Skip to main content
PMC home page
Molecular Plant Pathology logoLink to Molecular Plant Pathology
. 2014 Aug 24;16(3):301–307. doi: 10.1111/mpp.12175

Amino acid substitution in P3 of Soybean mosaic virus to convert avirulence to virulence on Rsv4‐genotype soybean is influenced by the genetic composition of P3

Y Wang 1,2, B Khatabi 1,3, M R Hajimorad 1,✉
PMCID: PMC6638367  PMID: 25040594

Summary

The modification of avirulence factors of plant viruses by one or more amino acid substitutions converts avirulence to virulence on hosts containing resistance genes. Limited experimental studies have been conducted on avirulence/virulence factors of plant viruses, in particular those of potyviruses, to determine whether avirulence/virulence sites are conserved among strains. In this study, the Soybean mosaic virus (SMV)–Rsv4 pathosystem was exploited to determine whether: (i) avirulence/virulence determinants of SMV reside exclusively on P3 regardless of virus strain; and (ii) the sites residing on P3 and crucial for avirulence/virulence of isolates belonging to strain G2 are also involved in virulence of avirulent isolates belonging to strain G7. The results confirm that avirulence/virulence determinants of SMV on Rsv4‐genotype soybean reside exclusively on P3. Furthermore, the data show that sites involved in the virulence of SMV on Rsv4‐genotype soybean vary among strains, with the genetic composition of P3 playing a crucial role.

Keywords: genetic background, Glycine max, potyvirus, resistance gene, virulence determinants

Plants have evolved diverse strategies to resist pathogens including viruses (Hull, 2002). One strategy involves classical nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) resistance (R) genes (Maule et al., 2007). Resistance mediated by this group of R genes operates on the basis of a ‘gene‐for‐gene’ model, in which an R‐gene‐encoded protein in a host recognizes, directly or indirectly, a pathogen avirulence gene product, termed an avirulence factor (Flor, 1971; Jones and Dangl, 2006). In the majority of viral pathosystems, R‐gene‐mediated recognition activates host defence signalling pathways leading to the expression of a hypersensitive response (HR) (Dangl and Jones, 2001; Palukaitis and Carr, 2008). However, there are instances involving the NBS‐LRR class of R genes in which the resistance phenotype is expressed as extreme resistance (ER) and HR expression is not required to limit infection (Bendahmane et al., 1999; Hajimorad and Hill, 2001).

Interestingly, unusual R genes against plant viruses have been reported that differ from the NBS‐LRR class and the mechanism of resistance does not involve HR or ER. These include RTM1/RTM2/RTM3 in Arabidopsis against Tobacco etch virus (TEV), Scmv2 in maize against Sugarcane mosaic virus (SCMV) and Rsv4 in soybean against Soybean mosaic virus (SMV) (Chisholm et al., 2000; Cosson et al., 2010; Ingvardsen et al., 2010; Kuntze et al., 1997; Saghai Maroof et al., 2010; Whitham et al., 2000). The RTM genes prevent systemic, but not cell‐to‐cell, movements of TEV within the inoculated leaves (Chisholm et al., 2001; Decroocq et al., 2009; Mahajan et al., 1998). Similarly, the mechanism of resistance against SCMV in Scmv2‐genotype maize seems to operate at the level of long‐distance movement (Ingvardsen et al., 2010; Kuntze et al., 1997). However, Rsv4, which belongs to a previously uncharacterized type or class of R gene, restricts SMV movement and accumulation in inoculated leaves and also prevents systemic movement (Gunduz et al., 2004; Khatabi et al., 2012; Saghai Maroof et al., 2010).

Regardless of the class or type of R gene, resistance phenotypes or the mechanism of resistance, experimental modification of a single virus‐encoded protein by one or more mutations converts avirulent viruses to virulent or R‐genotype plants (Decroocq et al., 2009; Hajimorad et al., 2011; Meshi et al., 1989; Wen et al., 2013). Avirulence/virulence function has been assigned to a number of viral‐encoded proteins of non‐potyviruses on R‐genotype hosts; however, studies on potyviral‐encoded proteins serving as avirulence factors are limited (Hull, 2002). Genome organization and gene expression strategy vary widely among plant viruses. In some, such as tobamoviruses and potexviruses, viral‐encoded proteins are expressed from single open reading frames (ORFs) and avirulence/virulence function dissociated from virus infection has been assigned to diverse proteins of these groups of viruses (Bendahmane et al., 1999; Malcuit et al., 1999; Meshi et al., 1989; Padgett and Beachy, 1993; Pfitzner and Pfitzner, 1992). In contrast, a potyviral genome contains one single long ORF that is expressed as a precursor polypeptide, which is subsequently cleaved co‐translationally and post‐translationally by three virus‐encoded proteases to produce 8–10 functional proteins (Urcuqui‐Inchima et al., 2001). Thus, at any given time, a number of functional potyviral‐encoded proteins, which are produced following processing in trans, are present in different polypeptide contexts within the infected cells. Although some in trans cleavage events occur rapidly and efficiently, others are slow events, which seem to have biological significance (Urcuqui‐Inchima et al., 2001). Despite this fundamental difference in genome expression strategy, it has been demonstrated that a single virus‐encoded protein serves as the avirulence/virulence factor in a number of potyviral pathosystems involving R genes. However, the majority of these studies were conducted in the context of virus infection and in the presence of other viral functional proteins (Chowda‐Reddy et al., 2011a; Jenner et al., 2000; Khatabi et al., 2012; Kim et al., 2010; Seo et al., 2009a; Zhang et al., 2009). Interestingly, there are a few other potyviral pathosystems involving R genes in which avirulence/virulence function has been attributed to more than one virus protein (Chowda‐Reddy et al., 2011b; Chu et al., 1997; Eggenberger et al., 2008; Hajimorad et al., 2008, 2011; Jenner et al., 2002a; Krause‐Sakate et al., 2005). In two of these pathosystems, Turnip mosaic virus–Brassica napus line 165 and SMV–Rsv1 pathosystems, subsequent studies have shown the presence of more than one R gene against each virus (Jenner et al., 2002a; Wen et al., 2013).

SMV is a species within the genus Potyvirus. A number of SMV strains, G1–G7, have been reported in North America (Cho and Goodman, 1979). Expression of the SMV genome results in the production of a number of multifunctional proteins, including helper‐component proteinase (HC‐Pro), P3 and cytoplasmic inclusion (CI) (Urcuqui‐Inchima et al., 2001). In addition, a small ORF (i.e. pipo) embedded in the P3 cistron encodes a protein in the +2 frame that is involved in virus movement (Chung et al., 2008; Wen and Hajimorad, 2010). Rsv4, a single atypical R gene in V94‐5152 and PI88788 soybean, confers resistance against SMV (Buss et al., 1997; Gunduz et al., 2004; Khatabi et al., 2012; Saghai Maroof et al., 2010). V94‐5152 (Rsv4) confers resistance against a larger number of SMV strains relative to PI88788 (Rsv4) (Gunduz et al., 2004; Khatabi et al., 2012). The source of the difference in resistance effectiveness between these two genotypes is unknown. Regardless, the phenotype of resistance conferred by Rsv4 is not associated with ER or HR (Gunduz et al., 2004; Khatabi et al., 2012).

It has been shown previously that P3 of SMV isolates belonging to the G2 strain serves as an avirulence/virulence factor on V94‐5152 (Chowda‐Reddy et al., 2011a; Khatabi et al., 2012). Experimentally, substitution at polyprotein position 1033 [glutamine (Gln) to lysine (Lys)] or 1054 [glycine (Gly) to arginine (Arg)] of SMV isolates and variants derived from the G2 strain converted avirulence to virulence on V94‐5152 (Chowda‐Reddy et al., 2011a; Khatabi et al., 2012). Recently, the virulence site of an SMV isolate originating from Iran, unassigned to a specific strain, was mapped experimentally to polypeptide position 1053 located on P3, where a serine (Ser) to asparagine (Asn) substitution converted avirulence to virulence (Ahangaran et al., 2013). In contrast, simultaneous mutations in multiple SMV proteins (HC‐Pro, P3, CI) have been reported to be essential for the gain of virulence of an isolate belonging to the G7 strain (SMV‐G7) on V94‐5152 (Chowda‐Reddy et al., 2011b).

Our main objective in this study was to evaluate the gain of virulence (i.e. ability to move systemically) by two isolates of the G7 strain, SMV‐G7 and SMV‐G7d, on V94‐5152 and PI88788. Two hypotheses were tested: (i) avirulence/virulence determinants of SMV on Rsv4‐genotype soybean reside exclusively on P3; and (ii) the site on P3 essential for gain of virulence on Rsv4‐genotype soybean is influenced by the genetic composition of P3. In this article, we have defined virulence as the capability of an SMV strain, or an SMV variant, to move systemically in Rsv4‐genotype soybean regardless of the symptom severity displayed.

When SMV‐G7 and SMV‐G7d tagged with β‐glucuronidase (GUS) were inoculated biolistically on attached primary leaves of V94‐5152 and PI88788, SMV‐G7‐GUS and SMV‐G7d‐GUS both established local infection (Fig. 1). However, unlike infection in inoculated leaves of Essex (rsv4), a universally susceptible cultivar to SMV, the infected foci in the inoculated leaves of both Rsv4‐genotype soybeans did not extend to the veins (Fig. 1). Furthermore, in contrast with infection in Essex, in which both viruses moved systemically, none was detected in non‐inoculated leaves of the two Rsv4‐genotype soybeans regardless of mechanical or biolistic inoculation and the presence or absence of the uidA gene (Fig. 1; Table 1).

Figure 1.

figure

Open in a new tab

β‐Glucuronidase (GUS) expression in soybean leaf tissues following biolistic inoculation. Fully expanded attached primary leaves of soybean seedlings were inoculated with plasmids containing the full‐length infectious cDNA clone of Soybean mosaic virus (SMV) strains G7 or G7d tagged with GUS (SMV‐G7‐GUS or SMV‐G7d‐GUS). The inoculated plants were analysed histochemically at 21 days post‐inoculation. Scale bar, 2 cm.

Table 1.

Abilities of Soybean mosaic virus (SMV) strains G7 and G7d and derivative P3 mutants to infect systemically rsv4‐ and R sv4‐genotype soybeans following biolistic or mechanical inoculationsa

Viruses/mutants/chimeras Soybean genotypes
Essex (rsv4) PI88788 (Rsv4) V94‐5152 (Rsv4)
Viruses
SMV‐G7 (5/5)b 10/11c (0/7) 0/22 (0/8) 0/11
SMV‐G7d (5/5) 9/9 (0/7) 0/12 (0/9) 0/12
SMV‐G7‐derived mutants
SMV‐G7Q1034K (10/10) 13/22 (4*d/4) 3/17 (3*/4) 0/25
SMV‐G7S1055R (6**/6) 18/18 (0/6) 0/11 (0/6) 0/12
SMV‐G7Q1034K+S1055R (12/13) 13/16 (3/7) 5/15 (6*/7) 3/23
SMV‐G7H1054N (9**/9) 5/5 (0/9) 0/9 (0/9) 0/18
SMV‐G7d‐derived mutants
SMV‐G7dQ1034K (4**/12) 8/13 (0/3) 0/12 (0/3) 0/10
SMV‐G7dS1055R (8**/8) 13/13 (0/3) 0/21 (0/3) 0/24
SMV‐G7dQ1034K+S1055R (11/11) 14/27 (2/6) 3/16 (1*/6) 3/20
SMV‐G7dH1054N (6**/6) 9/9 (0/4) 0/32 (0/3) 0/23
SMV‐G7d‐derived chimeras
SMV‐G7d/G7P3 (4**/4) 10/10 (0/5) 0/10 (0/7) 0/10
SMV‐G7d/G7P3Q1034K (6/6) 8/8 (2/3) 3/5 (1*/3) 0/14
SMV‐G7d/G7P3Q1034K+S1055R (6/6) 5/5 (3/3) 5/5 (3*/3) 9/10
Open in a new tab
a

For biolistic inoculation, plasmid containing full‐length infectious cDNA clones was delivered into fully expanded attached primary leaves of soybean seedlings. For mechanical inoculation, sap derived from biolistically inoculated Essex was used as inoculum and rub inoculated onto fully expanded primary leaves of soybean seedlings. The inoculated plants were evaluated for infection based on symptom expression at 21 days post‐inoculation. Absence of virus in asymptomatic plants was confirmed by enzyme‐linked immunosorbent assay (ELISA) using antiserum to recombinant coat protein of SMV‐G7d (see Materials and Methods S1).

b

Number of plants systemically infected/number of plants inoculated biolistically.

c

Number of plants systemically infected/number of plants sap inoculated mechanically.

d

The asterisks indicate that total RNA was extracted from one of the infected Rsv4‐genotype plants and subjected to reverse transcription‐polymerase chain reaction (RT‐PCR). The stability of the introduced mutation(s) and the lack of any newly emerged mutation in helper‐component proteinase (HC‐Pro), P3 and cytoplasmic inclusion (CI) proteins of progeny viruses were confirmed by sequencing the entire cistron (*). For non‐virulent mutants on Rsv4‐genotype plants, total RNA was extracted from one of the systemically infected Essex plants and subjected to RT‐PCR, but only the entire P3 cistron was sequenced (**). Infection of Essex with SMV‐G7dQ1034K was associated with an additional mutation in the P3 cistron, which resulted in K1034N substitution. No newly emerged mutation was detected in P3, HC‐Pro or CI derived from other progenies of molecularly cloned mutant viruses recovered from Rsv4‐genotype soybean.

It should be noted that interactions of SMV‐G7 and SMV‐G7d with V94‐5152 are no different from that of SMV‐N, an isolate belonging to the G2 strain; however, SMV‐N is virulent on PI88788, whereas both SMV‐G7 and SMV‐G7d are avirulent (Khatabi et al., 2012). A number of other field isolates of SMV are also virulent on PI88788, but not on V94‐5152 (Khatabi et al., 2012; Fig. S1, see Supporting Information). Previously, we have shown experimentally that substitution of glutamine (Gln) to Lys or Gly to Arg at polyprotein positions 1033 and 1054 of SMV‐N, respectively, results in the virulence of both SMV‐NQ1033K and SMV‐NG1054R on V94‐5152 (Khatabi et al., 2012). Alignment of P3 of SMV‐G7 and SMV‐G7d with that of SMV‐N shows that Gln is also present at polypeptide position 1034 of SMV‐G7 and SMV‐G7d, which corresponds to position 1033 on the SMV‐N genome (Fig. S2, see Supporting Information). However, at polypeptide position 1055 of SMV‐G7 and SMV‐G7d, corresponding to position 1054 of SMV‐N, Ser instead of Gly is present (Fig. S2). To evaluate the impact of amino acid substitutions at polyprotein positions 1034 and 1055 on the virulence of SMV‐G7, we synthesized SMV‐G7Q1034K and SMV‐G7S1055R (the details of the creation of point mutations, reverse transcription‐polymerase chain reaction (RT‐PCR) and sequencing are listed in Materials and Methods S1 and the primers used are listed in Table S1, see Supporting Information). Biolistic inoculation of SMV‐G7Q1034K resulted in virulence on PI88788 and V94‐5152, but SMV‐G7S1055R remained avirulent on both Rsv4‐genotype soybeans regardless of biolistic or mechanical inoculations (Table 1; Fig. 2). SMV‐G7S1055R infected systemically Essex and the mutation was stable (Table 1). When Q1034K + S1055R were introduced simultaneously into P3 of SMV‐G7, the rate of systemic movement of SMV‐G7Q1034K+S1055R on Rsv4‐genotype soybean was similar to that of SMV‐G7Q1034K (Table 1; Fig. 2). In general, biolistic inoculation with plasmids was more efficient than mechanical inoculation using progeny viruses derived from biolistically inoculated Essex (Table 1). To rule out the emergence of any new mutation in planta, the entire HC‐Pro, P3 and CI cistrons of progeny viruses were RT‐PCR amplified from one of the systemically infected Rsv4‐genotype soybeans inoculated biolistically with each of the SMV‐G7‐derived P3 mutants. Sequence analysis showed the absence of any new mutation in HC‐Pro, P3 and CI cistrons, and verified the stability of the introduced mutations (Table 1). To determine whether any additional adaptive or compensatory mutation emerges following multiple passages of progeny viruses derived from SMV‐G7Q1034K+S1055R in V94‐5152, progeny viruses were passaged five times in V94‐5152. All inoculated V94‐5152 were systemically infected in the last passage (data not shown). The full‐length HC‐Pro, P3 and CI cistrons of progeny viruses derived from two systemically infected V94‐5152 were RT‐PCR amplified and sequenced. Analyses showed the absence of any newly emerged mutation in HC‐Pro or CI cistrons; however, a single non‐synonymous mutation was present in P3 of progeny viruses derived from both plants at polypeptide position 938 changing phenylalanine (Phe) with Ser. This mutation was synonymous in the pipo ORF.

Figure 2.

figure

Open in a new tab

Phenotypic differences in the systemic responses of soybean genotypes following biolistic inoculation of fully expanded attached primary leaves with plasmids containing the full‐length infectious cDNA clone of Soybean mosaic virus (SMV)‐G7‐derived P3 mutants. Representative leaflets from non‐inoculated trifoliate leaves were photographed at 21 days post‐inoculation. Note that the phenotypic responses of Rsv4‐genotype soybeans to biolistic inoculation with molecularly cloned SMV‐G7 are similar to those expressed by SMV‐G7S 1055 R. Scale bar, 2 cm.

Surprisingly, substitution at position 1034 or 1055 of SMV‐G7d had no impact on virulence, as SMV‐G7dQ1034K and SMV‐G7dS1055R remained avirulent on Rsv4‐genotype soybean regardless of biolistic or mechanical inoculations (Table 1). However, when both mutations were introduced simultaneously into P3, SMV‐G7dQ1034K+S1055R gained the ability to move systemically in both Rsv4‐genotype soybeans, but inefficiently (Table 1). No newly emerged mutation was present in HC‐Pro, P3 and CI of progeny viruses derived from a single systemically infected V94‐5152 (Table 1). Attempts to passage further the progeny viruses derived from this single infected V94‐5152 on additional V94‐5152 was unsuccessful (data not shown).

P3 of SMV‐G7d differs from that of SMV‐G7 by four amino acid substitutions (Hajimorad et al., 2003; Fig. S2). To determine whether the genetic composition of P3 as a whole has an influence on virulence on Rsv4‐genotype plants, P3 of SMV‐G7d was replaced precisely with that of SMV‐G7 (Hajimorad et al., 2005). As expected, SMV‐G7d/G7P3, similar to parental viruses, remained avirulent on both Rsv4‐genotype soybeans (Table 1). When Q1034K was introduced at position 1034, SMV‐G7d/G7P3Q1034K moved inefficiently systemically in both Rsv4‐genotype soybeans (Table 1). However, when both Q1034K + S1055R mutations were introduced simultaneously into P3 of the chimera, SMV‐G7d/G7P3Q1034K+S1055R was highly efficient in infecting systemically both Rsv4‐genotype soybeans regardless of the method of inoculation (Table 1). Sequence analyses of HC‐Pro, P3 and CI cistrons derived from one of the systemically infected V94‐5152 with each of the SMV‐G7d‐derived chimeras showed the stability of the mutations and the absence of any newly emerged substitution.

Ahangaran et al. (2013) have shown experimentally that a Ser to Asn substitution at position 1053 of an avirulent SMV isolate converts avirulence to virulence on V94‐5152. We introduced this mutation into P3 of SMV‐N. SMV‐NS1053N remained avirulent on V94‐5152 and, similar to parental SMV‐N, was virulent on PI88788 (Table S2, see Supporting Information). Interestingly, both SMV‐G7 and SMV‐G7d encode histidine (His), instead of Ser, at position 1054, which corresponds to position 1053 of SMV‐N (Fig. S2). Replacement of His with Asn did not confer virulence to SMV‐G7H1054N and SMV‐G7dH1054N as neither moved systemically in either V94‐5152 or PI88788 (Table 1). SMV‐G7H1054N, SMV‐G7dH1054N and SMV‐NS1053N infected systemically Essex and the mutations were stable in viral progenies (Tables 1 and S2). These observations further demonstrate the context dependence of the virulence site on P3 of SMV. It seems that the ability of SMV to move systemically in Rsv4‐genotype soybean depends on the overall P3 structure. Hence, the three‐dimensional context within which these residues reside is expected to be crucial for P3 gain of function. Unfortunately, the three‐dimensional structure of P3 has not been resolved from any of the potyviruses.

In most viral pathosystems involving typical or atypical R genes, a single viral‐encoded protein serves as the avirulence factor (Hull, 2002). However, it has been reported recently that gain of virulence of SMV on soybean genotypes containing Rsv1, Rsv3 or Rsv4 individually requires simultaneous mutations in HC‐Pro, P3 and CI (Chowda‐Reddy et al., 2011b). The Rsv1 locus is multigenic, whereas Rsv3 is a single R gene; nevertheless, the NBS‐LRR class of R genes is associated with both Rsv1‐ and Rsv3‐mediated resistance against SMV (Hayes et al., 2004; Suh et al., 2011; Wen et al., 2013). Chowda‐Reddy et al. (2011b) replaced P3 of SMV‐G7 with that of a SMV‐G2 virulent mutant that contained the G1054R mutation. In our study, we used the same SMV‐G7 clone (Hajimorad et al., 2003) and demonstrated that avirulence/virulence determinants of SMV‐G7 on Rsv4‐genotype soybean, as well as those of SMV‐G7d, reside exclusively on P3. However, P3 genetic composition plays a crucial role in virulence, as mutation at position 1034 (Gln to Lys), but not 1055 (Ser to Arg), converts avirulent SMV‐G7 to virulent on both Rsv4‐genotype soybeans. SMV‐G7 is not the only isolate with a preferred virulence mutation at position 1034 instead of 1055 (Khatabi et al., 2012; Fig. S3, see Supporting Information). It should be noted that the CI cistrons of SMV‐G7 and SMV‐G7d are both identical and there is only a single amino acid difference in the HC‐Pro cistron between these two viruses (Hajimorad et al., 2003). However, neither HC‐Pro nor CI cistrons played a role in the virulence of these viruses on Rsv4‐genotype soybean, because SMV‐G7Q1034K+S1055R and SMV‐G7d/G7P3Q1034K+S1055R were virulent on both Rsv4‐genotype soybeans. This is further supported by the absence of any newly emerged mutation in HC‐Pro or CI cistrons of progeny viruses derived from systemically infected V94‐5152.

Wen et al. (2013) have also recently demonstrated that the involvement of both HC‐Pro and P3 as avirulence/virulence factors of SMV on Rsv1‐genotype soybean is caused by the multigenic nature of the Rsv1 locus. Furthermore, no evidence for the involvement of CI in the virulence of SMV‐N on Rsv1‐genotype soybean has been reported (Eggenberger et al., 2008; Hajimorad et al., 2008, 2011; Wen et al., 2013). It should be noted that the selection of adaptive mutations in HC‐Pro of SMV‐N on Rsv1‐genotype soybean is influenced by the P3 sequence content, as different sites on HC‐Pro, clustered mostly at the N‐terminus, convert avirulent SMV‐N‐derived P3 chimeras to virulent (Hajimorad et al., 2008; Wen et al., 2013). The impact of the genetic background surrounding the avirulence gene has been reported previously for gain of virulence of Potato virus Y (PVY) on pepper pvr2 (Montarry et al., 2011). In the study by Chowda‐Reddy et al. (2011b), position 682 corresponding to that of an isolate of the G2 strain was altered [Arg to methionine (Met)]. The R682M substitution in HC‐Pro of SMV‐N plays a role in avirulence/virulence of the virus on Rsv1‐genotype soybean (Eggenberger et al., 2008; Hajimorad et al., 2008, 2011); however, this is not the preferred mutational pathway towards virulence (Hajimorad et al., 2011; Khatabi et al., 2013).

It also seems unlikely that the virulence of SMV on Rsv3‐genotype soybean involves more than one SMV‐encoded protein (Chowda‐Reddy et al., 2011b). Studies on the SMV–Rsv3 pathosystem by two groups identified CI as the avirulence/virulence factor; however, the avirulence/virulence site was not identical between the two isolates used (Seo et al., 2009a; Zhang et al., 2009). It is interesting to note that, unlike Rsv1 and Rsv4, Rsv3 has been defeated globally by SMV (Ahangaran et al., 2013; Choi et al., 2005; Khatabi et al., 2012; Seo et al., 2009b; Viel et al., 2009). If simultaneous mutations in multiple SMV genes are required for gain of virulence on Rsv3‐genotype soybean (Chowda‐Reddy et al., 2011b), it is expected that Rsv3 will remain durable. Naturally occurring virulent isolates of SMV on Rsv1 are not frequent (Ahangaran et al., 2013; Khatabi et al., 2013; Seo et al., 2009b; Viel et al., 2009); however, virulent isolates have been reported from South Korea (Choi et al., 2005). Unfortunately, the virulence determinants of the Korean isolates have not been characterized.

The deployment of R genes is the most effective, economically sound and environmentally friendly approach to control viral diseases of plants; however, concern about the durability of R genes remains. Avirulence determinants and their significance in the life cycle of pathogens are considered to be predictors of the durability of R genes (Lauge et al., 1998). The durability of the Ry gene in potato against PVY has been attributed to the targeting of nuclear inclusion protein a (NIa), which is a key factor in the processing of potyviral precursor polyproteins (Mestre et al., 2000). Other factors, such as the number of mutations required for gain of virulence, evolutionary constraints acting against amino acid substitutions and the high fitness cost associated with gain of virulence mutation(s), also contribute to R‐gene durability (Bornemann and Varrelmann, 2013; Goulden et al., 1993; Harrison, 2002; Janzac et al., 2009, 2010; Jenner et al., 2002b; Khatabi et al., 2013). Durability can also be enhanced by careful selection and deployment of the most appropriate R genes individually or in combination using a gene stacking approach. Knowledge of the avirulence determinants and fitness costs associated with gain of virulence mutations may contribute to the appropriate implementation of this approach. Thus, the precise identification of avirulence determinants of viruses has practical implications in the durable management of R genes. Unfortunately, our knowledge of the avirulence/virulence sites residing on plant viral proteins is limited and it is unknown how widely sites are conserved amongst isolates/strains of the same virus. In some pathosystems, such as PVY–Ry, this question cannot even be addressed as there is not even a single virulent isolate available worldwide (Mestre et al., 2000). The context dependence of the required mutation(s) for gain of virulence by avirulent viruses asks for caution in assigning avirulence/virulence function to particular sites on viral proteins or genotyping of viral isolates for resistance‐breaking capability.

Supporting information

Fig. S1 Alignment of deduced primary amino acid sequences (residues 1020–1060) of P3 derived from Soybean mosaic virus (SMV) isolates recovered from systemically infected PI88788 (Rsv4).

Fig. S2 Alignment of the full‐length deduced primary amino acid sequences of P3 of pSMV‐N (N), pSMV‐G7 (G7) and pSMV‐G7d (G7d).

Fig. S3 Alignment of the deduced primary amino acid sequences (residues 1020–1060) of P3 from isolates of Soybean mosaic virus (SMV) virulent on V94‐5152 (Rsv4).

Table S1 Sequences of sense and antisense oligonucleotide primers used for site‐directed mutagenesis, reverse transcription‐polymerase chain reaction (RT‐PCR) and sequencing.

Table S2 Ability of Soybean mosaic virus (SMV)‐derived P3 mutant (SMV‐NS1053N) to infect systemically rsv4‐ and Rsv4‐genotype soybeans following biolistic or mechanical inoculations.

Materials and Methods S1 The details of viruses, soybean genotypes, inoculation, detection, construction of point mutants, RT‐PCR, sequencing and generation of polyclonal antibodies against bacterially expressed coat protein of SMV‐G7d.

Click here for additional data file. (39.3KB, zip)

Acknowledgements

Dr Y. Wang wishes to express his gratitude to the China Scholarship Council and Jilin Provincial Government for partial financial support. This project was supported in part by The University of Tennessee Agricultural Experimental Station, Knoxville, TN, North Central Soybean Research Program and Kentucky Soybean Promotion Board. The authors are grateful to Drs A. E. Eggenberger and J. H. Hill for a copy of the cloned pSMV‐G7‐GUS.

References

  1. Ahangaran, A. , Habibi, M.K. , Mosahebi Mohammadi, G.‐H. , Winter, S. and Garcia‐Arenal, F. (2013) Analysis of Soybean mosaic virus genetic diversity in Iran allows the characterization of a new mutation resulting in overcoming Rsv4‐resistance. J. Gen. Virol. 94, 2557–2568. [DOI] [PubMed] [Google Scholar]
  2. 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bornemann, K. and Varrelmann, M. (2013) Effect of sugar beet genotype on the Beet necrotic yellow vein virus P25 pathogenicity factor and evidence for a fitness penalty in resistant‐breaking strains. Mol. Plant Pathol. 14, 356–364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buss, G.R. , Ma, G. , Chen, P. and Tolin, S.A. (1997) Registration of V94‐5152 soybean germplasm resistant to soybean mosaic potyvirus. Crop Sci. 37, 1987–1988. [Google Scholar]
  5. 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chisholm, S.T. , Parra, M.A. , Anderberg, R.J. and Carrington, J.C. (2001) Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long‐distance movement of tobacco etch virus. Plant Physiol. 127, 1667–1675. [PMC free article] [PubMed] [Google Scholar]
  7. Cho, E.‐K. and Goodman, R.M. (1979) Strains of soybean mosaic virus: classification based on virulence in resistant soybean cultivars. Phytopathology, 69, 467–470. [Google Scholar]
  8. Choi, B.K. , Koo, J.M. , Ahn, H.J. , Yum, H.J. , Choi, C.W. , Ryu, K.H. , Chen, P. and Tolin, S.A. (2005) Emergence of Rsv‐resistance breaking Soybean mosaic virus isolates from Korean soybean cultivars. Virus Res. 112, 42–51. [DOI] [PubMed] [Google Scholar]
  9. Chowda‐Reddy, R.V. , Sun, H. , Chen, H. , Poysa, V. , Ling, H. , Gijzen, M. and Wang, A. (2011a) Mutations in the P3 protein of Soybean mosaic virus G2 isolates determine virulence on Rsv4‐genotype soybean. Mol. Plant–Microbe Interact. 24, 37–43. [DOI] [PubMed] [Google Scholar]
  10. Chowda‐Reddy, R.V. , Sun, H. , Hill, J.H. , Poysa, V. and Wang, A. (2011b) Simultaneous mutations in multi‐viral proteins are required for Soybean mosaic virus to gain virulence on soybean genotypes carrying different R genes. PLoS ONE, 6, e28342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chu, M. , Lopez‐Moya, J.L. , Llave‐Correas, C. and Pirone, T.P. (1997) Two separate regions in the genome of the tobacco etch virus contain determinants of the wilting response of Tabasco pepper. Mol. Plant–Microbe Interact. 10, 472–480. [DOI] [PubMed] [Google Scholar]
  12. Chung, B.Y.‐W. , Miller, W.A. , Atkins, J.F. and Firth, A.E. (2008) An overlapping essential gene in the potyviridae. Proc. Natl. Acad. Sci. USA, 105, 5897–5902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cosson, P. , Sofer, L. , Le, Q.H. , Leger, V. , Schurdi‐Levraud, V. , Whitham, S.A. , Yamamoto, M.L. , Gopalan, S. , Gall, O.L. , Candresse, T. , Carrington, J.C. and Reverse, F. (2010) RTM3, which controls long‐distance movement of potyviruses, is a member of a new plant gene family encoding a meprin and TRAF homology domain‐containing protein. Plant Physiol. 154, 222–232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dangl, J.L. and Jones, J.D.G. (2001) Plant pathogens and integrated defense responses to infection. Nature, 411, 826–833. [DOI] [PubMed] [Google Scholar]
  15. Decroocq, V. , Salvador, B. , Sicard, O. , Glasa, M. , Cosson, P. , Svanella‐Dumas, L. , Reverse, F. , Garcia, J.A. and Candresse, T. (2009) The determinant of potyvirus ability to overcome the RTM resistance of Arabidopsis thaliana maps to the N‐terminal region of the coat protein. Mol. Plant–Microbe Interact. 22, 1302–1311. [DOI] [PubMed] [Google Scholar]
  16. Eggenberger, A.L. , Hajimorad, M.R. and Hill, J.H. (2008) Gain of virulence on Rsv1‐genotype soybean by an avirulent Soybean mosaic virus requires concurrent mutations in both P3 and HC‐Pro. Mol. Plant–Microbe Interact. 21, 931–936. [DOI] [PubMed] [Google Scholar]
  17. Flor, H.H. (1971) Current status of the gene‐for‐gene concept. Annu. Rev. Phytopathol. 9, 275–296. [Google Scholar]
  18. Goulden, M.G. , Kohm, 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. [DOI] [PubMed] [Google Scholar]
  19. Gunduz, I. , Buss, G.R. , Chen, P. and Tolin, S.A. (2004) Genetic and phenotypic analysis of Soybean mosaic virus resistance in PI88788 soybean. Phytopathology, 94, 687–692. [DOI] [PubMed] [Google Scholar]
  20. Hajimorad, M.R. and Hill, J.H. (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. [DOI] [PubMed] [Google Scholar]
  21. Hajimorad, M.R. , Eggenberger, A.L. and Hill, J.H. (2003) Evolution of Soybean mosaic virus‐G7 molecularly cloned genome in Rsv1‐genotype soybean results in emergence of a mutant capable of evading Rsv1‐mediated recognition. Virology, 314, 497–509. [DOI] [PubMed] [Google Scholar]
  22. Hajimorad, M.R. , Eggenberger, A.L. and Hill, J.H. (2005) Loss and gain of elicitor function of Soybean mosaic virus G7 provoking Rsv1‐mediated lethal systemic hypersensitive response maps to P3. J. Virol. 79, 1215–1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hajimorad, M.R. , Eggenberger, A.L. and Hill, J.H. (2008) Adaptation of Soybean mosaic virus avirulent chimeras containing P3 sequences from virulent strains to Rsv1‐genotype soybeans is mediated by mutations in HC‐Pro. Mol. Plant–Microbe Interact. 21, 937–946. [DOI] [PubMed] [Google Scholar]
  24. Hajimorad, M.R. , Wen, R.‐H. , Eggenberger, A.L. , Hill, J.H. and Saghai Maroof, M.A. (2011) Experimental adaptation of an RNA virus mimics natural evolution. J. Virol. 85, 2557–2564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Harrison, B.D. (2002) Virus variation in relation to resistance‐breaking in plants. Euphytica, 124, 181–192. [Google Scholar]
  26. Hayes, A.J. , Jeong, S.C. , Gore, M.A. , Yu, Y.G. , Buss, G.R. , Tolin, S.A. and Saghai Maroof, M.A. (2004) Recombination within a nucleotide‐binding‐site/leucine‐rich‐repeat gene cluster produces new variants conditioning resistance to soybean mosaic virus in soybeans. Genetics, 166, 493–503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hull, R. (2002) Matthew's Plant Virology. New York: Academic Press. [Google Scholar]
  28. Ingvardsen, C.R. , Xing, Y. , Frei, U.K. and Lubberstedt, T. (2010) Genetic and physical fine mapping of Scmv2, a potyvirus resistance gene in maize. Theor. Appl. Genet. 120, 1621–1634. [DOI] [PubMed] [Google Scholar]
  29. Janzac, B. , Fabre, F. , Palloix, A. and Moury, B. (2009) Constraints on evolution of virus avirulence factors predict the durability of corresponding plant resistances. Mol. Plant Pathol. 10, 599–610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Janzac, B. , Montarry, J. , Palloix, A. , Navaud, O. and Moury, B. (2010) A point mutation in the polymerase of Potato virus Y confers virulence toward the Pvr4 resistance of pepper and a high competitiveness cost in susceptible cultivar. Mol. Plant–Microbe Interact. 23, 823–830. [DOI] [PubMed] [Google Scholar]
  31. Jenner, C.E. , Sanchez, F. , Nettleship, S.B. , Foster, G.D. , Ponz, F. and Walsh, J.A. (2000) The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the Brassica resistance gene TuRB01 . Mol. Plant–Microbe Interact. 13, 1102–1108. [DOI] [PubMed] [Google Scholar]
  32. Jenner, C.E. , Tomimura, K. , Ohshima, K. , Hughes, S.L. and Walsh, J.A. (2002a) Mutations in Turnip mosaic virus P3 and cylindrical inclusion proteins are separately required to overcome two Brassica napus resistance genes. Virology, 300, 50–59. [DOI] [PubMed] [Google Scholar]
  33. Jenner, C.E. , Wang, X. , Ponz, F. and Walsh, J.A. (2002b) A fitness cost for Turnip mosaic virus to overcome host resistance. Virus Res. 86, 1–6. [DOI] [PubMed] [Google Scholar]
  34. Jones, J.D.G. and Dangl, J.L. (2006) The plant immune system. Nature, 444, 323–329. [DOI] [PubMed] [Google Scholar]
  35. Khatabi, B. , Fajolu, O.L. , Wen, R.‐H. and Hajimorad, M.R. (2012) Evaluation of North American isolates of Soybean mosaic virus for gain of virulence on Rsv‐genotype soybeans with special emphasis on resistance‐breaking determinants on Rsv4 . Mol. Plant Pathol. 13, 1077–1088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Khatabi, B. , Wen, R.‐H. and Hajimorad, M.R. (2013) Fitness penalty in susceptible host is associated with virulence of Soybean mosaic virus on Rsv1‐genotype soybean: a consequence of perturbation of HC‐Pro and not P3. Mol. Plant Pathol. 14, 885–897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kim, B.M. , Suehiro, N. , Natsuaki, T. , Inukai, T. and Masuta, C. (2010) The P3 protein of Turnip mosaic virus can alone induce hypersensitive response‐like cell death in Arabidopsis thaliana carrying TuN1 . Mol. Plant–Microbe Interact. 23, 144–152. [DOI] [PubMed] [Google Scholar]
  38. Krause‐Sakate, R. , Redondo, E. , Richard‐Forget, F. , Jadao, A.S. , Houvenaghel, M.‐C. , German‐Retana, S. , Pavan, M.A. , Candresse, T. , Zerbini, F.M. and Le Gall, O. (2005) Molecular mapping of the viral determinants of systemic wilting induced by a Lettuce mosaic virus (LMV) isolate in some lettuce cultivars. Virus Res. 109, 175–180. [DOI] [PubMed] [Google Scholar]
  39. Kuntze, L. , Fuchs, E. , Gruntzig, M. , Schulz, B. , Klein, D. and Melchinger, A.E. (1997) Resistance of early‐maturing European maize germplasm to sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV). Plant Breed. 116, 499–501. [Google Scholar]
  40. Lauge, R. , Joosten, M.H.A. , Haanstra, J.P.W. , Goodwin, P.H. , Lindhout, P. and De Wit, P.J.G.M. (1998) Successful search for a resistance gene in tomato targeted against a virulence factor of a fungal pathogen. Proc. Natl. Acad. Sci. USA, 95, 9014–9018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mahajan, S.K. , Chisholm, S.T. , Whitham, S.A. and Carrington, J.C. (1998) Identification and characterization of a locus (RTM1) that restricts long‐distance movement of tobacco etch virus in Arabidopsis thaliana . Plant J. 14, 177–186. [DOI] [PubMed] [Google Scholar]
  42. Malcuit, I. , Marano, M.R. , Kavanagh, T.A. , De Jong, W. , Forsyth, A. and Baulcombe, D.C. (1999) The 25‐kDa movement protein of PVX elicits Nb‐mediated hypersensitive cell death in potato. Mol. Plant–Microbe Interact. 12, 536–543. [Google Scholar]
  43. Maule, A.J. , Caranta, C. and Boulton, M.I. (2007) Sources of natural resistance to plant viruses: status and prospects. Mol. Plant Pathol. 8, 223–231. [DOI] [PubMed] [Google Scholar]
  44. Meshi, T. , Motoyoshi, F. , Maeda, T. , Yoshiwoka, S. , Watanabe, H. and Okada, Y. (1989) Mutations in the tobacco mosaic virus 30‐kD protein gene overcome Tm‐2 resistance in tomato. Plant Cell, 1, 515–522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mestre, P. , Brigneti, G. and Baulcombe, D.C. (2000) An Ry‐mediated resistance response in potato requires the intact active site of the NIa proteinase from potato virus Y. Plant J. 23, 653–661. [DOI] [PubMed] [Google Scholar]
  46. Montarry, J. , Doumayrou, J. , Simon, V. and Moury, B. (2011) Genetic background matters: a plant–virus gene‐for‐gene interaction is strongly influenced by genetic contexts. Mol. Plant Pathol. 12, 911–920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Padgett, H.S. and Beachy, R.N. (1993) Analysis of tobacco mosaic virus strain capable of overcoming N gene‐mediated resistance. Plant Cell, 5, 577–586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Palukaitis, P. and Carr, J.P. (2008) Plant resistance responses to viruses. J. Plant Pathol. 90, 153–171. [Google Scholar]
  49. Pfitzner, U.M. and Pfitzner, A.J.P. (1992) Expression of a viral avirulence gene in transgenic plants is sufficient to induce the hypersensitive defense reaction. Mol. Plant–Microbe Interact. 5, 318–321. [DOI] [PubMed] [Google Scholar]
  50. Saghai Maroof, M.A. , Tucker, D.M. , Skoneczka, J.A. , Bowman, B.C. , Tripathy, S. and Tolin, S.A. (2010) Fine mapping and candidate gene discovery of the Soybean mosaic virus resistance gene, Rsv4 . Plant Genome, 3, 14–22. [Google Scholar]
  51. Seo, J.‐K. , Lee, S.‐H. and Kim, K.‐H. (2009a) Strain‐specific cylindrical inclusion protein of Soybean mosaic virus elicits extreme resistance and a lethal systemic hypersensitive response in two resistant soybean cultivars. Mol. Plant–Microbe Interact. 22, 1151–1159. [DOI] [PubMed] [Google Scholar]
  52. Seo, J.‐K. , Ohshima, K. , Lee, H.‐G. , Son, M. , Choi, H.‐S. , Lee, S.‐H. , Sohn, S.‐H. and Kim, K.‐H. (2009b) Molecular variability and genetic structure of the population of Soybean mosaic virus based on the analysis of complete genome sequences. Virology, 393, 91–103. [DOI] [PubMed] [Google Scholar]
  53. Suh, S.J. , Bowman, B.C. , Jeong, N. , Yang, K. , Kastl, C. , Tolin, S.A. , Saghai Maroof, M.A. and Jeong, S.‐C. (2011) The Rsv3 locus conferring resistance to Soybean mosaic virus is associated with a cluster of coiled‐coil nucleotide‐binding leucine‐rich repeat genes. Plant Genome, 4, 55–64. [Google Scholar]
  54. Urcuqui‐Inchima, S. , Haenni, A.L. and Bernardi, F. (2001) Potyvirus proteins: a wealth of functions. Virus Res. 74, 157–175. [DOI] [PubMed] [Google Scholar]
  55. Viel, C. , Ide, C. , Cui, X. , Wang, A. , Farsi, M. , Michelutti, R. and Stromvik, M. (2009) Isolation, partial sequencing, and phylogenetic analyses of Soybean mosaic virus (SMV) in Ontario and Quebec. Can. J. Plant Pathol. 31, 108–113. [Google Scholar]
  56. Wen, R.‐H. and Hajimorad, M.R. (2010) Mutational analysis of the putative pipo of soybean mosaic virus suggests disruption of PIPO protein impedes movement. Virology, 400, 1–7. [DOI] [PubMed] [Google Scholar]
  57. Wen, R.‐H. , Khatabi, B. , Ashfield, T. , Saghai Maroof, M.A. and Hajimorad, M.R. (2013) The HC‐Pro and P3 cistrons of an avirulent Soybean mosaic virus are recognized by different genes at the complex Rsv1 locus. Mol. Plant–Microbe Interact. 26, 203–215. [DOI] [PubMed] [Google Scholar]
  58. Whitham, S.A. , Anderberg, R.J. , 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zhang, C. , Hajimorad, M.R. , Eggenberger, A.L. , Tsang, S. , Whitham, S.A. and Hill, J.H. (2009) Cytoplasmic inclusion of Soybean mosaic virus serves as a virulence determinant on Rsv3‐genotype soybean and a symptom determinant. Virology, 391, 240–248. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 Alignment of deduced primary amino acid sequences (residues 1020–1060) of P3 derived from Soybean mosaic virus (SMV) isolates recovered from systemically infected PI88788 (Rsv4).

Fig. S2 Alignment of the full‐length deduced primary amino acid sequences of P3 of pSMV‐N (N), pSMV‐G7 (G7) and pSMV‐G7d (G7d).

Fig. S3 Alignment of the deduced primary amino acid sequences (residues 1020–1060) of P3 from isolates of Soybean mosaic virus (SMV) virulent on V94‐5152 (Rsv4).

Table S1 Sequences of sense and antisense oligonucleotide primers used for site‐directed mutagenesis, reverse transcription‐polymerase chain reaction (RT‐PCR) and sequencing.

Table S2 Ability of Soybean mosaic virus (SMV)‐derived P3 mutant (SMV‐NS1053N) to infect systemically rsv4‐ and Rsv4‐genotype soybeans following biolistic or mechanical inoculations.

Materials and Methods S1 The details of viruses, soybean genotypes, inoculation, detection, construction of point mutants, RT‐PCR, sequencing and generation of polyclonal antibodies against bacterially expressed coat protein of SMV‐G7d.

Click here for additional data file. (39.3KB, zip)

Articles from Molecular Plant Pathology are provided here courtesy of Wiley

RESOURCES