Increased Occurrence of Target Spot of Soybean Caused by Corynespora cassiicola in the Southeastern United States.

Target spot of soybean (Glycine max (L.) Merr.) caused by Corynespora cassiicola (Berk. & Curt.), although found in most soybean-growing countries, is considered to be a disease of limited importance (1) and has never been reported to cause soybean yield loss in the southeastern United States (2,3). Soybean plants submitted to the North Carolina Plant Disease and Insect Clinic (NCPDIC) in August 2004 from Beaufort, Robeson, Wilson, and Johnston counties, NC had symptoms consistent with target spot. Symptoms consisted of roughly circular, necrotic leaf lesions from minute to 11 mm in diameter, though typically approximately 4 to 5 mm in diameter, and with a yellow margin. Large lesions occasionally exhibited a zonate pattern often associated with this disease. Microscopic examination of the lesions revealed the presence of spores (conidia) typical of C. cassiicola (1). Conidia were mostly three to five septate with a central hilum at the base and ranged in size from 7 to 22 wide × 39 to 520 µm long. Three commercial soybean fields near Blackville, SC (Barnwell County) were severely affected by this disease and it caused premature defoliation. Nineteen of twenty-seven maturity group VII and VIII genotypes in the 2004 Clemson University soybean variety trial near Blackville, SC had visible symptoms of target spot. Heavy rainfall associated with hurricanes during September 2004 probably enhanced the incidence of this disease, and yield suppression due to target spot was estimated at 20 to 40% in some fields. In 2005, 20 of 161 soybean samples submitted to the NCPDIC or collected in surveys from 16 counties were positive for target spot on the basis of microscopic examination. Target spot also was diagnosed in six counties (Baldwin, DeKalb, Elmore, Fayette, Macon, and Pickens) in Alabama and in four additional counties (Bamberg, Hampton, Orange-burg, and Calhoun) in South Carolina in 2005. Records from the NCPDIC indicate that target spot had not been diagnosed on soybean in North Carolina since 1981. The large increase in incidence of target spot in the southeast may be related to changes in weather patterns, changes in pathogen virulence, and/or the introduction of more susceptible host genotypes. References: (1) J. B. Sinclair. Target spot. Page 27 in: Compendium of Soybean Diseases. G. L. Hartman et al. eds. The American Phytopathological Society, St. Paul, MN, 1999. (2) J. A. Wrather et al. Plant Dis. 79:1076. 1995. (3) J. A. Wrather et al. On-line publication. doi:10.1094/PHP-2003-0325-01-RV. Plant Health Progress, 2003.

Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells.

Exploiting the lytic life cycle of viruses has gained recent attention as an anticancer strategy (oncolysis). To explore the utility of adenovirus (Ad)-mediated oncolysis for rhabdomyosarcoma (RMS), we tested RMS cell lines for Ad gene transduction and infection. RMS cells were variably transduced by Ad. Compared with control cells, RMS cells were less sensitive or even resistant to oncolysis by wild-type virus. RMS cells expressed the Ad internalization receptors, alpha(v) integrins, but had low or undetectable expression of the major attachment receptor, coxsackievirus-Ad receptor (CAR). Mutant Ads with ablated CAR binding exhibited only 5-20% of transgene expression in RMS cells seen with a wild-type vector, suggesting that residual or heterogeneous CAR expression mediated the little transduction that was detectable. Immunohistochemical analysis of archived clinical specimens showed little detectable CAR expression in five embryonal and eight alveolar RMS tumors. Stable transduction of the cDNA for CAR enabled both efficient Ad gene transfer and oncolysis for otherwise resistant RMS cells, suggesting that poor CAR expression is the limiting feature. Gene transfer to RMS cells was increased >2 logs using Ads engineered with modified fiber knobs containing either an integrin-binding RGD peptide or a polylysine peptide in the exposed HI loop. The RGD modification enabled increased oncolysis for RMS cells by a conditionally replicative Ad, Ad delta24RGD, harboring a retinoblastoma-binding mutation in the E1A gene. Thus, the development of replication-competent vectors targeted to cell surface receptors other than CAR is critical to advance the use of Ad for treating RMS.

Reciprocal enhancement of gene transfer by combinatorial adenovirus transduction and plasmid DNA transfection in vitro and in vivo.

The addition of replication-defective recombinant adenovirus to plasmid transfection (termed here "adenofection") has been shown to increase plasmid transgene expression in limited studies. Similarly, the addition of cationic liposomes to adenovirus increases adenovirus-mediated gene transduction (termed here "lipoduction"). Here we demonstrate that adenofection was effective at enhancing transgene expression when used in conjunction with a variety of different transfection reagents, including a monocationic liposome, a polycationic liposome, an activated dendrimer, a large multilamellar liposomal vesicle, and a protein/amphipathic polyamine complex. The effect was seen regardless of the cellular expression of the adenovirus receptor, CAR, in three different human cancer cell lines derived from rhabdomyosarcomas (Rh18 and RD, CAR-) and cervical carcinoma (HeLa, CAR+). The protein/amphipathic polyamine complex showed an adenofection effect but did not show a lipoduction effect, consistent with different mechanisms of action for adenofection and lipoduction. Using dual-color flow cytometric analysis of cells transfected with a plasmid expressing the enhanced blue fluorescent protein (pEBFP) and a recombinant adenovirus expressing the green fluorescent protein (Ad5-GFP), we demonstrate that adenofection works primarily by increasing gene expression within a cell, whereas lipoduction increases the percentage of cells expressing the transgene. In addition, these studies show that both adenofection and lipoduction can occur simultaneously, further increasing gene transfer. The combination of lipofection and adenovirus transduction also prolonged the duration of transient gene expression and was generally no more toxic than lipofection alone. The enhancement of gene transfer was also seen after injection of complexes directly into subcutaneous human xenograft tumors. Therefore, more effective gene transfer in vitro and in vivo of either plasmid DNA, adenovirus DNA, or both can be achieved by combining liposomal transfection with adenoviral transduction.

Regulated expression of the diphtheria toxin A chain by a tumor-specific chimeric transcription factor results in selective toxicity for alveolar rhabdomyosarcoma cells.

Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was approximately 250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3-6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3-6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)- or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion protein-regulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.

Spatial and temporal control of gene therapy using ionizing radiation.

Activation of transcription of the Egr-1 gene by X-rays is regulated by the promoter region of this gene. We linked the radiation-inducible promoter region of the Egr-1 gene to the gene encoding the radiosensitizing and tumoricidal cytokine, tumour necrosis factor-alpha (TNF-alpha) and used a replication-deficient adenovirus to deliver the Egr-TNF construct to human tumours growing in nude mice. Combined treatment with Ad5.Egr-TNF and 5,000 cGy (rad) resulted in increased intratumoral TNF-alpha production and increased tumour control compared with treatment with Ad5.Egr-TNF alone or with radiation alone. The increase in tumour control was achieved without an increase in normal tissue damage when compared to tissue injury from radiation alone. Control of gene transcription by ionizing radiation in vivo represents a novel method of spatial and temporal regulation of gene-based medical treatments.

Expression of the polymorphic human DNA repair gene XRCC1 does not correlate with radiosensitivity in the cells of human head and neck tumor cell lines.

The human X-ray repair cross-complementing gene 1 (XRCC1) is highly polymorphic in the cells of human head and neck squamous cell carcinoma lines and in a variety of other human cell lines. We describe six patterns seen in Southern analysis of EcoRI-digested genomic DNA. Levels of XRCC1 mRNA vary in cells of human head and neck cell lines as seen in northern blots. Expression of this gene in cell culture does not correlate with radiobiological parameters, clinical outcome, or the DNA restriction fragment length polymorphisms.

Epidermal growth factor receptor gene amplification and expression in head and neck cancer cell lines.

We studied epidermal growth factor receptor (EGFR) gene amplification and expression in 11 early passage human head and neck carcinoma cell lines. Three cell lines demonstrated EGFR gene amplification and 10 lines showed an increase in EGFR mRNA when compared with normal keratinocytes, placenta, and a human skin carcinoma cell line. The effects of EGF on growth in 6 head and neck carcinoma cell lines was also studied. Growth inhibition at a concentration of 20 ng/mL was observed in one cell line but had no effect on growth in 5 cell lines. An increase in EGFR may be important in the etiology of, or progression of, head and neck carcinoma although the mechanisms need to be elucidated by further study.