Cis and Trans Acting Genetic Factors Relevant to the Expression of Biocontrol Genes

Lorito et al. (1996a) first used an in vitro approach to detect cis-acting motifs on the ch42 promoter being involved in mycoparasitism. They confronted Botrytis cinerea on agar plates with T. atroviride P1, prepared crude protein extracts from mycelia harvested at different phases during myco-parasitism, and used them in electrophoretic mobility shift assays. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Crel (5'-SYGGRG-3'; Kulmburg et al. 1993) provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Crel to both fragments of the ech-42 promoter. These findings are consistent with the presence of two and three consensus sites, respectively, for binding of Crel in the two ech-42 promoter fragments used. In contrast, the protein-DNA complex from mycoparasitic mycelia does not involve Crel, as its formation is unaffected by the addition of the competing oligonucleo-tides. Based on these findings, they offered a preliminary model for regulation of ech-42 expression in T. harzianum, which subsequently involves: (a) binding of Cre1 to two single sites in the ech-42 promoter; (b) binding of a "mycoparasitic" protein/protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (c) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the "mycoparasitic" protein-DNA complex (Lorito et al. 1996a). The cre1 gene from T. harzianum has been cloned (Ilmen et al. 1996), but no demonstration of its effect on biocontrol in vivo was as yet presented.

Cis Trans Genetics

Figure 1 Scheme illustrating the hypothesis how chitinase gene expression could be triggered in T. atroviride, based on results from Brunner et al. (2002); Mach et al. (1999); Kullnig et al. (2000); Peterbauer et al. (2002a,b); Zeilinger et al. (1999). Circled plus and minus indicate activation and inactivation of a process, respectively, without implying the underlying mechanism. Proteins A, B and C refer to the Zn(6) cluster protein (Peterbauer et al. 2002a), the mycoparasitic regulator (Lorito et al. 1996a) and the BrlA-box binding starvation response repressor (see text), respectively. The black triangles indicate NAcGlc molecules, and symbolize NAcGlc, (NAcGlc)2, and (NAcGlc)3, respectively.

Figure 1 Scheme illustrating the hypothesis how chitinase gene expression could be triggered in T. atroviride, based on results from Brunner et al. (2002); Mach et al. (1999); Kullnig et al. (2000); Peterbauer et al. (2002a,b); Zeilinger et al. (1999). Circled plus and minus indicate activation and inactivation of a process, respectively, without implying the underlying mechanism. Proteins A, B and C refer to the Zn(6) cluster protein (Peterbauer et al. 2002a), the mycoparasitic regulator (Lorito et al. 1996a) and the BrlA-box binding starvation response repressor (see text), respectively. The black triangles indicate NAcGlc molecules, and symbolize NAcGlc, (NAcGlc)2, and (NAcGlc)3, respectively.

Another cis-acting element was recently identified that may contribute to the regulation of ech42 gene expression: the ech42 promoter sequence contains two short nucleotide sequences which resemble the consensus for binding of the Aspergillus nidulans brlA (bristle) regulator (5'-MRAGGGR-3'; Chang and Timberlake 1992). The encoded BrlA protein is a general regulator of conidial development, which itself responds to carbon starvation (Skromne et al. 1995). Cell-free extracts of T. atroviride, prepared from mycelia subjected to carbon starvation, form a specific, consensus-dependent complex with BrlA site-containing oligonucleotide fragments of the ech42 promoter (K Brunner, CK Peterbauer, and CP Kubicek, unpublished data). Deletion of the promoter areas containing the BrlA sites in vivo resulted in a derepression of the starvation induced expression of ech42, but had no effect on the expression of ech42 during sporulation. This motif therefore likely binds a new repressor of Trichoderma rather than a sporulation specific regulator.

The induction of nag1 by chitin oligomers has been studied in more detail, using a combination of promoter deletion, in vivo footprinting, and EMSA experiments, proteins binding to an AGGGG-element, to a CCAGN13-CTGG motif and to a CCAAT-box were identified (Peterbauer et al. 2002a,b). Disruption of either of the two former binding sites in vivo resulted in an almost complete reduction of induction of nag1 expression by N-acetylgluco-samine. The nature of the proteins binding to these three motifs is only partially understood: the spatial organization of the CCAGN13CTGG motif would be compatible with the binding of a Zn(II)2Cys6-type zinc finger protein (Todd and Andrianopoulos 1997), whereas the CCAAT-box binds a protein complex consisting of at least three proteins Hap2, Hap3, and Hap5, which were originally described in S. cerevisiae and more recently characterized from T. reesei (Zeilinger et al. 2001). According to Narendja et al. (1999); Zeilinger et al. (2003), their function is the establishment of an open chromatin structure at the promoter.

The AGGGG-box is a motif which has been studied in detail in Saccharomyces cerevisiae and identified as a binding site for the Cys2His2 zinc finger proteins Msn2p and Msn4p, which are key regulators of the transcription of a number of genes coding for proteins with stress-protective functions (Ruis and Schiiller 1995). In Trichoderma, the occurrence of the AGGGG-box is not restricted to the nag1 promoter but also occurs in two other chitinase promoters, ech42 and chit33 (Lorito et al. 1996a; de las Mercedes Dana et al. 2001), consistent with a potential role in chitinase regulation. This would be compatible with a regulation of the expression of chitinase genes by metabolic stress, as shown both for ech42 and chit33 (see earlier). However, nag1 is not upregulated by stress (CK Peterbauer, unpublished data), and the presence of this motif must therefore serve another function. In this context it is interesting to note that an AGGGG-motif was also identified in the cutinase promotor of Haematonectria haematococca, where it appeared to be involved in maintaining the basal expression level (Kamper et al. 1994). In Yarrowia lipolytica, the AGGGG motif is bound by the Mhylp protein, whose transcription is dramatically increased during the yeast-to-hypha transition (Hurtado and Rachubinski 1999).

In order to study whether a homologue of S. cerevisiae MSN2/4 and H. haematococca AGGGG-binding protein encoded by the open reading frame AAB04132 (which we call seb1, = stress element binding) has recently been cloned from T. atroviride (Peterbauer et al. 2002a,b). Its zinc finger domain has high amino acid sequence identity with S. cerevisiae Msn2/4 and the H. haematococca AGGGG-binding protein, and specifically recognizes the AGGGG sequence of the ech42 and nag1 promoter in band shift assays. However, a cDNA clone of seb1 was unable to complement a MSN2/4 delta mutant of S. cerevisiae. Despite the presence of AGGGG elements in the promoter of the chitinase gene nag1, no differences in its expression were found between the parent and a seb1-delta-strain. The EMSA analyses with cell-free extracts of the seb1 -delta still showed the presence of proteins binding to the AGGGG-element in nag1 and ech42, and thus seb1 does not encode the protein binding to this sequence in the chitinase promoters. Rather, seb1 appears to be involved in osmotic stress response: seb1-mRNA accumulation was increased under conditions of osmotic stress (sorbitol, NaCl)—but not under other stress conditions (cadmium sulfate, pH, membrane perturbance), and growth of the delta-seb1 strain was significantly more inhibited by the presence of 1 M sorbitol and 1 M NaCl than that of the wild-type strain (Peterbauer et al. 2002a,b).

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