Secretome of Trichoderma interacting with maize roots: role in induced systemic resistance

Abstract

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.

Fig. 1.

Effect of T. virens on ISR in leaves of maize seedlings challenged with the maize pathogen C. heterostrophus. A, The hydroponic system of 11 day-old maize seedlings colonized for 4 days with T. virens. B, Lesion development in leaves of ± T. virens-induced maize, 2 days after pathogen challenge. C, Lesion size measured from photographed leaves of plants colonized with T. virens or control plants. Number of lesions measured in control plants, 48; with T. virens, 34. Bars indicate the S.E., unpaired t test, one-tailed, *** indicates significance at p < 0.001).

Fig. 2.

ISR activity of secreted proteins (SP). The secreted protein fractions from T. virens alone (Tv SP) or T. virens interacting with maize roots (Tv+M SP) were applied to the roots of plants for 24 h, followed by pathogen challenge of the leaves (C. heterostrophus). Lesion sizes were measured and were compared with the mock treatment, the growth medium. Number of lesions measured in mock-treated plants, 41; Tv SP, 48; Tv+M SP, 45. Bars indicate the S.E., unpaired t test, one-tailed, * indicates significance at p < 0.05, *** indicates significance at p < 0.001).

Fig. 3.

SDS-PAGE of T. virens secreted proteins ± maize roots. Three biological replicates of T. virens secreted proteins ± maize roots (Tv SP, Tv+M SP) were separated by SDS-PAGE (15% acrylamide). 40 μl of concentrated samples were loaded to compare the pattern of secreted proteins, which was further analyzed by mass spectrometry. Protein concentrations from left lane to right lane: 157, 294, 266, 425, 360, and 282 μg/ml.

Fig. 4.

Pie charts of the proteins whose abundance was significantly different (p < 0.05) between the two treatments. A, Proteins that were found in higher abundance in the Tv treatment and were reduced by maize roots, B, Proteins that were found in higher abundance in the Tv+M treatment and were induced by maize roots. Full list of all proteins identified, with the detailed proteomics data is available in supplemental Table S1.

Fig. 5.

Scatter plot of all proteins identified. We compared the Log_{2_(}intensity) (iBAQ, see supplemental Table S1) in each of the treatments (Tv or Tv+M) as calculated from the mass-spectrometry analysis. Proteins that are found near the dashed black line are proteins that did not change in their abundance between treatments (Sm1 for example), whereas proteins that are found above or below the dashed line are proteins that were found in higher abundance in the Tv or Tv+M treatment respectively. All the SSCPs identified are marked with red. The four SSCPs that were chosen as candidates for knock-out are marked with their JGI protein IDs.

Fig. 6.

ISR ability and root colonization by SSCPs mutants. A, Lesion development on leaves of T. virens (WT and mutants)-induced maize, 2 days after pathogen challenge. Number of lesions measured from left column to right; 88, 58, 44, 42, and 18. Note that the lesion numbers for the various fungal lines differ because all experiments performed were included (thus, for example, the number of WT control lesions is highest). B, Colonization of maize roots by the SSCPs mutants, relative to the colonization by WT. Number of repeats from three to four independent experiments from left column to right; 20, 16, 16, 13, and 21. C, Lesion development in leaves of maize seedlings whose roots were dipped for 24 h in the secreted protein fraction (SP) collected from each T. virens line grown alone. Images were recorded 2 days after pathogen challenge. Number of lesions measured from left column to right: 19, 19, 19, 18, and 17. Bars indicate the S.E., unpaired t test, one-tailed, * indicates significance at p < 0.05, *** indicates significance at p < 0.001.