Universität Marburg, Fachbereich Biologie, Karl-von-Frisch-Strasse 8, D-35032 Marburg, Germany1
Tel: +49 6421 2821536. Fax: +49 6421 2828971. e-mail: boelker{at}mailer.uni-marburg.de
Keywords: pathogenicity, plant pathogen, cAMP signalling, mating, MAP kinase signalling
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Overview |
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This favourable situation has allowed diverse approaches to identify components of the regulatory network that controls dimorphism and virulence in U. maydis. The ultimate goal is the understanding of pathogenic development at all levels of genetic regulation and biochemical action. U. maydis has been the subject of a number of reviews. A comprehensive overview, which covers the early literature, was given by Christensen (1963) . More recent reviews focus on the molecular mechanism of mating type determination (Banuett, 1995
; Kahmann & Bölker, 1996
) and the importance of signalling pathways during mating and pathogenesis (Banuett, 1998
; Kahmann et al., 1999
; Kronstad et al., 1998
; Lengeler et al., 2000
).
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Life cycle of U. maydis |
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The most conspicuous symptom of the disease caused by U. maydis is the formation of plant tumours by induction of cell proliferation in meristematic plant tissue (Fig. 1). It was always tempting to speculate that phytohormones produced by the fungus are responsible for this reaction (Wolf, 1952
). This hypothesis was strongly supported by the observation that in liquid cultures the fungus releases significant amounts of the plant hormone auxin (indole-3-acetic acid) (Wolf, 1952
). In addition, infected tumour tissue exhibits a 5- to 20-fold elevated concentration of auxin. As yet, however, it is unclear whether this auxin is produced by the fungus or by the plant as a response to the infection. Recently, an attempt has been made to abolish auxin production of U. maydis by disrupting the gene for an indole-3-acetaldehyde dehydrogenase suspected to be involved in the biosynthetic pathway (Basse et al., 1996
). However, in the absence of glucose as a carbon source, the resulting strain was still able to produce auxin, indicating the existence of an additional indole-3-acetaldehyde dehydrogenase whose expression might be subject to catabolite repression. Consequently, these mutant strains were not affected in pathogenicity and tumour induction (Basse et al., 1996
). Thus, no conclusive evidence for a crucial role of fungus-derived phytohormone production during pathogenic development has yet been achieved.
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Genetic control of mating and pathogenesis in U. maydis |
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Signalling affects morphology and virulence in U. maydis |
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Surprisingly, additional suppressors of uac1 turned out to be members of a MAP kinase (MAPK) signalling cascade, with Ubc3 acting as a MAPK, Ubc5 as a MAPKK and Ubc4 as a MAPKKK (Andrews et al., 2000 ; Mayorga & Gold, 1998
, 1999
). Interestingly, two of these components have also been identified as members of the pheromone signalling pathway. Ubc5 is identical to Fuz7 (Banuett & Herskowitz, 1994
), which has been suggested to be involved in pheromone signalling, and Ubc3 is identical to Kpp2, which has been identified as MAPK, being part of the pheromone response pathway (Müller et al., 1999
).
The cAMP and MAPK pathways involved in environmental and pheromone signalling appear to be connected at two levels. The first level is a G subunit, Gpa3, which was originally identified as part of the pheromone response pathway but which is apparently also involved in cAMP signalling. Its essential function for mating could be replaced by the addition of exogenous cAMP (Krüger et al., 1998
). This could indicate that either Gpa3 is not directly coupled to the pheromone receptors or that Gpa3 is coupled to multiple receptors, perhaps in combination with different ß/
heterodimers. If one assumes that activation of the MAPK kinase cascade occurs by the ß/
heterodimer, stimulation of the pheromone receptors could result in activation of both the cAMP and the MAPK signalling pathway (Fig. 3
). To explain why gpa3 mutant cells cannot mate if exogenous cAMP is added, one has to assume that cell fusion may require the activated receptor for directing the growth of conjugation tubes and the spatial co-ordination of membrane fusion events. This could indicate that either the ß/
heterodimer triggers these events or that the receptor itself interacts with components of the cell fusion apparatus.
The second level of cross-talk between cAMP and MAPK signalling occurs at the pivotal transcription factor Prf1, which regulates the pheromone-induced expression of the a and b mating type genes. The Prf1 protein carries several sequence motifs specific for PKA-and MAPK-dependent phosphorylation and the presence of these sites is essential for Prf1 function (Hartmann et al., 1999 ). In addition, expression of the prf1 gene itself is subject to nutritional stimulation and to positive feedback control after pheromone stimulation (Hartmann et al., 1999
). One has to assume that basal activity of Prf1 requires phosphorylation by low-level constitutive activity of the MAPK Kpp2/Ubc3. Nutritional signalling results in stimulation of Prf1 activity at two different levels, the induction of expression and additional phosphorylation of PKA sites. The fact that members of the MAPK cascade have been identified as suppressors of mutants lacking adenylylcyclase indicates that the basal level of MAPK phosphorylation of Prf1 (or another transcription factor) is required for the filamentous growth of cells lacking cAMP.
The existence of pathogenic haploid strains indicates that recognition of pheromones is not essential during infection. Nevertheless, both the cAMP signalling pathway and the MAPK cascade are required for virulence. This has been taken as evidence that these signalling networks are also engaged in sensing specific plant signals that trigger discrete stages of pathogenic development. This was supported by the observation that expression of a constitutive active variant of the G subunit, Gpa3Q260L, still allows tumour induction but interferes with fungal proliferation and spore production (Krüger et al., 2000
).
The genetic dissection of signal transduction cascades in U. maydis has added another fascinating piece to the growing puzzle of these networks and their function in fungal development and virulence. Although similar elements occur in a number of different fungi, their specific functions appear to vary significantly between these species. A comprehensive overview of this topic has been given recently by Lengeler et al. (2000) .
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Identification of virulence genes in U. maydis |
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However, not every gene whose deletion results in attenuated virulence necessarily plays a specific role during this stage. Many enzymes involved in the biosynthesis of amino acids and nucleotides are not required on complete media. During propagation inside the host, genes for such biosynthetic enzymes may become essential since amino acids and nucleotides are not, or at least not in sufficient amounts, provided by the host. Thus, many such viable mutants, which are reduced in their general fitness, may also be unable to infect the host plant. Mutations leading to loss of virulence are therefore not necessarily informative about functions specifically involved in pathogenic development. Discrimination between whether such a candidate gene can be regarded as a specific virulence gene, or as just a fitness gene, is rather difficult.
On the other hand, genes that do play important roles during infection processes are often present in several copies. Fungal species that attack plants or animals often contain batteries of genes encoding isoenzymes involved in, for example, cell-wall degradation or proteolysis (Hube, 1996 ; Koller et al., 1995
). Disruption of one, or even several, of these genes does not necessarily reduce virulence. Thus, in a genetic screen for avirulent mutants this important set of pathogenicity genes may escape detection. These genes may be identified by their specific expression patterns. Many different methods to identify infection-specific genes of pathogenic organisms have been described, such as differential display of RNA, cDNA-amplified fragment length polymorphism (cDNA-AFLP) and in vivo expression technology (IVET) (for a review, see Hensel & Holden, 1996
).
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Generation of avirulent mutants by plasmid insertion |
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Isolation of pathogenicity genes by their specific expression pattern |
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The existence of a single master control locus for pathogenicity allows a top-down approach |
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The b gene products belong to the family of homeodomain transcription factors and thus are believed to be either activators or repressors of other specific promoters (Gillissen et al., 1992 ; Schulz et al., 1990
). Until recently, all attempts to identify a direct DNA target for the active bW/bE protein complex have been unsuccessful. At least one target has now been isolated by the combination of genetic and biochemical approaches: transcription of the genes encoding pheromone precursors and pheromone receptors is induced upon recognition of pheromone of opposite mating type (Urban et al., 1996b
). After cell fusion, expression is turned down to a low, but constitutive level due to the presence of the active bW/bE heterodimer. Within the a2 mating type allele an ORF, lga2, exists, to which a biological function has not yet been assigned (Urban et al., 1996a
). Expression of lga2 is not only induced upon pheromone stimulation, but is even further increased after cell fusion has occurred. The expression of lga2 is also enhanced in cells that carry two different alleles of the b locus (Urban et al., 1996b
). Thus, this gene is a prime candidate for direct regulation through the bW/bE homeodomain complex. It has been successfully used to identify a short DNA sequence in its promoter region that is required for b-dependent regulation. In vitro DNA binding studies clearly demonstrated direct recognition of this sequence by a chimeric bW/bE fusion protein (Romeis et al., 2000
). However, lga2 cannot be the only target of the bW/bE heterodimer since it is not required for pathogenicity. The identification of a functional binding site for the bW/bE heterodimer will clearly help to study the interaction of the many different bE/bW protein complexes with DNA in more detail. In addition, this finding might lead to the isolation of further promoter sequences which contain similar sequence elements.
To identify genes that act further downstream of the master regulator, a screen has been performed for mutants that allow the expression of dikaryon-specific genes, for example the endoglucanase egl1, in the absence of an active bW-bE heterodimer. By this approach a gene, rum1, was identified which encodes a polypeptide highly similar to the human retinoblastoma binding protein 2 (Quadbeck-Seeger et al., 2000 ). From its domain structure Rum1 is supposed to interact with histone deacetylases. Histone deacetylases act as corepressors and decrease the concentration of acetylated histone, which correlates with actively transcribed DNA regions (Ng & Bird, 2000
). This indicates that the switch from the nonpathogenic to the pathogenic phase might be accompanied by a rearrangement of the heterochromatin structure. It is still unclear how this alteration of chromatin structure may be induced by the bW/bE heterodimer, which is supposed to act as a transcriptional regulator. In addition, it would be interesting to know which set of genes is affected by this global regulation and how these genes are involved in the control of pathogenicity.
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Outlook |
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