Silencing of a meristematic gene using geminivirus-derived vectors.

Geminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome-mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana. Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down-regulated throughout meristematic tissues. Our results demonstrate that geminivirus-derived vectors can be used to study genes involved in meristem function in intact plants.

Proliferating cell nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves.

The geminivirus tomato golden mosaic virus (TGMV) amplifies its DNA genome in differentiated plant cells that lack detectable levels of DNA replication enzymes. Earlier studies showed that TGMV induces the accumulation of proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerase delta, in mature cells of Nicotiana benthamiana. We sought to determine if PCNA protein accumulation reflects transcriptional activation of the host gene. RNA gel blot analysis detected an approximately 1200-nucleotide PCNA transcript in young leaves. The same RNA was found in mature leaves of infected but not healthy plants. Reporter gene analysis showed that a 633-bp promoter fragment of the N. benthamiana PCNA gene supports high levels of expression in cultured cells and in young but not mature leaves of healthy transgenic plants. In contrast, PCNA promoter activity was detected in both young and mature leaves of TGMV-infected plants. Developmental studies established a strong relationship between symptom severity, viral DNA accumulation, PCNA promoter activity, and endogenous PCNA mRNA levels. Mutation of an E2F consensus element in the PCNA promoter had no effect on its activity in young leaves but increased transcription in healthy mature leaves. Unlike the wild-type PCNA promoter, TGMV infection had no detectable effect on the activity of the mutant E2F promoter. Together, these results demonstrate that geminivirus infection induces the accumulation of a host replication factor by activating transcription of its gene in mature tissues, most likely by overcoming E2F-mediated repression.

Biosynthetic studies on the alpha-glucosidase inhibitor acarbose in Actinoplanes sp.: source of the maltose unit.

To investigate the source of the maltose unit in acarbose, feeding experiments using 3H- or 2H-labeled maltose or maltotriose were carried out with resting cells of Actinoplanes sp. SN223/29. It was found by experiments with [6"-3H]- and [1-3H]maltotriose that a maltose unit from the nonreducing end of maltotriose is incorporated into acarbose more efficiently than from the reducing end. However, experiments with [6"-2H]- and [2-2H]maltotriose showed that maltose from either the reducing end or from the nonreducing end of maltotriose was incorporated into acarbose. The results established that acarbose is formed from maltotriose by two routes; (1) Sixty percent of the acarbose are formed by attachment of maltose, produced by removing a glucose exclusively from the nonreducing end of maltotriose, to the pseudodisaccharide core unit. (2) The other 40% of the acarbose are formed by direct attachment of maltotriose to the core unit followed by loss of the terminal glucose from the reducing end. Furthermore, it was observed that there is no scrambling of label between the two glucose moieties of acarbose, that maltotriose is a comparably efficient precursor of acarbose as is maltose, and that the core unit is enriched up to 50% from the 2H-glucose liberated from the deuterated maltotrioses.

A family of wound-induced genes in Populus shares common features with genes encoding vegetative storage proteins.

Two wound-inducible cDNAs from poplar leaves show sequence identity to vegetative storage proteins (VSP) that accumulate seasonally in poplar bark tissues. We have compared the genomic organization, cDNA sequences and expression of the genes encoding the wound-inducible cDNAs (win4) with that of a bark VSP (called bark storage protein, or BSP). There appear to be several win4 genes in the poplar genome which segregate as a single locus and are therefore likely to be clustered. The same is true of the BSP genes. The win4 locus is linked (map distance of 5 cM) to the BSP locus, consistent with a common evolutionary origin of the genes. A near full-length win4 cDNA shows 75% sequence identity to BSP cDNAs. Both win4 and BSP are systemically wound-inducible; win4 transcripts accumulate in leaves and stems, whereas BSP transcripts accumulate almost exclusively in stems. A phloem transport-dependent signaling mechanism appears to be involved in systemic win4 expression after wounding. In contrast to BSP gene expression, win4 genes are not expressed in response to short day conditions. The data indicate win4 and BSP genes are differentially regulated, and their products may play important roles in the storage and reallocation of nitrogen in perennial plants.