Model System-Guided Protein Interaction Mapping for Virus Isolated from Phloem Tissue

Phloem localization of plant viruses is advantageous for acquisition by sap-sucking vectors but hampers host–virus protein interaction studies. In this study, Potato leafroll virus (PLRV)–host protein complexes were isolated from systemically infected potato, a natural host of the virus. Comparing two different co-immunoprecipitation (co-IP) support matrices coupled to mass spectrometry (MS), we identified 44 potato proteins and one viral protein (P1) specifically associated with virus isolated from infected phloem. An additional 142 proteins interact <i>in complex</i> with virus at varying degrees of confidence. Greater than 80% of these proteins were previously found to form high confidence interactions with PLRV isolated from the model host <i>Nicotiana benthamiana</i>. Bioinformatics revealed that these proteins are enriched for functions related to plasmodesmata, organelle membrane transport, translation, and mRNA processing. Our results show that model system proteomics experiments are extremely valuable for understanding protein interactions regulating infection in recalcitrant pathogens such as phloem-limited viruses

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Interaction between the cyclophilin protein and CYDV-RPV and PLRV purified virus.

<p>His-tag cyclophilin vector (CV) and nonvector (CNV) isoforms were expressed <i>in vitro</i> in <i>E. coli</i> and co-immunoprecipitated with CYDV-RPV or PLRV. Co-immunoprecipitated proteins were detected with anti-his antibodies. First lane shows the synthesized cyclophilin protein. Interactions were notable between both isoforms and CYDV-RPV but not between PLRV and the vector isoform.</p

Transmission efficiency is correlated to presence of vectoring cyclophilin allele in field-collected aphid biotypes.

<p>Transmission efficiency is calculated as the number of plants infected with virus out of the number of plants infested with viruliferous aphids (five aphids per plant, 12 plants used). +/- indicates the detection of the vectoring allele. Biotypes NY and H were heterozygous. Biotypes NY, F, G, and H efficiently transmitted CYDV-RPV whereas Biotyoes B, I, and Fl did not transmit at all. Biotypes C, K and Ks transmitted with poor efficiencies.</p

Normalized peak areas from two cyclophilin A peptides show enrichment in co-immunoprecipitation experiments using aphid proteins and purified CYDV-RPV.

<p>Whole insects of genotypes A3, C2 and biotype WY-10A (efficient vector biotype recently collected from a field in Wyoming) were subjected to cryogenic cell lysis and protein extraction. The extracted proteins were co-immonoprecipitated with purified CYDV-RPV using anti-RPV antibodies. Two peptides from cyclophilin were enriched in gentoypes A3, C2 and the field collected biotype WY-10A as compared to the control co-immunoprecipitation with no virus (aphid proteins incubated with beads and antibodies). Peptide FFDMTADGEQLR (2+ precursor <i>m/z</i> 793.369) was higher in intensity than the HTGPGILSMANAGANTNGSQFFTTVK peptide (3+ precursor <i>m/z</i> 912.871). This is not a good indicator of differences in relative abundance between the two peptides, which could result from different ionization efficiencies. However comparison of peak areas for each peptide across the various samples is an accurate way to measure relative abundance of the peptide in each sample. Both peptides showed similar trends in the experimental co-IPs compared to the control. Both peptides were more abundant in the co-IP reactions with virus. Although the peak areas showed an overall lower abundance in biotype WY10-A, which might reflect a lower overall expression of cyclophilin in this biotype as compared to the lab-reared F2 genotypes, this difference was not significant using a Kruskal Wallis test.</p

Discovery and Targeted LC-MS/MS of Purified Polerovirus Reveals Differences in the Virus-Host Interactome Associated with Altered Aphid Transmission

<div><p>Circulative transmission of viruses in the <em>Luteoviridae</em>, such as cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus, plant, and aphid proteins. Natural selection has favored these viruses to be retained in the phloem to facilitate acquisition and transmission by aphids. We show that treatment of infected oat tissue homogenate with sodium sulfite reduces transmission of the purified virus by aphids. Transmission electron microscopy data indicated no gross change in virion morphology due to treatments. However, treated virions were not acquired by aphids through the hindgut epithelial cells and were not transmitted when injected directly into the hemocoel. Analysis of virus preparations using nanoflow liquid chromatography coupled to tandem mass spectrometry revealed a number of host plant proteins co-purifying with viruses, some of which were lost following sodium sulfite treatment. Using targeted mass spectrometry, we show data suggesting that several of the virus-associated host plant proteins accumulated to higher levels in aphids that were fed on CYDV-infected plants compared to healthy plants. We propose two hypotheses to explain these observations, and these are not mutually exclusive: (a) that sodium sulfite treatment disrupts critical virion-host protein interactions required for aphid transmission, or (b) that host infection with CYDV modulates phloem protein expression in a way that is favorable for virus uptake by aphids. Importantly, the genes coding for the plant proteins associated with virus may be examined as targets in breeding cereal crops for new modes of virus resistance that disrupt phloem-virus or aphid-virus interactions.</p> </div

Summary of <i>R. padi</i> transmission and virion detection following membrane feeding of different cereal yellow dwarf virus<i>-</i>RPV preparations used for RT-PCR and TEM evaluations.

a<p>Purified virus that was used for RT-PCR and TEM analysis was tested for transmissibility using aphid transmission assays. Number of plants infected/total number of plants inoculated; total percent infection indicated.</p>b<p>Number of aphids with detectable amount of viral RNA/total number of aphids tested.</p>c<p>Number of aphids with virus in the HG cell/total number of aphids evaluated.</p