Plant surface cues prime Ustilago maydis for biotrophic development.

Infection-related development of phytopathogenic fungi is initiated by sensing and responding to plant surface cues. This response can result in the formation of specialized infection structures, so-called appressoria. To unravel the program inducing filaments and appressoria in the biotrophic smut fungus Ustilago maydis, we exposed cells to a hydrophobic surface and the cutin monomer 16-hydroxy hexadecanoic acid. Genome-wide transcriptional profiling at the pre-penetration stage documented dramatic transcriptional changes in almost 20% of the genes. Comparisons with the U. maydis sho1 msb2 double mutant, lacking two putative sensors for plant surface cues, revealed that these plasma membrane receptors regulate a small subset of the surface cue-induced genes comprising mainly secreted proteins including potential plant cell wall degrading enzymes. Targeted gene deletion analysis ascribed a role to up-regulated GH51 and GH62 arabinofuranosidases during plant penetration. Among the sho1/msb2-dependently expressed genes were several secreted effectors that are essential for virulence. Our data also demonstrate specific effects on two transcription factors that redirect the transcriptional regulatory network towards appressorium formation and plant penetration. This shows that plant surface cues prime U. maydis for biotrophic development.

A seven-WD40 protein related to human RACK1 regulates mating and virulence in Ustilago maydis.

In mammalian cells RACK1 serves as a scaffold protein that has a role in integrating inputs from different signalling pathways and affects translation through association with ribosomes. Ustilago maydis contains a seven-WD40 repeat motif protein designated Rak1, which shows 68% identity to RACK1 and 51% identity to Asc1p of Saccharomyces cerevisiae. An asc1 mutant could be complemented by introduction of U. maydis rak1. The deletion of rak1 affected cell growth, cell wall integrity and specifically attenuated cell fusion. This latter defect was caused by reduced expression of prf1 encoding the regulator for pheromone (mfa) and pheromone-receptor genes. Rak1 interacts with a variety of ribosomal proteins and microarray analysis revealed that the deletion of rak1 led to severely reduced expression of rop1, a transcriptional activator of prf1. The constitutive expression of rop1 could rescue the defect of mfa1 expression as well as conjugation tube formation in response to pheromone induction in the rak1 mutant. Moreover, a solopathogenic rak1 mutant failed to respond to plant-derived stimuli, resulting in attenuated filamentation and pathogenicity. This could be partially rescued by constitutive expression of the b heterodimer. These data suggest that rak1 is a regulator of rop1 expression with additional roles after cell fusion.

The AGC Ser/Thr kinase Aga1 is essential for appressorium formation and maintenance of the actin cytoskeleton in the smut fungus Ustilago maydis.

On the plant surface the dimorphic fungus Ustilago maydis switches from budding to hyphal growth and differentiates appressoria. To get more insight into these highly regulated processes we report on the role of a conserved Ser/Thr kinase of the AGC kinase family, Aga1. U. maydis Aga1 could functionally replace Ypk1p in Saccharomyces cerevisiae. aga1 deletion mutants were affected in growth, cell wall integrity, mating as well as the ability to form appressoria and showed defects in actin organization and actin-dependent endocytosis. With respect to appressorium formation and endocytosis, the aga1 deletion phenotype could be mimicked by inhibiting the formation of actin filaments with Latrunculin A. These data suggest a critical role of Aga1 in F-actin organization during the morphological changes accompanying the development of appressoria.

Physical-chemical plant-derived signals induce differentiation in Ustilago maydis.

Ustilago maydis is able to initiate pathogenic development after fusion of two haploid cells with different mating type. On the maize leaf surface, the resulting dikaryon switches to filamentous growth, differentiates appressoria and penetrates the host. Here, we report on the plant signals required for filament formation and appressorium development in U. maydis. In vitro, hydroxy-fatty acids stimulate filament formation via the induction of pheromone genes and this signal can be bypassed by genetically activating the downstream MAP kinase module. Hydrophobicity also induces filaments and these resemble the dikaryotic filaments formed on the plant surface. With the help of a marker gene that is specifically expressed in the tip cell of those hyphae that have formed an appressorium, hydrophobicity is shown to be essential for appressorium development in vitro. Hydroxy-fatty acids or a cutin monomer mixture isolated from maize leaves have a stimulatory role when a hydrophobic surface is provided. Our results suggest that the early phase of communication between U. maydis and its host plant is governed by two different stimuli.

Trehalose metabolism is important for heat stress tolerance and spore germination of Botrytis cinerea.

To analyse the role of trehalose as stress protectant and carbon storage compound in the grey mould fungus Botrytis cinerea, mutants defective in trehalose-6-phosphate synthase (TPS1) and neutral trehalase (TRE1) were constructed. The Deltatps1 mutant was unable to synthesize trehalose, whereas the Deltatre1 mutant showed elevated trehalose levels compared to the wild-type and was unable to mobilize trehalose during conidial germination. Both mutants showed normal vegetative growth and were not affected in plant pathogenicity. Growth of the Deltatps1 mutant was more heat sensitive compared to the wild-type. Similarly, Deltatps1 conidia showed a shorter survival under heat stress, and their viability at moderate temperatures was strongly reduced. In germinating wild-type conidia, rapid trehalose degradation occurred only when germination was induced in the presence of nutrients. In contrast, little trehalose breakdown was observed during germination on hydrophobic surfaces in water. Here, addition of cAMP to conidia induced trehalose mobilization and accelerated the germination process, probably by activation of TRE1. In accordance with these data, both mutants showed germination defects only in the presence of sugars but not on hydrophobic surfaces in the absence of nutrients. The data indicate that in B. cinerea trehalose serves as a stress protectant, and also as a significant but not essential carbon source for germination when external nutrients are low. In addition, evidence was obtained that trehalose 6-phosphate plays a role as a regulator of glycolysis during germination.

Different signalling pathways involving a Galpha protein, cAMP and a MAP kinase control germination of Botrytis cinerea conidia.

Conidial germination of the grey mould fungus Botrytis cinerea was found to be induced by different chemical and physical signals, namely the amount and quality of nutrients as well as the hydrophobicity and rigidity of the surface. A B. cinerea Deltabcg3 mutant disrupted in the Galpha3 subunit of the heterotrimeric G protein was specifically defective in germination induced by carbon sources. A similar germination defect of an adenylate cyclase mutant, and the complementing effect of cAMP addition to conidia of these mutants confirmed the involvement of cAMP. In contrast, a Deltabmp1 MAP kinase mutant was delayed in carbon source-induced germination, but completely unable to germinate on hydrophobic surfaces. Based on these data, it is proposed that the germination response of B. cinerea conidia is controlled by three signalling pathways: Germination induction by rich media is weakly dependent on BMP1; induction by carbon sources requires BCG3, cAMP and BMP1; and induction by contact to hydrophobic surfaces is absolutely dependent on BMP1. Other defects of the Deltabcg3 mutant, such as low conidiation, excessive formation of sclerotia and delayed host infection, were also restored by cAMP. Microscopical studies of germling growth and differentiation on host cuticles revealed that the delayed infection of the Deltabcg3 mutant was due to a surface sensing defect leading to a reduced penetration. Thus, in addition to their role in germination, Galpha3, cAMP as well as BMP1 are required also for proper host surface recognition and penetration ability of germinated conidia.