Summary auto-generated
This study investigated two non-structural proteins encoded by pea early browning virus (PEBV) RNA2 that are involved in transmission by root-feeding nematode vectors. Researchers produced antibodies against the 29 kDa (29K) and 23 kDa (23K) proteins by expressing them in bacteria. Western blotting confirmed both proteins are present in leaves and roots of infected plants, with the 23K protein appearing to be glycosylated. A PEBV mutant lacking the entire 23K gene was constructed and found to cause striking chlorotic ringspot symptoms and stunting, unlike wild-type virus. This mutant remained capable of systemic infection but showed dramatically reduced nematode transmissibility (6 of 39 plants versus 18 of 20 for wild-type). The reduction in transmission was not due to decreased virus accumulation in roots or impaired 29K protein expression. Co-infection with wild-type virus partially restored transmission frequency and suppressed the chlorotic phenotype. These findings indicate the 23K protein contributes to but is not essential for vector transmission, suggesting it plays a supporting role in the nematode transmission process.
Key findings
- The PEBV 29K and 23K transmission proteins are expressed in both leaves and roots of infected plants, with temporal expression patterns consistent with their transmission function
- The 23K protein appears to be glycosylated in plant-infected tissue, as determined by periodate treatment of blotted proteins
- Complete deletion of the 23K gene drastically reduces nematode transmission frequency (15% versus 90% for wild-type) while maintaining systemic infection capability
- The 23K protein is important but not essential for nematode transmission, indicating it has a helper protein role rather than being absolutely required
- Reduced transmission of the 23K deletion mutant is not explained by decreased viral replication or impaired 29K protein expression
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Abstract
Pea early browning virus (PEBV) is transmitted between plants by root- feeding trichodorid nematodes. Mutagenesis studies have implicated two non-structural viral proteins in the transmission process. These two proteins [the 29 kDa ('29K') protein and the 23K protein] were expressed in bacteria and used to raise antibodies. In Western blotting experiments, the antibodies detected both of these virus proteins in leaves and roots of infected Nicotiana bethamiana and N. clevelandii plants. Periodate treatment of proteins transferred to nitrocellulose membranes suggested that the PEBV 23K protein may be glycosylated. A PEBV mutant was constructed lacking the complete 23K coding sequence. The mutant was able systemically to infect Nicotiana spp. but caused striking chlorotic ringspot leaf symptoms and stunting of both leaves and roots. These symptoms were absent in plants doubly-infected with the mutant and wild-type PEBV. The 23K gene deletion mutant was transmitted by nematodes at a much reduced frequency compared to wild- type virus, indicating that the 23K protein is involved in but not essential for vector transmission. Western immuno-blot and ELISA experiments revealed that the reduction in the nematode- transmissibility of PEBV carrying mutations in the 23K gene did not result from interference in the expression of the 29K transmission protein or from gross changes in the titre of virus in the roots of infected plants.