Investigation of the Effects of the Yr-18 Durable Adult-Plant Resistance Gene in the Near Isogenic Line of Spring Wheat to Stripe Rust with Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) is a revolutionary advance in the field of light microscopy which, in recent years, has been used on interaction studies between host and pathogen. This study carried out on the flag leaves of the susceptible spring wheat cultivar (Triticum aestivum L.) known as Thatcher and its near isogenic line with the Yr-18 adult-plant resistance gene to stripe rust fungus (Puccinia striiformis f. sp. tritici), by confocal laser scanning microscopy. A suspension of fresh urediniospores of stripe rust, isolate SR99-UA (race, 70E 128) in a light mineral oil (Soltrol, 170) was sprayed on the flag leaves in vitro, then four segments were sampled at 2, 4, 8, 12 and 18 days after inoculation. These segments were further divided into 1-2 cm. For detailed observation, two fluorescent dyes Acridin orange and X-Rodamin-1, known to be nucleic acid and calcium binding probes, were used, respectively. CLSM made it possible to visualize host/pathogen interaction in serial without sectioning at a three dimensional level from adaxial to aboxial leaves. According to the results, no difference in pre-penetration behavior of the fungus in the two genotypes was observed. During the early stages of penetration, host cell necrosis was occasionally seen only on the resistant genotype. Few dead cells were observed on the susceptible host 12-18 days after inoculation. However, by this time, the resistant host had a markedly high number of dead cells; there were numerous necrotic areas or pustules resembling hypersensitive response. In the infected areas and around the pustules of the resistant host a remarkable red color was observed by the presence of a thick fluorescence rich calcium layer. The intensity of calcium fluorescence in the non-infected areas of both genotypes and in the infected areas of the susceptible host were nearly the same, indicating calcium mobilization as part of the defensive response in resistant near isogenic lines containing the gene Yr-18. This result indicated that calcium mobilization at the point of challenge by the pathogen played an important role in the near isogenic resistance line of Thatcher in the wheat stripe rust pathosystem. This is the first report on calcium mobilization in a cereal rust pathosystem as a part of the resistance response.