Signal transduction in the barley powdery mildew fungus
Barley powdery mildew disease is caused by the highly specialised phytopathogenic fungus, Blumeria graminis f.sp. hordei. Disease is spread prolifically by the production of asexual conidia. Following contact with the barley leaf surface, a short, primary germ tube (PGT) emerges, followed by elaboration of a second-formed germ tube, the appressorial germ tube (AGT). This second-formed germ tube elongates, swells and hooks to form an appressorium, which allows direct penetration of the barley cuticle and infection of the host. Infection structure differentiation in B. graminis is a highly regulated and complex process. It demands the coordinated perception of multiple external signals, but little is known about how these signals are integrated and transduced within the fungus. Protein kinase A (PKA) and cAMP signalling are known to play important, but complex, roles during infection structure development. However, signalling via cAMP alone is not sufficient to promote progression through infection structure differentiation. This study describes the characterization of two B. graminis protein kinase C genes, pkc1 and pkc-like. PKC activity was identified in B. graminis protein extracts. Efforts to find an inhibitor specific for B. graminis PKC were unsuccessful. However, phorbol ester, a PKC agonist, invoked both appressorium formation when applied to spores in vivo and PKC activity in protein extracts. In addition, real-time PCR confirmed the differentially regulated transcript profiles of both pkc1 and pkc-like, revealing a peak in transcript levels just prior to PGT emergence for pkc1, and during PGT differentiation for the pkc-like gene. Two mitogen-activated protein (MAP) kinases, mpk1 and mpk2, were characterized. MAP kinase activity was detected in conidial protein extracts. The MAP kinase kinase (MEK) inhibitor, PD98059, inhibited B. graminis germling morphogenesis. However, a MAP kinase agonist failed to show any effect on germling differentiation. In addition, real-time PCR confirmed the differentially regulated transcript profiles of both mpk1 and mpk2, and revealed a peak in transcript levels during appressorial germ tube elongation and swelling for both genes. The "model" phytopathogenic fungus, Magnaporthe grisea, was employed as a "surrogate host" for the functional analysis of the B. graminis MAP kinase gene, mpk1. Firstly, the mpk1 promoter was sequenced and a plasmid construct made comprising the mpk1 gene under the control of the mpk1 promoter. This, and the control construct, that is the M. grisea PMK1 gene under the control of the PMK1 promoter, were transformed into M. grisea Δpmk1. Southern analysis identified transformants for phenotypic studies. These showed that whereas Δpmk1 was complemented by M. grisea PMK1 in the control experiments, B. graminis mpk1 failed to complement Δpmk1. Expression studies showed that there was no expression of mpk1 in an mpk1 transformant.