Involvement of Trichoderma trichothecenes in the biocontrol activity and induction of plant defense-related genes

Abstract

Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studied.

Fig 1

(Left) Ion chromatograms of the trichodiene standard (A), extracted TaΔTri4 supernatant (B), supernatant extracted from xanthotoxin-treated strain Ta37 (C), and supernatant extracted from Ta37 without xanthotoxin (D). (Right) Schematic HA biosynthetic pathway. Dotted lines indicate the production of 12,13-epoxytrichoene-2-ol from trichodiene by the strains within the gray box. Note that this compound was detected in Ta37 cultures only after xanthotoxin treatment.

Fig 2

Effect of tri4 disruption or silencing on relative tri gene expression levels. (A) Comparison between the tri gene expression levels of TaΔTri4 and those of Ta37 determined by using REST2009 software. (B) TaS4-9 versus Ta37. Dotted-line squares indicate gene expression levels statistically different from those under the control conditions (n = 3; P < 0.05).

Fig 3

Fungal growth inhibition of R. solani after 10 days of incubation on plates prepared by growing Trichoderma strains for 2 days on cellulose membranes. As a control, a plate without previous Trichoderma growth (R.s. control) was included.

Fig 4

Relative tri gene expression levels of Ta37 in confrontation assays against B cinerea (A) and R. solani (B), determined by using REST2009 software. Dotted-line squares indicate gene expression levels statistically different from those under the control conditions (strain Ta37 grown without the pathogen) (n = 3; P < 0.05).

Fig 5

Effect of the presence of tomato roots on relative tri gene expression levels of Ta37 (A) and TaΔTri4 (B). Data were analyzed with REST2009 software. Dotted-line squares indicate gene expression levels statistically different from those under the control conditions (n = 3; P < 0.05). A schematic representation of a hydroponic culture is shown.

Fig 6

(A) Relative expression levels of defense-related genes belonging to the SA and JA pathways in 4-week-old tomato plants infected (+Bc) or not (−Bc) with B. cinerea, when seeds were coated with Ta37. The expression ratio data were obtained and represented by using REST2009 software. Black-line squares (SA genes) and dotted-line squares (JA genes) indicate gene expression levels statistically different from those under the control conditions (n = 3; P < 0.05). (B) Relative expression levels of defense-related genes belonging to the SA and JA pathways in 4-week-old tomato plants infected (+Bc) or not (−Bc) with B. cinerea when seeds were coated with TaΔTri4. The expression ratio data were obtained and represented by using REST2009 software. Black-line squares (SA genes) and dotted-line squares (JA genes) indicate gene expression levels statistically different from those under the control conditions (n = 3; P < 0.05). Note that the top left panels of panels A and B correspond to different repetitions of the same experiment. Thus, they are almost identical, as they represent the effect of B. cinerea when Trichoderma is not present (control condition).

Fig 7

Schematic representation of the network of interactions established among Ta37, B. cinerea, and tomato plants deduced from the present work. Arrows indicate response stimulation or gene upregulation, and blunt-ended lines indicate gene repression or growth inhibition. Red, blue, and green lines indicate interactions mediated by B. cinerea, tomato plant, and strain Ta37, respectively. a, sensitizing effect of Ta37-pretreated tomato plants mediated by HA; b, coupled action of HA and extracellular hydrolytic enzymes to inhibit B. cinerea growth; c, other metabolites produced by T. arundinaceum that, in addition to HA, would also affect its interaction with plants and with its fungal targets.