Differential gene expression in a disease resistant colonial x creeping bentgrass hybrid relative to its creeping parent

Huaijun Mike Li, David Rotter and Faith C. Belanger

Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ

Creeping bentgrass (Agrosstis stolonifera L.), an important turfgrass species, is highly susceptible to dollar spot disease caused by Sclerotinia homoeocarpa F. T. Bennett. Colonial bentgrass (A. capillaris L.), a closely related species, has good resistance. Several interspecific hybrids between creeping bentgrass and colonial bentgrass showed excellent dollar spot resistance in field tests. In order to investigate the mechanism of dollar spot resistance in the interspecific hybrids, we are using suppression subtractive hybridization (SSH) to detect genes unique or over-expressed in either resistant hybrids or the creeping parent following fungal inoculation. Two subtraction libraries were created from resistant hybrid (#15) and its creeping parent cDNAs. Nine hundred sixty colonies from the colonial specific library and 480 colonies from the creeping specific library were screened for differential expression using reverse northern dot blotting. On average, 14% of the colonies screened from both libraries were differentially expressed. Differentially expressed clones were sequenced and compared to the GenBank database. Most of the clones sequenced are homologous to genes related to stress resistance in other plants, and so may be relevant to the disease response of the bentgrasses. Further studies will utilize either Northern analysis or real time RT-PCR to confirm the differential expression of these cDNAs. The sequence of these genes can be used in future mapping project as DNA markers. A disease-responsive gene was identified from two of the clones from the creeping bentgrass-specific library. The full-length sequence of the cDNA was obtained through RACE PCR. The amino acid sequence of the protein was homologous to a wheat benzothiazol-induced protein, a wheat Hessian fly response protein, a maize beta-glucosidase aggregating factor, a barley jasmonate-regulated gene, and a wheat vernalization-related gene. Northern blot analysis confirmed its expression was increased in creeping bentgrass upon dollar spot infection. No expression was detected in hybrid #15 or colonial bentgrass from either greenhouse or field samples. These results validated the effectiveness of the SSH method. Southern blot analysis indicated creeping bentgrass parent and another hybrid (#14) each had a single copy of this gene. Interestingly we didn't detect any homologs from related bentgrass species, such as colonial bentgrass, velvet bentgrass (A. canina), or redtop (A. gigantea). The apparent uniqueness of this gene to creeping bentgrass is very interesting regarding the evolution of creeping bentgrass.