Sm2, a paralog of the Trichoderma cerato-platanin elicitor Sm1, is also highly important for plant protection conferred by the fungal-root interaction of Trichoderma with maize

Abstract

Background: The proteins Sm1 and Sm2 from the biocontrol fungus Trichoderma virens belong to the cerato-platanin protein family. Members of this family are small, secreted proteins that are abundantly produced by filamentous fungi with all types of life-styles. Some species of the fungal genus Trichoderma are considered as biocontrol fungi because they are mycoparasites and are also able to directly interact with plants, thereby stimulating plant defense responses. It was previously shown that the cerato-platanin protein Sm1 from T. virens - and to a lesser extent its homologue Epl1 from Trichoderma atroviride - induce plant defense responses. The plant protection potential of other members of the cerato-platanin protein family in Trichoderma, however, has not yet been investigated.

Results: In order to analyze the function of the cerato-platanin protein Sm2, sm1 and sm2 knockout strains were generated and characterized. The effect of the lack of Sm1 and Sm2 in T. virens on inducing systemic resistance in maize seedlings, challenged with the plant pathogen Cochliobolus heterostrophus, was tested. These plant experiments were also performed with T. atroviride epl1 and epl2 knockout strains. In our plant-pathogen system T. virens was a more effective plant protectant than T. atroviride and the results with both Trichoderma species showed concordantly that the level of plant protection was more strongly reduced in plants treated with the sm2/epl2 knockout strains than with sm1/epl1 knockout strains.

Conclusions: Although the cerato-platanin genes sm1/epl1 are more abundantly expressed than sm2/epl2 during fungal growth, Sm2/Epl2 are, interestingly, more important than Sm1/Epl1 for the promotion of plant protection conferred by Trichoderma in the maize-C. heterostrophus pathosystem.

Figure 1

Gene expression of sm1 , sm2 and sm3 , analyzed with RT-PCR. (a) Biomass harvested from shake flask cultivations with glucose as carbon source (b) Biomass harvested from sporulating fungal cultures grown on agar plates (PDA). Tef1 was used as reference gene.

Figure 2

Colony growth of T. virens parental strain Δ tku70, sm1 and sm2 knockout strains on agar plates (PDA).

Figure 3

Gene expression (qPCR) of sm1 in the parental strain and sm2 knockout strain. Samples were taken at the indicated time points from shake flask cultivations with glucose as carbon source. All samples were normalized to the 24 h sample of the parental strain. Tef1 was used as reference gene. Bars indicate the SEM (*, ** and *** indicate significance at P < 0.05, 0.01 and 0.001).

Figure 4

Effect of T. virens (parental strain, ∆ sm1 and ∆ sm2 ) on plant protection in maize seedlings challenged with the maize pathogen C. heterostrophus . (a) Lesion development in leaves of T. virens-induced maize, two days after pathogen challenge. (b) Lesion size measured from photographed leaves. Number of lesions measured in control plant, 33; T. virens parental strain, 23; ∆sm1, 24; ∆sm2, 23. (c) Gene expression (qPCR) of sm1 and sm2 in maize seedlings infected with the pathogen C. heterostrophus and treated with the T. virens parental strain, sm1 and sm2 knockout strain. Number of lesions measured in control plant, 24; T. atroviride wild-type, 27; ∆epl1, 23; ∆epl2, 23; ∆epl1∆epl2, 24. Data from (b) and (d) represent the combined data from plants taken from two beakers. The experiment was carried out independently twice, with two biological repeats, each one containing 12 plants, with the same results within the variability. Bars indicate the SEM (standard error of the mean). *, ** and *** indicate significance at P < 0.05, 0.01 and 0.001. (d) Effect of T. atroviride (wild-type, ∆epl1, ∆epl2, and ∆epl1∆epl2) on plant protection in maize seedlings challenged with the maize pathogen C. heterostrophus. Lesion size measured from photographed leaves.