification, and sequencing of the candidates are listed in Table S1. The nucleotide sequence of the Glyma.14g204700 was deposited in GenBank, with accession number MF687340.
4. Materials and methods 4.1. Isolation of the candidate Rsv3 from soybean L29 To isolate Rsv3 candidates, we inoculated the first trifoliate of soybean L29 with the SMV avirulent strain G5H. Eight hours after inoculation, the total RNAs were extracted from the inoculated leaves using Triazol reagent (RNAiso Plus, Takara). The cDNA was obtained by reverse transcription using the oligodT (20 mer, Bioneer) and a reverse transcriptase (Goscripts, Promega). Because the sequences at the outermost ends of some candidates are identical, we designed specific internal primers to obtain the full-length cDNA clone via overlapping PCR (Fig. S5A). Two halves of the doublestranded cDNA Glyma.14g204700 were amplified with internally specific primers (Fig. S5B) and outermost primers (Fig. S6, panels A and B). Partial regions homologous to Glyma.14g204500 and Glyma.14g204600 were detected and confirmed in the genomic DNA but not in the cDNA of L29 (Fig. S6C). The two candidates Glyma.14g205000 and Glyma.14g205300 were not detected in either the genomic DNA or cDNA by partial sequence amplifications (Fig. S6D); another primer set was designed and used to detect these two candidates, but no positive
4.2. SMV resistance assay To evaluate the effect of the cloned candidate on SMV resistance, the DNA plasmid of the empty vector pPZP, the expression clone of Glyma.14g204700, and infectious clones of SMV::GFP were prepared with a maxi-prep DNA plasmid purification kit (Nucleobond® Xtra 157
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Fig. 4. Effect of transient silencing of Glyma.14g204700 in soybean cultivar L29 on SMVG5H accumulation. A, Symptoms of the third trifoliate leaf of the soybean plants 2 weeks after the plants were inoculated with phosphate buffer, BPMV empty vector, or BPMV clones specific to GmPDS and Glyma.14g204700 (scale bar = 1 cm). B, Relative expression level of GmPDS and Glyma.14g204700 2 weeks after plants were inoculated with phosphate buffer (healthy), BPMV only (vector), and BPMV clones specific to the targets (VIGS). C, Time course accumulation of SMV-G5H in inoculated plants that were silenced (VIGS) or not silenced (vector). D, Relative expression level of Glyma.14g204700 within 1 week after silenced or non-silenced (vector) plants were inoculated with SMV. In panels B, C, and D, error bars are standard deviations of three biological replications. Asterisks indicate significant t-test results, * P < 0.05, ** P < 0.01.
used as a negative control for the non-silenced plants. The plasmid DNAs were prepared with the maxi-prep plasmid DNA purification kit. For delivery of the VIGS clones, the plasmid DNAs (0.5 µg/µl) corresponding to RNA1 and RNA2 of the BPMV were mixed at 1:1 ratios in 50 mM phosphate buffer (pH 7.4). The mixtures then were then used to inoculate the primary leaves of 10-day-old plants of soybean cultivar L29 (with 10 µg of total DNA plasmids per leaf, two leaves per plant, and three plants per experiment). Two weeks after the inoculation, the silencing of the target genes was assessed by quantifying the expression levels of the target gene in plants treated with VIGS clones and with the empty vector.
Maxi, MN). A 1:1 ratio mixture (0.5 µg/µl) of the candidate and infectious clone was prepared in 50 mM phosphate buffer (pH 7.4). The empty vector was used in the control instead of the candidate. These mixtures were then used to inoculate the primary leaves of 2-week-old soybean cultivar Lee74 (with 10 µg of total DNA plasmids per leaf, two leaves per plant, and three plants per experiment); Lee74 is susceptible to SMV G5H. Lee74. Detailed experimental steps were shown in Fig. S2A. After inoculation, the GFP images of the inoculated and upper non-inoculated leaves were captured under UV light with a digital camera (D700, Nikon) with a green filter. The microscopic images of the SMV::GFP in the inoculated leaves were recorded with a fluorescent microscope (Axio Imager A1, Carl Zeiss). To check the effect of the candidate on SMV infectivity, the trifoliate leaves of soybean Lee74 were first mechanically inoculated with the DNA plasmid of expression clones with 60 µg per trifoliate. Two days later, the same leaves were inoculated with “viral sap”, which was prepared by grinding 100 mg of soybean tissue infected by SMVG5H::GFP in 1 ml of 50 mM phosphate buffer (pH 7.4); each leaf was inoculated with 20 µl of the sap. Four days after inoculation with the virus, the expression of GFP was assessed as described above.
4.4. Immuno-blotting To evaluate the expression level of GFP, about 100 mg of tissue was collected and ground in liquid nitrogen. Total protein was extracted with a plant total protein extraction kit (Sigma). Protein concentration was measured by the Bradford assay using Bradford reagent (Bio-Rad). A 40-μg quantity of total protein for each sample was separated by 12% SDS-PAGE and electro-blotted onto PVDF membranes (GE Healthcare). Protein blots were probed with rabbit anti-GFP antibody (Santa Cruz, 1:20,000 dilution) followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad, 1:20,000 dilution). The probed blots were visualized using Clarity western ECL substrate (Bio-Rad) in a Fusion Fx imaging instrument (Vilber Lourmat).
4.3. Virus-induced gene silencing To down-regulate the expression of the candidate in soybean genotype L29, which contains Rsv3 and is resistant to G5H, we used a previously described BPMV-based system (Zhang et al., 2010). The experimental procedure was symbolically represented in the Fig. S2B. A 347-bp DNA fragment amplified from Glyma.14g204700 was inserted in reverse orientation into the BPMV RNA2 vector via BamHI-XhoI restriction enzyme sites. To test the applicability of the BPMV vector in this study, we used a similar insertion of a 327-bp DNA fragment amplified from the GmPDS gene (NM_001249840). Primers for amplification of the target fragments are listed in Table S1. The empty vector was
4.5. Real-time RT-PCR For quantification of target gene expression and SMV accumulation, total RNAs were extracted from leaves with Triazol reagent and were treated with DNase I (RQ1, Promega), and cDNA was synthesized using oligodT and reverse transcriptase. Real-time PCR was then carried out with biological triplicates and technically replicates in a real-time PCR 158
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system (CFX96, Bio-rad) using Sybr green master mix (IQ SYBR green supermix, Bio-rad) as described by the manufacturer. The soybean gene encoding CDPK-related protein kinase (AW396185) was used as reference for data normalization as described previously (Libault et al., 2008). Primers used for the qRT-PCRs are listed in Table S1. The expression levels of the target sequence were normalized based on Ct values of the target or the reference and were statistically calculated with Microsoft Excel 2010 software (Schmittgen, 2006). Differences between the tested group and the control group were accessed by an unpaired two-tailed t-test. Acknowledgements This research was supported in part by grants from the NextGeneration BioGreen 21 Program (no. PJ01101401) and the Rural Development Administration and the Vegetable Breeding Research Center (no. 710001-3) through the Agriculture, the Food and Rural Affairs Research Center Support Program from the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. PT and KW were supported by research fellowships from the Brain Korea 21 Plus Project. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2017.10.014. References Babu, M., Gagarinova, A.G., Brandle, J.E., Wang, A., 2008. Association of the transcriptional response of soybean plants with soybean mosaic virus systemic infection. J. Gen. Virol. 89, 1069–1080. Bej, A., Sahoo, B.R., Swain, B., Basu, M., Jayasankar, P., Samanta, M., 2014. LRRsearch: an asynchronous server-based application for the prediction of leucine-rich repeat motifs and an integrative database of NOD-like receptors. Comput. Biol. Med. 53, 164–170. Chen, P., Buss, G., Roane, C., Tolin, S., 1991. Allelism among genes for resistance to soybean mosaic virus in strain-differential soybean cultivars. Crop Sci. 31, 305–309. Chen, P., Buss, G., Tolin, S., 1993. Resistance to soybean mosaic virus conferred by two independent dominant genes in PI 486355. J. Hered. 84, 25–28. Chisholm, S.T., Coaker, G., Day, B., Staskawicz, B.J., 2006. Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124, 803–814. Cho, E.-K., Goodman, R.M., 1979. Strains of soybean mosaic virus: classification based on virulence in resistant soybean cultivars. Phytopathology 69, 467–470. Chung, B.Y.-W., Miller, W.A., Atkins, J.F., Firth, A.E., 2008. An overlapping essential gene in the Potyviridae. Proc. Natl. Acad. Sci. USA 105, 5897–5902. Combet, C., Blanchet, C., Geourjon, C., Deleage, G., 2000. NPS@: network protein sequence analysis. Trends Biochem. Sci. 25, 147–150. Cui, M.-Y., Wei, W., Gao, K., Xie, Y.-G., Guo, Y., Feng, J.-Y., 2017. A rapid and efficient Agrobacterium-mediated transient gene expression system for strawberry leaves and the study of disease resistance proteins. Plant Cell Tiss. Org. 130, 1–14. Dangl, J.L., Jones, J.D., 2001. Plant pathogens and integrated defence responses to infection. Nature 411, 826–833. Diaz‐Pendon, J.A., Truniger, V., Nieto, C., Garcia‐Mas, J., Bendahmane, A., Aranda, M.A., 2004. Advances in understanding recessive resistance to plant viruses. Mol. Plant Pathol. 5, 223–233. Ellis, J., Dodds, P., Pryor, T., 2000. Structure, function and evolution of plant disease resistance genes. Curr. Opin. Plant Biol. 3, 278–284. Ellis, J.G., Dodds, P.N., Lawrence, G.J., 2007. Flax rust resistance gene specificity is based on direct resistance-avirulence protein interactions. Annu. Rev. Phytopathol. 45, 289–306. Flor, H.H., 1971. Current status of the gene-for-gene concept. Annu. Rev. Phytopathol. 9, 275–296. Hajimorad, M., Hill, J., 2001. Rsv1-mediated resistance against soybean mosaic virus-N is hypersensitive response-independent at inoculation site, but has the potential to initiate a hypersensitive response-like mechanism. Mol. Plant-Microbe Interact. 14, 587–598. Hill, J.H., Whitham, S.A., 2014. Control of virus diseases in soybeans. Adv. Virus Res. 90, 355–390. Ilut, D.C., Lipka, A.E., Jeong, N., Bae, D.N., Kim, D.H., Kim, J.H., Redekar, N., Yang, K., Park, W., Kang, S.-T., 2016. Identification of haplotypes at the Rsv4 genomic region in soybean associated with durable resistance to soybean mosaic virus. Theor. Appl. Genet. 129, 453–468. Jeong, S., Kristipati, S., Hayes, A., Maughan, P., Noffsinger, S., Gunduz, I., Buss, G., Maroof, M., 2002. Genetic and sequence analysis of markers tightly linked to the resistance gene, Rsv3. Crop Sci. 42, 265–270.
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