Infection of Rrs1 barley by an incompatible race of the fungus Rhynchosporium secalis expressing the green fluorescent protein

Scald disease of barley, caused by the fungal pathogen Rhynchosporium secalis, is one of the most serious diseases of this crop worldwide. Disease control is achieved in part by deployment of major resistance (Rrs) genes in barley. However, in both susceptible and resistant barley plants, R. secalis is able to complete a symptomless infection cycle. To examine the R. secalis infection cycle, Agrobacterium tumefaciens-mediated transformation was used to generate R. secalis isolates expressing the green fluorescent protein or DsRed fluorescent protein, and that were virulent on an Rrs2 plant (cv. Atlas), but avirulent on an Rrs1 plant (cv. Atlas 46). Confocal laser scanning microscopy revealed that R. secalis infected the susceptible cultivar and formed an extensive hyphal network that followed the anticlinal cell walls of epidermal cells. In the resistant cultivar, hyphal development was more restricted and random in direction of growth. In contrast to earlier models of R. secalis infection, epidermal collapse was not observed until approximately 10 days post-inoculation in both cultivars. Sporulation of R. secalis was observed in both susceptible and resistant interactions. Observations made using the GFP-expressing isolate were complemented and confirmed using a combination of the fluorescent probes 5-chloromethylfluorescein diacetate and propidium iodide, in the non-transformed wild-type isolate. The findings will enable the different Rrs genes to be better characterized in the effect they exert on pathogen growth and may aid in identification of the most effective resistance.