Virus-induced gene silencing (VIGS) as a reverse genetic tool to study development of symbiotic root nodules.

Virus-induced gene silencing (VIGS) can provide a shortcut to plants with altered expression of specific genes. Here, we report that VIGS of the Nodule inception gene (Nin) can alter the nodulation phenotype and Nin gene expression in Pisum sativum. PsNin was chosen as target because of the distinct non-nodulating phenotype of nin mutants in P. sativum, Lotus japonicus, and Medicago truncatula. The vector based on Pea early browning virus (PEBV) was engineered to carry one of three nonoverlapping fragments (PsNinA, PsNinB, and PsNinC) derived from the PsNin cDNA. Vector inoculation was mediated by agroinfiltration and, 2 weeks later, a Rhizobium leguminosarum bv. viceae culture was added in order to induce root nodulation. At this time point, it was estimated that systemic silencing was established because leaves of reference plants inoculated with PEBV carrying a fragment of Phytoene desaturase displayed photo bleaching. Three weeks after Rhizobium spp. application, plants inoculated with a control vector nodulated normally, whereas nodulation was almost eliminated in plants inoculated with a vector carrying PsNinA and PsNinC. For plants inoculated with a vector carrying PsNinB, nodulation was reduced by at least 45%. Down-regulation of PsNin transcripts in plants inoculated with vectors carrying PsNin cDNA fragments was confirmed and these plants displayed a relative increase in the root/shoot ratio, as expected if nitrogen fixation had been impaired.

The same allele of translation initiation factor 4E mediates resistance against two Potyvirus spp. in Pisum sativum.

Pathogenicity of two sequenced isolates of Bean yellow mosaic virus (BYMV) was established on genotypes of Pisum sativum L. reported to carry resistance genes to BYMV and other potyviruses. Resistance to the white lupin strain of BYMV (BYMV-W) is inherited as a recessive gene named wlv that maps to linkage group VI together with other Potyvirus resistances. One of these, sbm1, confers resistance to strains of Pea seedborne mosaic virus and previously has been identified as a mutant allele of the eukaryotic translation initiation factor 4E gene (eIF4E). Sequence comparison of eIF4E from BYMV-W-susceptible and -resistant P. sativum genotypes revealed a polymorphism correlating with the resistance profile. Expression of eIF4E from susceptible plants in resistant plants facilitated BYMV-W infection in inoculated leaves. When cDNA of BYMV-W was agroinoculated, resistance mediated by the wlv gene frequently was overcome, and virus from these plants had a codon change causing an Arg to His change at position 116 of the predicted viral genome-linked protein (VPg). Accordingly, plants carrying the wlv resistance gene were infected upon inoculation with BYMV-W derived from cDNA with a His codon at position 116 of the VPg coding region. These results suggested that VPg determined pathogenicity on plants carrying the wlv resistance gene and that wlv corresponded to the sbm1 allele of eIF4E.

Mutations in pea seedborne mosaic virus genome-linked protein VPg after pathotype-specific virulence in Pisum sativum.

Pisum sativum plant introduction (PI) line 269818 is resistant to potyvirus pea seedborne mosaic virus (PSbMV) isolates, categorized as pathotype P1, and is susceptible to pathotype P4 isolates. This difference in infectivity is determined by the viral genome-linked protein (VPg) cistron. Mutational analysis of VPg of PSbMV isolates DPD1 and NY representing pathotypes P1 and P4 revealed that codon changes affecting amino acids 105 to 117 in the central region of VPg influenced virulence on PI 269818. In contrast, infectivity on pea cultivar Dark Skinned Perfection, which is susceptible to both pathotypes, was not affected by the mutations. Mutants overcoming resistance in PI 269818 were analyzed for changes in the VPg coding region upon passage through PI 269818 and Dark Skinned Perfection. Adaptive changes were observed only upon passage through PI 269818 and only at codons from amino acid 105 to 117. Expression of DPD1 VPg in PI 269818 did not affect infection by NY, which suggests that VPg from DPD1 is not an elicitor of a general resistance response. The results are compatible with the hypothesis that viral amplification depends upon the interaction between VPg and a host factor.

Recessive resistance in Pisum sativum and potyvirus pathotype resolved in a gene-for-cistron correspondence between host and virus.

Pea seed-borne mosaic potyvirus (PSbMV) isolates are divided into pathotypes P-1, P-2, and P-4 according to their infection profile on a panel of Pisum sativum lines. P. sativum PI 269818 is resistant to P-1 and P-2 isolates and is susceptible to P-4 isolates. Resistance to P-1 is inherited as a single recessive gene, denoted sbm-1, and the pathogenicity determinant has previously been mapped to the virus-coded protein VPg. In the cultivar Bonneville, a second recessive gene, sbm-2, confers specific resistance to P-2. By exchanging cistrons between a P-2 and a P-4 isolate, the P3-6k1 cistron was identified as the PSbMV host-specific pathogenicity determinant on Bonneville. Exchange of P3-6k1 did not affect infection on PI 269818, and infection of Bonneville was not altered by substitution of the VPg cistron, indicating that P3-6k1 and VPg are independent determinants of pathotype-specific infectivity. On PI 269818 the pathogenicity determinant of both P-1 and P-2 mapped to the N terminus of VPg. This suggests that VPg from the P-1 and P-2 isolates are functionally similar on this host and that resistance to P-1 and P-2 in PI 269818 may operate by the same mechanism. Identification of VPg-sbm-1 and P3-6k1-sbm-2 as independent pairs of genetic interactors between PSbMV and P. sativum provides a simple explanation of the three known pathotypes of PSbMV. Furthermore, analysis of beta-glucuronidase-tagged P-2 virus indicated that sbm-2 resistance affected an early step in infection, implying that the P3-6k1 region plays a critical role in potyvirus replication or cell-to-cell movement.

Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus.

The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.

A single conserved amino acid in the coat protein gene of pea seed-borne mosaic potyvirus modulates the ability of the virus to move systemically in Chenopodium quinoa.

Two isolates of pea seed-borne mosaic potyvirus, DPD1 and NY, which both infect pea (Pisum sativum) systemically, differ in their ability to move long distance in Chenopodium quinoa. DPD1 spreads to uninoculated leaves, whereas NY is restricted to the inoculated leaves. The NY isolate was found to move from cell to cell infecting all parts of the inoculated leaves, including the petiole. The coat protein (CP) coding region was identified as the determinant of long-distance movement. Virus chimeras containing the CP coding sequence of NY were restricted to inoculated leaves, whereas chimeras containing the CP coding sequence of DPD1 infected C. quinoa systemically. Mutational analysis of the CP demonstrated that changing the serine at position 47 of the NY CP to proline was sufficient to permit systemic spread of the NY(S47P) mutant. The reverse mutant, DPD1(P47S), in which the proline at position 47 of the CP was changed to serine, was restricted to inoculated leaves. The movement characteristics and CP sequences of 10 additional PSbMV isolates were determined. All isolates caused systemic infection in pea. In C. quinoa 6 of the isolates that were restricted to inoculated leaves had a serine at position 47. Two isolates that infected C. quinoa systemically had a proline at position 47. Two isolates, S6 and NEP-1, infected C. quinoa systemically, but had a serine at position 47 of the CP. This shows that although a proline/serine difference at position 47 of the CP determined systemic spread of the isolates DPD1 and NY, this amino acid alone does not govern the spread of PSbMV in C. quinoa.

Potyvirus genome-linked protein (VPg) determines pea seed-borne mosaic virus pathotype-specific virulence in Pisum sativum.

The mechanism of Pisum sativum pathotype-specific resistance to pea seed-borne mosaic potyvirus (PSbMV) was investigated and the coding region determinant of PSbMV virulence was defined. Homozygous recessive sbm-1 peas are unable to support replication of PSbMV pathotype 1 (P-1), whereas biochemically and serologically related pathotype 4 (P-4) is fully infectious in the sbm-1/sbm-1 genotype. We were unable to detect viral coat protein or RNA with double antibody sandwich-enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction in sbm-1/sbm-1 P-1-inoculated protoplasts and plants. Lack of viral coat protein or RNA in P-1 transfected sbm-1/sbm-1 protoplasts suggests that sbm-1 resistance is occurring at the cellular level and that inhibition of cell-to-cell virus movement is not the operating form of resistance. In addition, because virus products were not detected at any time post-inoculation, resistance must either be constitutive or expressed very early in the virus infection process. P-1-resistant peas challenged with full-length, infectious P-1/P-4 recombinant clones demonstrated that a specific P-4 coding region, the 21-kDa, genome-linked protein (VPg), was capable of overcoming sbm-1 resistance, whereas clones containing the P-1 VPg coding region were noninfectious to sbm-1/sbm-1 peas. VPg is believed to be involved in potyvirus replication and its identification as the PSbMV determinant of infectivity in sbm-1/sbm-1 peas is consistent with disruption of an early P-1 replication event.

Specificity of resistance to pea seed-borne mosaic potyvirus in transgenic peas expressing the viral replicase (Nlb) gene.

Transgenic pea lines carrying the replicase (NIb) gene of pea seed-borne mosaic potyvirus (PSbMV) were generated and used in experiments to determine the effectiveness of induced resistance upon heterologous isolates. Three pea lines showed inducible resistance in which an initial infection by the homologous isolate (PSbMV-DPD1) was followed by a highly resistant state. Resistance was observed in plants in either the homozygous or hemizygous condition and resulted in no overall yield loss despite the initial infection. Resistance was associated with a loss of both viral and transgene RNA, which is indicative of a mechanism based upon post-transcriptional gene silencing. There was no correlation between the steady-state levels of transgene RNA and ability of the plants to show resistance. To test the specificity of the resistance, plants were also inoculated with the most distantly related sequenced PSbMV isolate, NY. PSbMV-NY varied between experiments in its ability to induce resistance, suggesting that the sequence identity in the NIb gene is borderline for the specificity required for triggering gene silencing. Upon challenge inoculation of virus-free recovered leaves, the specificity of the induced resistance varied between the two isolates and indicated that the virus and transgene additively determined the resistant state. These results suggest that the sequence requirements for triggering gene silencing may differ from those involved in the degradation process.

Multiple viral determinants affect seed transmission of pea seedborne mosaic virus in Pisum sativum.

Two pea seedborne mosaic potyvirus (PSbMV) isolates, P-1 DPD1 (P-1), which is highly seed-transmitted, and P-4 NY (P-4), which is rarely seed-transmitted, and chimeras between P-1 and P-4 were analysed to map the viral genetic determinants of seed transmission. Infectivity of chimeric viruses was evaluated by inoculating Pisum sativum with RNA transcribed in vitro from recombinant full-length cDNA clones. The chimeric viruses that were used demonstrated that a genomic segment encoding the 49 kDa protease and putative RNA polymerase was responsible for symptom induction. Attempts to determine transmission of the chimeric viruses in P. sativum cultivars known to transmit P1 at high frequencies showed that seed transmission is a quantitative character influenced by multiple viral determinants. Seed transmission frequency did not correlate with accumulation of virus in vegetative tissue. The 5' 2.5 kb of the 10 kb PSbMV genome had a major influence on the seed transmission frequency and was analysed further. This showed that, while the helper-component protease was a major determinant of seed transmission, the potyviral P1 -protease exerted no measurable influence.

Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo.

Insertion of introns into cloned cDNA of two isolates of the plant potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E. coli. Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, were stable and easy to amplify in E. coli if one or more introns were inserted into the virus sequence. These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves. Examination of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.

Biological and molecular properties of a pathotype P-1 and a pathotype P-4 isolate of pea seed-borne mosaic virus.

Two isolates of pea seed-borne mosaic potyvirus, DPD1 and NY, were identified as pathotypes P-1 and P-4, respectively, by their infectivity on Pisum sativum L. lines homozygous for the recessive resistance genes sbm-1 and sbm-4. The two isolates differed in several biological characteristics. DPD1 induced transient vein clearing, downward rolling of leaflets and internode shortening on P. sativum, whereas NY only caused a slight growth reduction. DPD1 moved systemically in Chenopodium quinoa whereas NY was restricted to inoculated leaves. DPD1 was frequently transmitted by seeds whereas NY was rarely seed-transmitted: 24% and 0.3%, respectively, in P. sativum '549'. Both DPD1 and NY were transmitted by aphids (Myzus persicae), though a DAG triplet was not present in the N terminus of the coat protein. The nucleotide sequence and deduced amino acid sequence of NY were determined and compared to the corresponding sequences of DPD1.