Altered HC-toxin reductase activity in transgenic BMS cultures
--Bob Meeley, Keith Lowe, Dave Songstad, Guri Johal , Joanie Phillips and Steve Briggs

In maize, tolerance to the cyclic peptide HC-toxin is the basis of resistance to the fungal pathogen Cochliobolus carbonum Nelson race 1. The toxin is required by the pathogen for successful colonization of susceptible maize tissues. The resistance gene Hm1 (located on 1L) encodes the enzyme HC-toxin reductase, which inactivates HC-toxin by carbonyl reduction. Due to the action of HCTR, resistant maize lines are 100-fold more tolerant to HC-toxin, and are able to avoid infection by C. carbonum. HC-toxin induces a variety of biological responses in maize. In callus cultures, effective doses of HC-toxin cause necrosis and cell death. This toxin is being tested for use as a selectable marker in maize transformation. Nearly all commercially relevant maize lines carry the Hm1 allele, therefore our selection method relies on over-expression of HCTR activity.

The Hm1 gene has been cloned and sequenced. A vector containing the Hm1 cDNA fused to the CaMV 35S promoter was used in conjunction with a second plasmid containing the BAR (herbicide "Basta" resistance) and GUS (ÿ-glucuronidase) genes. BMS co-transformants containing these constructs were generated by microprojectile bombardment. HC-toxin reductase activities were measured in 22 independent Basta-resistant transformants and 4 controls (transformed with BAR only). Relative to the controls, over-expression of HCTR activity was detected in 19 out of 22 transformants. The range of HCTR expression was grouped into classes: transformants expressing 10- to 20-fold higher HCTR activity (3), 5- to 10-fold higher (9), and 2- to 5-fold higher (7). Two transformants had no detectable increase in HCTR activity. The final transformant from the group of 22 had a five-fold reduction in HCTR activity compared to controls. This transformant is a candidate for the phenomenon of co-suppression.

Representatives from each transformant class, including the co-suppressed colony and controls, were tested for sensitivity to HC-toxin in a callus bioassay. Samples (50mg) from each colony were suspended in MS salts containing Basta (5µg/mL), and varying doses of purified HC-toxin. After several days in suspension at 30 C, the toxin response was evaluated by necrosis, a reduction in the suspension culture fresh weight, and a loss of GUS activity. Based on these criteria, transformants displayed tolerance to elevated levels of HC-toxin, consistent with the findings from the HCTR assays. The co-suppressed colony was considerably more sensitive to HC-toxin than the controls. Presently, HC-toxin is being tested as the primary selection agent for a fresh set of 35S-Hm1 transformants. 

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