Trichoderma virens is a biocontrol agent used in agriculture to antagonize pathogens of crop plants. In addition to direct mycoparasitism of soil-borne fungal pathogens,
T. virens interacts with roots. This interaction induces systemic resistance (ISR), which reduces disease in above-ground parts of the plant. In the molecular dialog between fungus and plant leading to ISR, proteins secreted by
T. virens provide signals. Only a few such proteins have been characterized previously. To study the secretome, proteins were characterized from hydroponic culture systems with
T. virens alone or with maize seedlings, and combined with a bioassay for ISR in maize leaves infected by the pathogen
Cochliobolus heterostrophus. The secreted protein fraction from coculture of maize roots and
T. virens (Tv+M) was found to have a higher ISR activity than from
T. virens grown alone (Tv). A total of 280 fungal proteins were identified, 66 showing significant differences in abundance between the two conditions: 32 were higher in Tv+M and 34 were higher in Tv. Among the 34 found in higher abundance in Tv and negatively regulated by roots were 13 SSCPs (small, secreted, cysteine rich proteins), known to be important in the molecular dialog between plants and fungi. The role of four SSCPs in ISR was studied by gene knockout. All four knockout lines showed better ISR activity than WT without affecting colonization of maize roots. Furthermore, the secreted protein fraction from each of the mutant lines showed improved ISR activity compared with WT. These SSCPs, apparently, act as negative effectors reducing the defense levels in the plant and may be important for the fine tuning of ISR by
Trichoderma. The down-regulation of SSCPs in interaction with plant roots implies a revision of the current model for the
Trichoderma-plant symbiosis and its induction of resistance to pathogens.Fungi belonging to the genus
Trichoderma are used as biocontrol agents in agriculture. In addition to direct antagonism of soil-borne pathogens, these fungi intimately interact with plant roots, and are thus considered rhizosphere-competent (
1). The interaction is, in general, a beneficial one, promoting plant growth as well as inducing systemic resistance (ISR)
1 to pathogens (
2–
6). The elicitation of defense response in the leaves of plants whose roots are colonized with
Trichoderma enhances the plant''s resistance to foliar pathogens. This clear potential for application in agriculture is already beginning to be realized (
7–
9).Secreted proteins are central to the molecular dialog between fungi and their plant hosts. Recent studies addressed, for example, the molecular basis for mutualistic interactions between soil fungi and plants in mycorrhizae, a fungus-root symbiosis of widespread importance for nutrient acquisition. Specific secreted proteins were found to have targets in the plant (
10,
11). The
Trichoderma-root mutualism is distinct from these well-studied mycorrhizal symbioses, but some of the principles may be shared. Proteomic studies on several
Trichoderma species have been reviewed (
12). These studies employed total protein extracts from
Trichoderma interacting with plants, or the three-way
Trichoderma-plant-pathogen interaction (
13,
14), and led to the identification of some secreted proteins expressed during the interaction with plant and fungal hosts. Indeed, the first studies of secreted proteins demonstrated an abundant
Trichoderma secreted protein, belonging to the ceratoplatanins, which are a fungal family of secreted elicitors and toxins. This protein, Sm1 (in
T. virens)/Epl1 (
in T. atroviride (
15–
20) was shown to elicit ISR. The ceratoplatanin Sm1/Epl1 also belongs to a larger class of fungal proteins defined as SSCPs or SSPs: small, secreted (cysteine rich) proteins (
21–
23). There are no sequence motifs or domains common to the members of the entire SSCP class. Within the wide definition, though, there are subfamilies of proteins that do share sequence homology, for example the ceratoplatanin family to which Sm1 belongs.A bioinformatic survey of the SSCPs encoded in the genomes of three
Trichoderma species,
T. virens (Tv),
T. atrovirde (Ta), and
T. reesei (Tr) revealed several hundred candidate SSCPs in each species (
24). Approximately half of the SSCPs from each species have homologs in the same and/or in the other two species, whereas the other half are unique and do not share homology in or between the species. This diversity between the three species suggests that SSCPs are evolving rapidly.Given the known importance of Sm1, and the wide host range of
Trichoderma species, it seemed likely that many SSCPs might be involved in the
Trichoderma-root interaction. Secreted proteins (with emphasis on SSCPs), whose abundance changes in response to association with plant roots, may function in the fungal-plant molecular dialog. To test the hypothesis that the abundance of specific SSCPs and other secreted proteins is regulated by the interaction with plant roots, we compared the secretome of
Trichoderma alone to the secretome of
Trichoderma cocultured with the roots of maize seedlings. Functional experiments using knock out mutants in the genes encoding some of the regulated SSCPs were carried out in order to shed light on their role in the molecular interaction between the plant and the fungus.
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