Host Invasion

It has been proposed that penetration of the host mycelium takes place by partial degradation of its cell wall (Elad et al. 1983b,c). Interaction sites have been stained by fluores-ceinisothiocyanate-conjugated lectins or calcofluor. The appearance of fluorescence indicated the presence of localized cell wall lysis at points of interaction between the antagonist and its host (Elad et al. 1983c). Furthermore,

Figure 1 Transmission electron micrographs of Trichoderma atroviride parasiting Rhizoctonia solani. A) T. atroviride (T) penetrates R. solani (R). B) Trichoderma (T) grows inside an R. solani (R) hyphae.

analysis by electron microscopy has shown that during the interaction of Trichoderma spp. with either S. rolfsii or R. solani the parasite hyphae contacted their host and perforated their cell walls. These observations led to the suggestion that Trichoderma produced and secreted mycoly-tic enzymes responsible for the partial degradation of the host's cell wall.

Indeed, Trichoderma produces a complex set of gluca-nases, chitinases, lipases, and proteases extracellularly when grown on cell walls of R. solani (Geremia et al. 1991; Vazquez-Garciduenas et al. 1998). Table 1 summarizes the currently available information on this complex set of lytic enzymes produced by Trichoderma. Most attention has been paid to chitinases and several have been studied to some extent in different isolates or even species of the genus. The purification and characterization of three endochitinases secreted by T. harzianum was first reported by De la Cruz et al. (1992). They reported the isozymes to be 37, 33, and 42kDa, respectively. Only the purified 42kDa chitinase hydrolyzed Botrytis cinerea purified cell walls in vitro, but this effect was heightened in the presence of either of the other two isoenzymes (De la Cruz et al. 1992). The 42kDa endochitinase has been found in most isolates. Recently, this enzyme has been proposed to play a major role in the regulatory circuits governing the expression of chitinases upon contact of Trichoderma with its host (Kubicek et al. 2001). Cloning of the genes coding for the 42kDa secreted chitinases has allowed the construction of a phylogenetic tree, which showed that they belong to family 18, class V of the glycosyl hydrolases. Interestingly, of the eight fungal species within this clade of the phylogenetic tree, all of them are either fungal or insect parasites and many of the corresponding genes have been implicated in their parasitic activity. However, the chitinolytic system of Trichoderma was recently found to be more complex. Two genes showing similarity to the one encoding the 33 kDa endochitinase described by De la Cruz et al. (1992) have been cloned from T. virens (Kim et al. 2002). These two genes are closely related, according to phylogenetic analysis, and belong to family 18, class III of the glycosyl hydrolases (Kim et al. 2002). Further, at least two types of N-acetyl-p-d-glucosaminidases belonging to family 20 of the glycosyl hydrolases have been identified in T. harzianum and T. virens (Draborg et al. 1995; Kim et al. 2002). Chit 36 is another antifungal chitinase recently isolated from T. harzianum TM. This 36 kDa protein shares no significant homology to either Chit33 or 42 (Viterbo et al. 2001). In addition, a 40kDa chitobiosidase and a 28 kDa exochitinase have been purified (Deane et al. 1998; Harman et al. 1993).

In 1995, Haran and co-workers identified six distinct intracellular chitinases by activity on gels. This intracellular set of chitinases is apparently composed of two p-1,4-N-acetylglucosaminidases of 102 and 73 kDa, respectively, and four endochitinases of 52, 42, 33, and 31 kDa, respectively. From this set, the 102 kDa and the 73 kDa N-acetyl glucosaminidases and the 42 kDa endochitinase, were

Table l Trichoderma genes encoding cell-wall degrading enzymes

Gene

Trichoderma spp.

Strain

Encoded protein

References

Th-En42

T.

atroviride

P1

42-kDa endochitinase

Hayes et al. (1994)

ech42

T.

atroviride

IMI206040

42-kDa endochitinase

Carsolio et al. (1994)

chit42

T.

harzianum

CECT241S

42-kDa endochitinase

Garcia et al. (1994)

ech1

T.

virens

Tv29-B

42-kDa endochitinase

Kim et al. (2002)

th-ch

T.

harzianum

Tam-61

42-kDa endochitinase

Fekete et al. (1996)

ENC1

T.

harzianum

T25-1

42-kDa endochitinase

Draborg et al. (1996)

ech2

T.

virens

Tv29-B

42-kDa endochitinase

Kim et al. (2002)

echS

T.

virens

Tv29-B

Endochitinase

Kim et al. (2002)

chitSS

T.

harzianum

CECT241S

33-kDa endochitinase

Limon et al. (1995)

cht1

T.

virens

Tv29-B

33-kDa endochitinase

Kim et al. (2002)

cht2

T.

virens

Tv29-B

33-kDa endochitinase

Kim et al. (2002)

chitS6

T.

harzianum

TM

36-kDa endochitinase

Viterbo et al. (2001)

nag1

T.

atroviride

P1

73-kDa N-acetyl-b-d-glucosaminidase

Peterbauer et al. (1996)

exc1

T.

harzianum

T25-1

73-kDa N-acetyl-b-d-glucosaminidase

Draborg et al. (1995)

nag1

T.

virens

Tv29-B

N-acetyl-b-d-glucosaminidase

Kim et al. (2002)

exc2

T.

harzianum

T25-1

N-acetyl-b-d-glucosaminidase ??

Draborg et al. (1995)

nag2

T.

virens

Tv29-B

N-acetyl-b-d-glucosaminidase

Kim et al. (2002)

bgn1S.1

T.

harzianum

CECT 241S

78 kDa b-1,3-endoglucanase

De la Cruz et al. (1995)

bgn1

T.

virens

Tv29-B

78 kDa b-1,3-endoglucanase

Kim et al. (2002)

bgn2

T.

virens

Tv29-B

78 kDa b-1,3-endoglucanase

Kim et al. (2002)

gluc?B

T.

atroviride

P1

78 kDa exo-b-1,3-glucosidase

Donzelli et al. (2001)

bgnS

T.

virens

Tv29-B

b-1,6-endoglucanase

Kim et al. (2002)

prb1

T.

atroviride

IMI206040

31-kDa subtilisinlike protease

Geremia et al. (1993)

expressed differentially when Trichoderma was confronted with different hosts on plates (Haran et al. 1996; Inbar and Chet 1995). In conclusion, the complexity and diversity of the chitinolytic system of T. harzianum involves the complementary modes of action of a diversity of enzymes, all of which might be required for maximum efficiency against a broad spectrum of chitin containing plant pathogenic fungi.

Trichoderma atroviride also secretes ß-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of ß-1,3-glucanase activity secreted by T. atroviride was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular ß-1,3-glucanases upon induction with laminarin, a soluble ß-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60 to 80 kDa, and their pis are 5.0-6.8. In addition, a 35-kDa protein with a pi of 5.5 and a 39-kDa protein are also secreted (Vazquez-Garciduenas et al. 1998). bgn13, which encodes a 78 kDa protein from T. harzianum was the first endoglucanase gene identified (De la Cruz et al. 1995). Recently, two genes showing high homology to bgn13.1 have been identified in T. virens. These endoglucanases belong to family 55 of the glycosyl hydrolases (Kim et al. 2002). In addition, a 78 kDa exo-ß-1,3-glucanase from T. harzianum has been characterized (Donzelli and Harman 2001).

From the set of glucanases produced by Trichoderma two ß-1,6-endoglucanase genes have been identified, one in T. harzianum and one in T. virens (Kim et al. 2002; Lora et al. 1995). These two genes encode nearly identical proteins belonging to family 5 of the glycosyl hydrolases (Kim et al. 2002).

In 1993, Geremia and co-workers reported the isolation of a 31 kDa basic proteinase, which is secreted by T. harzianum during simulated mycoparasitism. The corresponding gene (prb1) was cloned and characterized (Geremia et al. 1993) and was the first report of the cloning of a mycoparasitism-related gene.

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