Biomedical subjects

Linda Hanley-Bowdoin

Publications and source records attributed to Linda Hanley-Bowdoin.

7 recordsLinked to original sources

PubMed2006

Geminivirus infection up-regulates the expression of two Arabidopsis protein kinases related to yeast SNF1- and mammalian AMPK-activating kinases.

Geminivirus Rep-interacting kinase 1 (GRIK1) and GRIK2 constitute a small protein kinase family in Arabidopsis (Arabidopsis thaliana). An earlier study showed that a truncated version of GRIK1 binds to the geminivirus replication protein AL1. We show here both full-length GRIK1 and GRIK2 interact with AL1 in yeast two-hybrid studies. Using specific antibodies, we showed that both Arabidopsis kinases are elevated in infected leaves. Immunoblot analysis of healthy plants revealed that GRIK1 and GRIK2 are highest in young leaf and floral tissues and low or undetectable in mature tissues. Immunohistochemical staining showed that the kinases accumulate in the shoot apical meristem, leaf primordium, and emerging petiole. Unlike the protein patterns, GRIK1 and GRIK2 transcript levels only show a small increase during infection and do not change significantly during development. Treating healthy seedlings and infected leaves with the proteasome inhibitor MG132 resulted in higher GRIK1 and GRIK2 protein levels, whereas treatment with the translation inhibitor cycloheximide reduced both kinases, demonstrating that their accumulation is modulated by posttranscriptional processes. Phylogenetic comparisons indicated that GRIK1, GRIK2, and related kinases from Medicago truncatula and rice (Oryza sativa) are most similar to the yeast kinases PAK1, TOS3, and ELM1 and the mammalian kinase CaMKK, which activate the yeast kinase SNF1 and its mammalian homolog AMPK, respectively. Complementation studies using a PAK1/TOS3/ELM1 triple mutant showed that GRIK1 and GRIK2 can functionally replace the yeast kinases, suggesting that the Arabidopsis kinases mediate one or more processes during early plant development and geminivirus infection by activating SNF1-related kinases.

AMP-Activated Protein Kinase Kinases↗

PubMed2006

Peptide aptamers that bind to a geminivirus replication protein interfere with viral replication in plant cells.

The AL1 protein of tomato golden mosaic virus (TGMV), a member of the geminivirus family, is essential for viral replication in plants. Its N terminus contains three conserved motifs that mediate origin recognition and DNA cleavage during the initiation of rolling-circle replication. We used the N-terminal domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 protein. Plant expression cassettes corresponding to the TrxA peptides and a TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA levels to 13 to 64% of those of the wild type. All of the interfering aptamers also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 20-mer peptides revealed that their sequences are not random. The alignments detected seven potential binding motifs, five of which are more highly represented among the interfering peptides. One motif was present in 18 peptides, suggesting that these peptides interact with a hot spot in the AL1 N terminus. The peptide aptamers characterized in these studies represent new tools for studying AL1 function and can serve as the basis for the development of crops with broad-based resistance to single-stranded DNA viruses.

Aptamers, Peptide↗

PubMed2005

Geminivirus C3 protein: replication enhancement and protein interactions.

Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants.

Amino Acid Sequence↗

PubMed2004

A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein.

The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) to modulate host gene expression. The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBR through an 80-amino-acid region that contains two highly predicted alpha-helices designated 3 and 4. Earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in pRBR binding. We generated a series of alanine substitutions across helix 4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays. These experiments showed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being a leucine at position 148 in the middle of the motif. Various amino acid substitutions at leucine-148 indicated that both structural and side chain components contribute to pRBR binding. The replication proteins of the geminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeast dihybrid assays. Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding. Together, these results suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface in begomovirus replication proteins.

Amino Acid Motifs↗

PubMed2002

A geminivirus replication protein interacts with a protein kinase and a motor protein that display different expression patterns during plant development and infection.

The geminivirus protein AL1 initiates viral DNA replication, regulates its own expression, and induces plant gene transcription. To better understand how AL1 interacts with host proteins during these processes, we used yeast two-hybrid library screening and a baculovirus protein interaction system to identify plant proteins that interact with AL1. These studies identified a Ser/Thr kinase, a kinesin, and histone H3 as AL1 partners. The kinase is autophosphorylated and can phosphorylate common kinase substrates in vitro. The kinesin is phosphorylated in insect cells by a cyclin-dependent kinase. Immunostaining of Nicotiana benthamiana and Arabidopsis showed that kinase protein levels and subcellular location are regulated during plant development and geminivirus infection. By contrast, the kinesin is ubiquitous even though it is associated with the spindle apparatus in mitotic cells. Together, our results establish that AL1 interacts with host proteins involved in plant cell division and development. Possible functions of these host factors in healthy and geminivirus-infected plants are discussed.

Animals↗

PubMed2002

Host DNA replication is induced by geminivirus infection of differentiated plant cells.

The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.

Aphidicolin↗

PubMed2002

Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development.

E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes. Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a Nicotiana benthamiana gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element (E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal element (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nuclear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites. Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported previously for the E2F2 mutation, the E2F1 and E2F1+2 mutations partially relieved the repression of the PCNA promoter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activity, whereas the E2F1+2 mutation had no detectable effect. The activity of E2F1+2 mutants was indistinguishable from that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the PCNA promoter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.

Base Sequence↗