E values for this analysis and the amino acid sequence identity between the N. crassa protein and the predicted polypeptide sequence for the E. festucae homologue are given. Proteins chosen for this analysis were based on those present in the N. crassa genome (Galagan et al. 2003). As this analysis focused on identification of homologues of the N. crassa two-component signalling proteins, there may be additional histidine kinases encoded in the E. festucae genome that were not identified by this analysis. E. festucae gene models are available on request
4.3.2 Response Regulator and Histidine Phosphotransfer Protein
Very little information is available on the role of fungal response regulators in phytopathogenesis. However, in Magnaporthe oryzae one RR (SSK1) and one RR-like gene (RIM15) were shown to be required for virulence (Motoyama et al. 2008). In addition, in C. heterostrophus SSK1 but not SKN7 is required for virulence, and in Gibberella zeae, sskl mutants show reduced pathogenicity (Oide et al. 2010). The genomes of E. festucae Fl1 and E2368 encode two response regulators, homologous to N. crassa genes for RRG-1 and RRG-2 (Fig. 8 and Table 3). Given the precedence for a role of RRs in virulence of the phytopathogenic fungi it is likely that at least one of the two E. festucae RRs will be required for symbiotic maintenance. To date there are no reports of a role for the HPt in phytopathogenesis, possibly because mutants of this gene are lethal as found for A. nidulans (Vargas-Perez et al. 2007). E. festucae contains one HPt gene, and like in A. nidulans it is predicted to be essential.
In order to sense and respond to internal and external environmental signals, cells have developed signalling systems that transmit signals from cell surface receptors to their intracellular targets using second messengers. Second messengers are small molecules whose cytoplasmic concentration is controlled in response to extracellu-lar/intracellular signals. This section reviews two of the most important fungal second messengers: cyclic adenosine monophosphate (cAMP) and calcium.
4.4.1 cAMP Signalling cAMP regulates a wide range of developmental, metabolic and pathogenic processes in fungi. However, despite regulating a diverse range of processes, components of cAMP signalling are highly conserved (D'Souza and Heitman 2001). Synthesis of cAMP is catalysed by adenylate cyclase and its degradation is facilitated by phosphodiesterases. The activity of these enzymes is regulated by various extracellular signals. Adenylate cyclase is directly regulated by heterotrimeric G-protein signalling through protein kinase A (PKA), a key enzyme involved in most cAMP regulated processes. In its inactive state, PKA is a tetramer composed of two regulatory and two catalytic subunits. When the cytoplasmic concentration of cAMP increases, cAMP binds to the regulatory subunits causing disassociation of the catalytic subunits. The two catalytic subunits act downstream on various transcription factors and metabolic enzymes.
In filamentous fungi, cAMP has a key role in the regulation of growth and developmental processes. Inactivation of the PKA regulatory subunit disrupts polarised growth in N. crassa (Bruno et al. 1996) and Aspergillus niger (Saudohar et al. 2002). cAMP is also important for regulating plant infection by fungal pathogens, likely due to a role in regulating morphological switching which is often required for pathogenesis. For example, in U. maydis cAMP is required for formation of the dikaryon and the subsequent morphological switch to filamentous growth required for infection (reviewed in Kahmann et al. 1999). Mutations in adenylate cyclase, uacl, or the catalytic subunit of PKA, adrl, induce filamentous growth of haploid cells while mutation in the PKA regulatory subunit, ubcl, as expected, has the opposite effect and results in a multiple budding phenotype (Gold et al. 1994; Durrenberger et al. 1998). Both adrl and ubcl are also involved in the second stage of the infection process, with adrl mutants being non-pathogenic and ubcl mutants able to penetrate the host but unable to induce tumours. These results suggest cAMP is required for the penetration process but inhibits later steps in infection, such as tumour development. In the maize pathogen Fusarium verticillioides, disruption of the PKA catalytic subunit CPKl or adenylate cyclase FACl leads to defects in radial growth, but only facl mutants show significantly reduced virulence, suggesting there may be another copy of the catalytic subunit gene which regulates the infection process (Choi and Xu 2010). In M. graminicola, disruption of either the regulatory or catalytic PKA subunit significantly reduces virulence (Mehrabi and Kema 2006). Disruption of adenylate cyclase in B. cinerea disrupts sporulation and reduces the rate of lesion development during the infection process (Klimpel et al. 2002). cAMP signalling has diverse roles in M. grisea. Deletion of the PKA catalytic subunit, CPKA, dramatically reduces appressorium formation (Mitchell and Dean 1995). Deletion of the adenylate cyclase gene, MACl, also reduces appressorium formation but it also affects vegetative growth, conidiation and conidial germination (Choi and Dean 1997). Mutations in the PKA regulatory subunit (SUMl) suppressed the growth and development phenotypes of macl, including appressorium formation (Adachi and Hamer 1998). Under non-inducing conditions SUMl mutants undergo precocious development of appressoria, suggesting CPKA is not the only catalytic subunit present in M. grisea. Homologues of adenylate cyclase and the PKA catalytic and regulatory subunits are found in the genomes of E.festucae Fl1 and E2368. Disruption of acyA (adenylate cyclase) in E. festucae reduced radial growth and increased conidiation (Voisey et al. 2007). The most pronounced effects in planta were a reduction in infection rate and an increase in hyphal branching, although host plants did not become stunted as was observed for noxA and racA mutants.
4.4.2 Ca2+ as a Second Messenger
Calcium is a universal secondary messenger, involved in a broad range of processes from gene expression to apoptosis (Bootman et al. 2001), and is proposed to be the most versatile biological messenger known (Cheng and Lederer 2008). Cytoplasmic calcium concentration is maintained at a very low level (100-500 nM) compared with that in the extracellular spaces and internal stores (1-5 mM) (Halachmi and Eilam 1989; Permyakov and Kretsinger 2009). A calcium signal is generated when the cytoplasmic Ca2+ concentration increases, which in turn activates Ca2+-regulated effectors. Calcium ions are transported from the cytoplasm by calcium pumps and ion exchangers.
Fungal calcium signalling has been best characterised in the budding yeast S. cerevisiae. While relatively little is known about calcium signalling in filamentous fungi, it appears to be more complex than in S. cerevisiae (Zelter et al. 2004). This section will provide an overview of the main components of fungal Ca2+ signalling pathways, including a bioinformatic survey of calcium signalling components present in the E. festucae genome (Table 4 and Fig. 4). Calcium signalling proteins are highly conserved across the fungal kingdom, but with some variation in copy number. While few of the identified calcium signalling genes have been functionally characterised in E. festucae, systematic functional analysis of Ca2+ genes in the phytopathogenic fungus M. oryzae using RNA silencing provides an insight into the importance of these pathways for plant-fungal interactions (Nguyen et al. 2008).
Three Ca2+ permeable channels have been characterised in S. cerevisiae: the voltage-gated channel Cch1p, stretch-activated Mid1p channel and the transient receptor potential-like Ca2+ channel Yvclp. Disruption of midl induces loss of cell viability after pheromone-induced cell differentiation, with the mutant displaying low Ca2+ uptake. This loss of viability can be restored by incubation in a high extracellular Ca2+ concentration medium (Iida et al. 1994). Dcchl mutants appear identical to Dmidl mutants, suggesting Cch1p and Mid1p are components of the same Ca2+ permeable channel (Paidhungat and Garrett 1997). The Yvc1p channel is located in the vacuolar membrane (Palmer et al. 2001), and is involved in the calcium response triggered by hyperosmotic shock (Denis and Cyert 2002; Zhou et al. 2003). Knock-down of M. oryzae Cch1p leads to reduced sporulation and appressorium formation but the strains remain pathogenic (Nguyen et al. 2008). Knock-down of Mid1p resulted in a similar phenotype, whereas knock-down of Yvc1p strongly affected conidia and appressoria formation (Nguyen et al. 2008). Interestingly, deletion of C. purpurea midl resulted in a complete loss of pathoge-nicity and production of appressoria-like structures not normally seen in C. purpurea (Bormann and Tudzynski 2009). Like other filamentous fungi, E. festucae contains homologues of the Ca2+ channel proteins Cch1, Mid1 and Yvc1 (Table 4). Given the close relationship between E. festucae and C. purpurea it is likely that E. festucae midl may be involved in regulating symbiosis.
The role of Ca2+ ATPases (Ca2+ pumps) and exchangers is to maintain low cytoplasmic Ca2+ concentrations by actively removing Ca2+ from the cytoplasm. In fungi, these proteins work together with the phosphatase calcineurin in regulating
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