Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic resistance

Abstract

The role of a mitogen-activated protein kinase (MAPK) TmkA in inducing systemic resistance in cucumber against the bacterial pathogen Pseudomonas syringae pv. lacrymans was investigated by using tmkA loss-of-function mutants of Trichoderma virens. In an assay where Trichoderma spores were germinated in proximity to cucumber roots, the mutants were able to colonize the plant roots as effectively as the wild-type strain but failed to induce full systemic resistance against the leaf pathogen. Interactions with the plant roots enhanced the level of tmkA transcript in T. virens and its homologue in Trichoderma asperellum. At the protein level, we could detect the activation of two forms reacting to the phospho-p44/42 MAPK antibody. Biocontrol experiments demonstrated that the tmkA mutants retain their biocontrol potential against Rhizoctonia solani in soil but are not effective against Sclerotium rolfsii in reducing disease incidence. Our results show that, unlike in many plant-pathogen interactions, Trichoderma TmkA MAPK is not involved in limited root colonization. Trichoderma, however, needs MAPK signaling in order to induce full systemic resistance in the plant.

FIG. 1.

Cucumber root colonization by T. virens wild type and tmkA mutant. (A) Roots were detached 48 h postinoculation and washed. After sterilization in 1% NaOCl for 2 min, the roots were homogenized in water and serial dilutions were plated on Trichoderma selective medium at 30°C. Bars: 1, WT; 2, ΔMAPK-72; 3, ΔMAPK-95; 4, ΔMAPK-7. (B) Expression of Trichoderma tmkA and the β-tubulin gene (βtub) was assessed 48 h after inoculation in cucumber roots by RT-PCR with specific primers. Actin of C. sativus was used as a reference for RNA equal loading. Noninoculated roots (R) were used as controls for primer specificity.

FIG. 2.

Effect of Trichoderma root inoculation on multiplication of P. syringae pv. lachrymans in challenged cotyledons, 96 h after infection. (A) Bars: 1, challenged plants, no Trichoderma inoculation; 2, inoculation with WT; 3, ΔMAPK-72; 4, ΔMAPK-95; 5, ΔMAPK-7. Columns represent means ± standard deviations of three independent experiments from which 10 cotyledons were randomly selected. (B) Bioassay comparing the antimicrobial activity of 2 to 10 μl of the aglycone fraction obtained by acid hydrolysis of crude phenolic extract of cucumber leaves 48 h postchallenge with P. syringae pv. lachrymans. Plants elicited with T. virens (black columns) or elicited with mutants ΔMAPK-95 (light gray columns) and ΔMAPK-7 (dark gray columns). The antimicrobial activity was assayed on P. syringae pv. lachrymans. Columns represent the mean inhibition diameters of three independent experiments ± standard deviations.

FIG. 3.

Systemic induction of pal1 and hpl by T. asperellum (T-203), T. virens (T.v) wild type, and a T. virens tmkA mutant (ΔM). (A) Expression of hpl and pal1 genes in cucumber leaves was assessed 48 h after Trichoderma inoculation of roots by RT-PCR with specific primers (24). Actin of C. sativus was used as a reference for RNA equal loading. (B) Quantification of mRNA fold induction. The values obtained for each band were normalized to actin. A value of 1 indicates no induction relative to basal expression in noninoculated plants. The results are averages of three independent experiments, and the error bars indicate standard deviations. Similar results were obtained with mutants ΔMAPK-72 and ΔMAPK-95.

FIG. 4.

tmkA/task1 transcipt induction by root contact. T. virens (TV, wild type; ΔM, mutants) and T. asperellum (T-203) were washed out from cucumber seedlings (+) 48 h postinoculation, and expression of tmkA/task1 was determined by RT-PCR with specific primers. RNA extracted from mycelia grown in SM with 0.05% glucose on nylon fibers was used as control (−). For the quantification of mRNA fold induction, the values obtained for each band were normalized to β-tubulin. A value of 1 indicates no induction relative to basal expression in the Trichoderma control. The results are the averages of three independent experiments.

FIG. 5.

Activation of Trichoderma MAPK by plant root interaction. Mycelia interacting with cucumber seedlings were harvested after 48 h (+). Mycelia grown in SM with 0.05% glucose were used as control (−). Cell lysates of T. asperellum (T-203), of the T. virens wild type (T.v-WT), and of the tmkA mutant (ΔMAPK-7) were analyzed by Western blotting using antibodies that specifically recognize either the dually phosphorylated forms of ERK1/2 or total ERK2 polypeptide.

FIG. 6.

Biocontrol activity of T. virens and tmkA mutant against R. solani (A) and S. rolfsii (B). The disease percentage was recorded 10 days after seed planting and inoculation. The results are averages of three independent experiments with 60 plants in each treatment; bars indicate standard deviations between experiments.