Replacement of P1 of soybean mosaic virus with P1 of clover yellow vein virus has no impact on virus viability and host specificity.

Potyvirus genomes are expressed as polyproteins that are autocatalytically cleaved to produce 10 to 12 multifunctional proteins, among which P1 is the most variable. It has long been hypothesized that P1 plays role(s) in host adaptation and host specificity. We tested this hypothesis using two phylogenetically distinct potyviruses: soybean mosaic virus (SMV), with a narrow host range, and clover yellow vein virus (ClYVV), with a broader host range. When the full-length P1 cistron of SMV-N was replaced with P1 from ClYVV-No.30, the chimera systemically infected only SMV-N-permissive hosts. Hence, there were no changes in the host range or host specificity of the chimeric viruses. Despite sharing only 20.3% amino acid sequence identity, predicted molecular models of P1 proteins from SMV-N and ClYVV-No.30 showed analogous topologies. These observations suggest that P1 of ClYVV-No.30 can functionally replace P1 of SMV-N. However, the P1 proteins of these two potyviruses are not determinants of host specificity and host range.

Detection and discovery of plant viruses in soybean by metagenomic sequencing.

BACKGROUND: Viruses negatively impact soybean production by causing diseases that affect yield and seed quality. Newly emerging or re-emerging viruses can also threaten soybean production because current control measures may not be effective against them. Furthermore, detection and characterization of new plant viruses requires major efforts when no sequence or antibody-based resources are available. METHODS: In this study, soybean fields were scouted for virus-like disease symptoms during the 2016-2019 growing seasons. Total RNA was extracted from symptomatic soybean parts, cDNA libraries were prepared, and RNA sequencing was performed using high-throughput sequencing (HTS). A custom bioinformatic workflow was used to identify and assemble known and unknown virus genomes. RESULTS: Several viruses were identified in single or mixed infections. Full- or nearly full-length genomes were generated for tobacco streak virus (TSV), alfalfa mosaic virus (AMV), tobacco ringspot virus (TRSV), soybean dwarf virus (SbDV), bean pod mottle virus (BPMV), soybean vein necrosis virus (SVNV), clover yellow vein virus (ClYVV), and a novel virus named soybean ilarvirus 1 (SIlV1). Two distinct ClYVV isolates were recovered, and their biological properties were investigated in Nicotiana benthamiana, broad bean, and soybean. In addition to infections by individual viruses, we also found that mixed viral infections in various combinations were quite common. CONCLUSIONS: Taken together, the results of this study showed that HTS-based technology is a valuable diagnostic tool for the identification of several viruses in field-grown soybean and can provide rapid information about expected viruses as well as viruses that were previously not detected in soybean.

Incidence, Geographical Distribution, and Genetic Diversity of Sugarcane Striate Virus in Saccharum Species in China.

A novel virus of the genus Mastrevirus, family Geminivirdae, has been reported in sugarcane germplasm collections in Florida, Guadeloupe, and Réunion, and was named sugarcane striate virus (SStrV). Although the full-length sequence of an SStrV isolate from China was obtained in 2015, the incidence, geographical distribution, and genetic diversity of this virus remained unclear. A single leaf sample from 2,368 sugarcane plants from main sugarcane-producing regions of China and germplasm collections were tested for SStrV by PCR. Average virus incidence was 25.1% for field-collected samples, and SStrV was detected in most Saccharum species and two sugarcane-related species, with the highest incidence in Saccharum officinarum (44.1%) followed by Saccharum spp. local varieties (33.3%) grown for chewing cane for a long time. The virus incidence was much lower (6.8%) in modern commercial cultivars (Saccharum spp. hybrids). Phylogenetic trees based on full-length genomes of 157 SStrV isolates revealed that Chinese isolates comprised strains A and B, but not C and D, that were reported in Florida, U.S.A. SStrV strain A was the most prominent (98.7%) and widespread strain in China and was further divided into eight subgroups. Almost half (45.6%) of the SStrV-positive samples from S. officinarum and Saccharum spp. local varieties were coinfected with sugarcane mosaic disease viruses or sugarcane yellow leaf virus. Interestingly, most of the plants infected by strain A of SStrV were asymptomatic. SStrV appears to be widespread in China, and its influence on chewing cane deserves further investigation.

Cultivated and Wild Grapevines in Tennessee Possess Overlapping but Distinct Virus Populations.

Viruses and viroids prevalent in a population of 42 wild grapevines (i.e., free-living, uncultivated grapevines; Vitis spp.) were compared with those in a population of 85 cultivated grapevines collected in Tennessee, United States by RNA sequencing analysis of pools of ribosomal RNA-depleted total RNA. The sequences of 10 viruses (grapevine fleck virus, grapevine leafroll-associated virus 2, grapevine rupestris stem pitting-associated virus, grapevine Syrah virus 1, grapevine vein-clearing virus, grapevine virus B, grapevine virus E, tobacco ringspot virus, tomato ringspot virus, and a novel nano-like virus) and two viroids (hop stunt viroid and grapevine yellow speckle viroid 1) were detected in both grapevine populations. Sequences of four viruses (grapevine associated tymo-like virus, grapevine leafroll-associated virus 3, grapevine red blotch virus, and grapevine virus H) were identified only from cultivated grapevines. High, moderate, and low numbers of sequence reads were identified only from wild grapevines for a novel caulimovirus, an enamovirus, and alfalfa mosaic virus, respectively. The presence of most virus sequences and both viroids was verified independently in the original samples by reverse-transcription PCR followed by Sanger sequencing. Comparison of viral sequences shared by both populations showed that cultivated and wild grapevines harbored distinct sequence variants, which suggests that there was limited virus movement between the two populations. Collectively, this study represents the first unbiased survey of viruses and viroids in both cultivated and wild grapevines within a defined geographic region.

Precise Exchange of the Helper-Component Proteinase Cistron Between Soybean mosaic virus and Clover yellow vein virus: Impact on Virus Viability and Host Range Specificity.

Soybean mosaic virus and Clover yellow vein virus are two definite species of the genus Potyvirus within the family Potyviridae. Soybean mosaic virus-N (SMV-N) is well adapted to cultivated soybean (Glycine max) genotypes and wild soybean (G. soja), whereas it remains undetectable in inoculated broad bean (Vicia faba). In contrast, clover yellow vein virus No. 30 (ClYVV-No. 30) is capable of systemic infection in broad bean and wild soybean; however, it infects cultivated soybean genotypes only locally. In this study, SMV-N was shown to also infect broad bean locally; hence, broad bean is a host for SMV-N. Based on these observations, it was hypothesized that lack of systemic infection by SMV-N in broad bean and by ClYVV-No. 30 in cultivated soybean is attributable to the incompatibility of multifunctional helper-component proteinase (HC-Pro) in these hosts. The logic of selecting the HC-Pro cistron as a target is based on its established function in systemic movement and being a relevant factor in host range specificity of potyviruses. To test this hypothesis, chimeras were constructed with precise exchanges of HC-Pro cistrons between SMV-N and ClYVV-No. 30. Upon inoculation, both chimeras were viable in infection, but host range specificity of the recombinant viruses did not differ from those of the parental viruses. These observations suggest that (i) HC-Pro cistrons from SMV-N and ClYVV-No. 30 are functionally compatible in infection despite 55.6 and 48.9% nucleotide and amino acid sequence identity, respectively, and (ii) HC-Pro cistrons from SMV-N and ClYVV-No. 30 are not the determinants of host specificity on cultivated soybean or broad beans, respectively.

Recessive Resistance Governed by a Major Quantitative Trait Locus Restricts Clover Yellow Vein Virus in Mechanically but Not Graft-Inoculated Cultivated Soybeans.

Clover yellow vein virus (ClYVV) infects and causes disease in legume plants. However, here, we found that ClYVV isolate No. 30 (ClYVV-No.30) inefficiently multiplied or spread via cell-to-cell movement in mechanically inoculated leaves of a dozen soybean (Glycine max) cultivars and resulted in failure to spread systemically. Soybean plants also had a similar resistance phenotype against additional ClYVV isolates. In contrast, all but one of 24 tested accessions of wild soybeans (G. soja) were susceptible to ClYVV-No.30. Graft inoculation of cultivated soybean TK780 with ClYVV-No.30-infected wild soybean B01167 scion resulted in systemic infection of the cultivated soybean rootstock. This suggests that, upon mechanical inoculation, the cultivated soybean inhibits ClYVV-No.30, at infection steps prior to the systemic spread of the virus, via vascular systems. Systemic infection of all F1 plants from crossing between TK780 and B01167 and of 68 of 76 F2 plants with ClYVV-No.30 indicated recessive inheritance of the resistance. Further genetic analysis using 64 recombinant inbred lines between TK780 and B01167 detected one major quantitative trait locus, designated d-cv, for the resistance that was positioned in the linkage group D1b (chromosome 2). The mapped region on soybean genome suggests that d-cv is not an allele of the known resistance genes against soybean mosaic virus.

A novel picornavirus-like genome from transcriptome sequencing of sugar beet cyst nematode represents a new putative genus.

Analysis of transcriptome sequence data from eggs and second-stage juveniles (J2s) of sugar beet cyst nematode (SBCN, Heterodera schachtii) identified the full-length genome of a positive-sense single-stranded RNA virus, provisionally named sugar beet cyst nematode virus 1 (SBCNV1). The SBCNV1 sequence was detected in both eggs and J2s, indicating its possible vertical transmission. The 9503-nucleotide genome sequence contains a single long open reading frame, which was predicted to encode a polyprotein with conserved domains for picornaviral structural proteins proximal to its amino terminus and RNA helicase, cysteine proteinase and RNA-dependent RNA polymerase (RdRp) conserved domains proximal to its carboxyl terminus, hallmarks of viruses belonging to the order Picornavirales. Phylogenetic analysis of the predicted SBCNV1 RdRp amino acid sequence indicated that the SBCNV1 sequence is most closely related to members of the family Secoviridae, which includes genera of nematode-transmitted plant-infecting viruses. SBCNV1 represents the first fully sequenced viral genome from SBCN.

The HC-Pro and P3 cistrons of an avirulent Soybean mosaic virus are recognized by different resistance genes at the complex Rsv1 locus.

The complex Rsv1 locus in soybean plant introduction (PI) 'PI96983' confers extreme resistance (ER) against Soybean mosaic virus (SMV) strain N but not SMV-G7 and SMV-G7d. Both the SMV helper-component proteinase (HC-Pro) and P3 cistrons can serve as avirulence factors recognized by Rsv1. To understand the genetics underlying recognition of the two cistrons, we have utilized two soybean lines (L800 and L943) derived from crosses between PI96983 (Rsv1) and Lee68 (rsv1) with distinct recombination events within the Rsv1 locus. L800 contains a single PI96983-derived member (3gG2) of an Rsv1-associated subfamily of nucleotide-binding leucine-rich repeat (NB-LRR) genes. In contrast, although L943 lacks 3gG2, it contains a suite of five other NB-LRR genes belonging to the same family. L800 confers ER against SMV-N whereas L943 allows limited replication at the inoculation site. SMV-N-derived chimeras containing HC-Pro from SMV-G7 or SMV-G7d gained virulence on L943 but not on L800 whereas those with P3 replacement gained virulence on L800 but not on L943. In reciprocal experiments, SMV-G7- and SMV-G7d-derived chimeras with HC-Pro replacement from SMV-N lost virulence on L943 but retained virulence on L800 whereas those with P3 replacement lost virulence on L800 while remaining virulent on L943. These data demonstrate that distinct resistance genes at the Rsv1 locus, likely belonging to the NB-LRR class, mediate recognition of HC-Pro and P3.

Experimental adaptation of an RNA virus mimics natural evolution.

Identification of virulence determinants of viruses is of critical importance in virology. In search of such determinants, virologists traditionally utilize comparative genomics between a virulent and an avirulent virus strain and construct chimeras to map their locations. Subsequent comparison reveals sequence differences, and through analyses of site-directed mutants, key residues are identified. In the absence of a naturally occurring virulent strain, an avirulent strain can be functionally converted to a virulent variant via an experimental evolutionary approach. However, the concern remains whether experimentally evolved virulence determinants mimic those that have evolved naturally. To provide a direct comparison, we exploited a plant RNA virus, soybean mosaic virus (SMV), and its natural host, soybean. Through a serial in vivo passage experiment, the molecularly cloned genome of an avirulent SMV strain was converted to virulent variants on functionally immune soybean genotypes harboring resistance factor(s) from the complex Rsv1 locus. Several of the experimentally evolved virulence determinants were identical to those discovered through a comparative genomic approach with a naturally evolved virulent strain. Thus, our observations validate an experimental evolutionary approach to identify relevant virulence determinants of an RNA virus.

Diagnostic Potential of Polyclonal Antibodies Against Bacterially Expressed Recombinant Coat Protein of Alfalfa mosaic virus.

Alfalfa mosaic virus (AMV), a pathogen of a wide range of plant species, including Glycine max (soybean), is poorly immunogenic. Polyclonal antibodies were generated against bacterially expressed recombinant coat proteins (rCPs) of two biologically distinct AMV strains in rabbits and compared with those raised against native and glutaraldehyde-treated virions of the same strains. Analyses showed that sera against rCPs had comparable antibody titers in indirect enzyme-linked immunosorbent assay with those raised against virions when soybean sap containing homologous viruses served as antigens. Polyclonal antibodies against rCPs were specific, sensitive, and detected all AMV isolates that originated from soybean fields from geographically different regions of the United States. Comparison of CP genes of these isolates showed 96 to 99 and 96 to 100% nucleotide and amino acid sequence identities, respectively, suggesting that they are all closely related. This was further confirmed by phylogenetic analysis where they were all clustered together along with representative AMV strains belonging to group I. Collectively, our data demonstrate that, despite poor immunogenicity of AMV, polyclonal antibodies against rCP are effective probes for detection and diagnosis of the virus.

Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation.

Despite their economic significance in agricultural cropping systems, a lack of suitable molecular tools for manipulating gene expression has hindered progress in the functional genomics of plant parasitic nematodes (PPN). Obligate sexual reproduction and the obligate nature of PPN-host interactions further complicate the development of in vivo gene delivery and expression systems in these pests. Methods such as microinjection and microprojectile bombardment have been developed for introducing gene constructs into the free-living nematode, Caenorhabditis elegans. However, these procedures can be laborious and inefficient. Electroporation has been used extensively to introduce macromolecules, including single-stranded RNAs, into eukaryotic and prokaryotic cells. The technique has also been used for the delivery of DNA and double-stranded RNA constructs into nematodes by whole-animal electroporation. Here, we describe methods for the expression of a nematode-optimized NanoLuc luciferase mRNA in the form of in vitro transcripts following whole-animal electroporation of Heterodera glycines, Meloidogyne incognita, and C. elegans. The ability to transiently express single-stranded RNA constructs in economically important PPN provides a rapid means to evaluate nematode and/or foreign genes for their biological significance and potential role in nematode management.

Molecular characterization of a new Tospovirus infecting soybean.

A new, widespread disease was recently observed in soybean in the United States. The disease, named Soybean vein necrosis, is manifested by intraveinal chlorosis and necrosis, and has been found in almost all of the 50 fields visited over a period of 3 years in the midwest and midsouth part of the United States. A virus was isolated from symptomatic material, and detection protocols were developed. More than 150 symptomatic specimens collected from seven US States were tested, and all were found positive for the virus unlike 75 asymptomatic samples, revealing the absolute association between virus and disease. Protein pairwise comparisons coupled with phylogenetic analyses indicate that the virus is a new member of the genus Tospovirus.

Occurrence of Alfalfa mosaic virus in Soybean in Tennessee.

Alfalfa mosaic virus (AMV), a member of the genus Alfamovirus in the family Bromoviridae, naturally infects a wide range of plant species (1). Soybean (Glycine max (L.) Merr.) has seldom been reported as a natural host of AMV and there are limited reports of detection of AMV in field-grown soybean plants (4). However, AMV incidence in soybean fields in the midwestern United States has been on the rise in recent years, which is partly attributed to the introduction of the soybean aphid (Aphis glycines) (1,4). In June 2009, soybean plants of cv. Lee68 exhibiting moderate leaf distortion, mottling, and stunting were observed at the East Tennessee Research and Education Center. Leaf samples from 18 symptomatic plants were collected and the sap was extracted and analyzed by antigen-coated indirect ELISA (3) with polyclonal antibodies against AMV, Soybean mosaic virus (SMV), and Bean pod mottle virus (BPMV). None of the samples tested positive for BPMV, but all were found to be infected with SMV. Sap extract from 1 of 18 plants tested positive for AMV and SMV. Sap from this infected plant ground in 10 mM phosphate buffer, pH 7.0, was mechanically inoculated to Carborundum-dusted unifoliate leaves of PI96983, which contains the dominant Rsv1-locus conferring functional immunity to a majority of SMV strains (2). AMV, not SMV, was detected by ELISA in the systemically infected trifoliolate leaves that exhibited moderate mottling, mild leaf distortion, and stunting 14 days postinoculation. Sap was extracted from the infected tissues and the virus was passaged four times through PI96983 before being inoculated to Phaseolus vulgaris cv. Blue Lake. A local lesion isolate was obtained following three successive passages in this host and the isolate was propagated in soybean cv. Williams82. The biologically purified isolate was capable of infecting soybean cvs. L78-379 (Rsv1), L81-4420 (Rsv1), L29 (Rsv3), V94-5152 (Rsv4), Lee68, and Colfax upon sap inoculation. The infected plants exhibited a range of systemic symptoms including mottling, leaf distortion, necrosis, chlorosis, and moderate stunting. To characterize the virus further, total RNA was extracted from infected Williams82 leaf tissues with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). The RNA served as a template for cDNA synthesis in the presence of random primers. The resultant cDNA served as a template in a PCR assay with primers 1193 (forward) (5'-AGCTGAATTCATGAGTTCTTCACAAC-3') and 1858 (reverse) (5'-GCTAGCGGCCGCTCAATGACGATC-3') corresponding to nucleotides 1,193 to 1,210 and 1,858 to 1,840 of RNA3 from AMV-Kr (GenBank Accession No. AB126032), respectively. The amplified fragments were purified and directly sequenced bidirectionally using the same primers. BLAST analysis of the resultant nucleotide sequences showed 98% identity to an AMV isolate from a naturally infected soybean plant in Illinois (GenBank Accession No. HQ185569), and 97% identity to an isolate described from P. vulgaris in the United States (GenBank Accession No. AY340070.1). To our knowledge, this is the first report of natural infection of soybean by AMV in Tennessee. References: (1) J. Bol. Mol. Plant Pathol. 4:1, 2003. (2) M. R. Hajimorad and J. H. Hill. Mol. Plant-Microbe Interact. 14:587, 2001. (3) M. Malapi-Nelson et al. Plant Dis. 93:1259, 2009. (4) E. E. Mueller and C. R. Grau. Plant Dis. 91:266, 2007.

De novo emergence of a novel satellite RNA of cucumber mosaic virus following serial passages of the virus derived from RNA transcripts.

Satellite RNA (satRNA) is often associated with cucumber mosaic virus (CMV); however, its origin remains unexplained and a subject for speculation. We passaged progeny of molecularly cloned CMV-Fny and CMV-LS in Nicotiana tabacum cv. Ky 14 under greenhouse conditions. A satRNA emerged after at least eight successive transfers of CMV-Fny, but no satRNA was recovered after eleven serial transfers of CMV-LS under the same conditions. The sequences of the newly emerged satRNA were determined, and an infectious cDNA clone was synthesized. Comparison of the sequences of the newly emerged satRNA with those of known CMV satRNAs showed that it is unique. This observation raises interesting questions regarding the enigmatic nature of the origin of CMV satRNAs.

Genetic variability and phylogenetic analysis of hosta virus X.

Triple gene block 1 (TGB1) and coat protein (CP) sequences of 30 hosta virus X (HVX) isolates from Tennessee (TN), USA, were determined and compared with available sequences in GenBank. The CPs of all known HVX isolates, including those from TN, shared 98.3-100% and 98.2-100% nucleotide and amino acid sequence identity, respectively, whereas TGB1 shared 97.4-100% nucleotide and 97-100% amino acid sequence identity. TGB1 of TN isolates were all longer by one codon from that of a Korean isolate, which is the only sequence publicly available. Phylogenetic analysis of nucleotide and amino acid sequences of TGB1 and CP of all known HVX isolates, separately or combined, revealed a close relationship, suggesting that all of them are derived from a common ancestor. Phylogenetic analysis with the type member of each genus of the family Flexiviridae confirmed that HVX is a member of a distinct species of the genus Potexvirus.

Co-infection of Soybean with Soybean mosaic virus and Alfalfa mosaic virus Results in Disease Synergism and Alteration in Accumulation Level of Both Viruses.

Co-infection of potyviruses with taxonomically diverse plant viruses results in disease synergism and elevation in the level of accumulation of non-potyviruses involved. In the majority of cases, however, the accumulation level of potyviruses remains essentially unaltered. A few potyviruses, such as Soybean mosaic virus (SMV), naturally infect soybean (Glycine max). Soybean is also a natural host to a number of non-potyviruses including Alfalfa mosaic virus (AMV), which causes mild symptoms often associated with symptom remission. We have now studied the interactions between AMV and SMV on symptom severity and accumulation level of each of the two viruses in soybean. Co-infection of soybean with AMV and SMV was established following mechanical inoculation, irrespective of simultaneous or sequential introduction of the two viruses. In multiple experiments, co-infection of soybean resulted in severe symptoms in doubly infected plants in a strain-independent manner, with enhancement in the level of AMV indicating that the interaction of AMV with SMV is synergistic. Conversely, the level of SMV accumulation was reduced. This suggests that in co-infection with AMV, SMV interacts antagonistically. The observation that co-infection of AMV and SMV results in disease synergism suggests enhancement of potential that AMV may become a serious viral disease of soybean.

Gain of virulence on Rsv1-genotype soybean by an avirulent Soybean mosaic virus requires concurrent mutations in both P3 and HC-Pro.

In soybean, Rsv1, a single dominant resistance gene, invokes extreme resistance (ER) against most Soybean mosaic virus (SMV) strains, including SMV-N, but not SMV-G7, which provokes a virulent lethal systemic hypersensitive response (LSHR). The elicitor functions of the two viruses provoking Rsv1-mediated ER and LSHR have been mapped to the N-terminal 271 amino acids of P3 from SMV-N and SMV-G7, respectively, which differ by nine residues between the two strains. To identify amino acids of P3 from SMV-N provoking Rsv1-mediated ER, the unique residues of SMV-G7 were substituted with those of SMV-N. Of the mutants tested on Rsv1-genotype soybean, only SMV-G7(I788R) and SMV-G7(T948A) lost virulence. However, substitution of amino acids of SMV-N, individually or in combination, with the reciprocal residues from SMV-G7 at these two positions failed to confer virulence to SMV-N. In the search for additional virulence determinants, a series of SMV-N chimeras was generated in which fragments within a region from near the middle of the helper-component proteinase (HC-Pro) cistron to the 5' end of the cytoplasmic inclusion cistron, nucleotides 1,605 to 3,787, were replaced with those of SMV-G7. Only SMV-N-derived chimeras harboring the 3' region of HC-Pro, at least from nucleotide 2,013, and the entire 5' end of P3 (nucleotides 2,430 to 3,237) from SMV-G7 were virulent whereas reciprocal exchanges resulted in loss of SMV-G7 virulence. This region of HC-Pro differs by three amino acids between SMV-N and SMV-G7. Analyses of SMV-G7-derived HC-Pro site-directed mutants showed that only SMV-G7(M683R) lost virulence on Rsv1-genotype soybean; however, SMV-N(R682M) failed to gain virulence. Nevertheless, an SMV-N derived mutant with three concurrent substitutions, R682M+R787I+A947T, gained virulence. The data indicate that both P3 and HC-Pro are involved in virulence of SMV on Rsv1-genotype soybean.

Adaptation of Soybean mosaic virus avirulent chimeras containing P3 sequences from virulent strains to Rsv1-genotype soybeans is mediated by mutations in HC-Pro.

In Rsv1-genotype soybean, Soybean mosaic virus (SMV)-N (an avirulent isolate of strain G2) elicits extreme resistance (ER) whereas strain SMV-G7 provokes a lethal systemic hypersensitive response (LSHR). SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. Thus, for Rsv1-genotype soybean, SMV-N is avirulent whereas SMV-G7 and SMV-G7d are both virulent. Exploiting these differential interactions, we recently mapped the elicitor functions of SMV provoking Rsv1-mediated ER and LSHR to the N-terminal 271 amino acids of P3 from SMV-N and SMV-G7, respectively. The phenotype of both SMV-G7 and SMV-G7d were rendered avirulent on Rsv1-genotype soybean when the part of the genome encoding the N-terminus or the entire P3 cistron was replaced with that from SMV-N; however, reciprocal exchanges did not confer virulence to SMV-N-derived P3 chimeras. Here, we describe virulent SMV-N-derived P3 chimeras containing the full-length or the N-terminal P3 from SMV-G7 or SMV-G7d, with or without additional mutations in P3, that were selected on Rsv1-genotype soybean by sequential transfers on rsv1 and Rsv1-genotype soybean. Sequence analyses of the P3 and helper-component proteinase (HC-Pro) cistrons of progeny recovered from Rsv1-genotype soybean consistently revealed the presence of mutations in HC-Pro. Interestingly, the precise mutations in HC-Pro required for the adaptation varied among the chimeras. No mutation was detected in the HC-Pro of progeny passaged continuously in rsv1-genotype soybean, suggesting that selection is a consequence of pressure imposed by Rsv1. Mutations in HC-Pro alone failed to confer virulence to SMV-N; however, reconstruction of mutations in HC-Pro of the SMV-N-derived P3 chimeras resulted in virulence. Taken together, the data suggest that HC-Pro complementation of P3 is essential for SMV virulence on Rsv1-genotype soybean.

Soybean mosaic virus: a successful potyvirus with a wide distribution but restricted natural host range.

TAXONOMY: Soybean mosaic virus (SMV) is a species within the genus Potyvirus, family Potyviridae, which includes almost one-quarter of all known plant RNA viruses affecting agriculturally important plants. The Potyvirus genus is the largest of all genera of plant RNA viruses with 160 species. PARTICLE: The filamentous particles of SMV, typical of potyviruses, are about 7500 Å long and 120 Å in diameter with a central hole of about 15 Å in diameter. Coat protein residues are arranged in helices of about 34 Å pitch having slightly less than nine subunits per turn. GENOME: The SMV genome consists of a single-stranded, positive-sense, polyadenylated RNA of approximately 9.6 kb with a virus-encoded protein (VPg) linked at the 5' terminus. The genomic RNA contains a single large open reading frame (ORF). The polypeptide produced from the large ORF is processed proteolytically by three viral-encoded proteinases to yield about 10 functional proteins. A small ORF, partially overlapping the P3 cistron, pipo, is encoded as a fusion protein in the N-terminus of P3 (P3N + PIPO). BIOLOGICAL PROPERTIES: SMV's host range is restricted mostly to two plant species of a single genus: Glycine max (cultivated soybean) and G. soja (wild soybean). SMV is transmitted by aphids non-persistently and by seeds. The variability of SMV is recognized by reactions on cultivars with dominant resistance (R) genes. Recessive resistance genes are not known. GEOGRAPHICAL DISTRIBUTION AND ECONOMIC IMPORTANCE: As a consequence of its seed transmissibility, SMV is present in all soybean-growing areas of the world. SMV infections can reduce significantly seed quantity and quality (e.g. mottled seed coats, reduced seed size and viability, and altered chemical composition). CONTROL: The most effective means of managing losses from SMV are the planting of virus-free seeds and cultivars containing single or multiple R genes. KEY ATTRACTIONS: The interactions of SMV with soybean genotypes containing different dominant R genes and an understanding of the functional role(s) of SMV-encoded proteins in virulence, transmission and pathogenicity have been investigated intensively. The SMV-soybean pathosystem has become an excellent model for the examination of the genetics and genomics of a uniquely complex gene-for-gene resistance model in a crop of worldwide importance.

Gain of virulence by Soybean mosaic virus on Rsv4-genotype soybeans is associated with a relative fitness loss in a susceptible host.

'Gene-for-gene' theory predicts that gain of virulence by an avirulent pathogen on plants expressing resistance (R) genes is associated with fitness loss in susceptible hosts. However, the validity of this prediction has been studied in only a few plant viral pathosystems. In this study, the Soybean mosaic virus (SMV)-Rsv4 pathosystem was exploited to test this prediction. In Rsv4-genotype soybeans, P3 of avirulent SMV strains provokes an as yet uncharacterized resistance mechanism that restricts the invading virus to the inoculated leaves. A single amino acid substitution in P3 functionally converts an avirulent to a virulent strain, suggesting that the genetic composition of P3 plays a crucial role in virulence on Rsv4-genotype soybeans. In this study, we examined the impact of gain of virulence mutation(s) on the fitness of virulent variants derived from three avirulent SMV strains in a soybean genotype lacking the Rsv4 gene. Our data demonstrate that gain of virulence mutation(s) by all avirulent viruses on Rsv4-genotype soybean is associated with a relative fitness loss in a susceptible host. The implications of this finding on the durable deployment of the Rsv4 gene in soybean are discussed.