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

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.

Plant phenolic acids induce programmed cell death of a fungal pathogen: MAPK signaling and survival of Cochliobolus heterostrophus.

Plant aromatic compounds provide signals and a nutrient source to pathogens, and also act as stressors. Structure-activity relationships suggest two pathways sensing these compounds in the maize pathogen Cochliobolus heterostrophus, one triggering a stress response, and one inducing enzymes for their degradation. Focusing on the stress pathway, we found that ferulic acid causes rapid appearance of TUNEL-positive nuclei, dispersion of histone H1:GFP, hyphal shrinkage, and eventually membrane damage. These hallmarks of programmed cell death (PCD) were not seen upon exposure to caffeic acid, a very similar compound. Exposure to ferulic acid dephosphorylated two MAP kinases: Hog1 (stress activated) and Chk1 (pathogenicity related), while increasing phosphorylation of Mps1 (cell integrity related). Mutants lacking Hog1 or Chk1 are hypersensitive to ferulic acid while Mps1 mutants are not. These results implicate three MAPK pathways in the stress response. Ferulic acid and the antifungal fludioxonil have opposite additive effects on survival and on dephosphorylation of Hog1, which is thus implicated in survival. The results may explain why some fungal pathogens of plants undergo cell death early in host invasion, when phenolics are released from plant tissue.

Host-specific transcriptomic pattern of Trichoderma virens during interaction with maize or tomato roots.

BACKGROUND: Members of the fungal genus Trichoderma directly antagonize soil-borne fungal pathogens, and an increasing number of species are studied for their potential in biocontrol of plant pathogens in agriculture. Some species also colonize plant roots, promoting systemic resistance. The Trichoderma-root interaction is hosted by a wide range of plant species, including monocots and dicots. RESULTS: To test the hypothesis that gene expression by the fungal partner in this beneficial interaction is modulated by the plant, Trichoderma virens was co-cultured with maize or tomato in a hydroponic system allowing interaction with the roots. The transcriptomes for T. virens alone were compared with fungus-inoculated tomato or maize roots by hybridization on microarrays of 11645 unique oligonucleotides designed from the predicted protein-coding gene models. Transcript levels of 210 genes were modulated by interaction with roots. Almost all were up-regulated. Glycoside hydrolases and transporters were highly represented among transcripts induced by co-culture with roots. Of the genes up-regulated on either or both host plants, 35 differed significantly in their expression levels between maize and tomato. Ten of these were expressed higher in the fungus in co-culture with tomato roots than with maize. Average transcript levels for these genes ranged from 1.9 fold higher on tomato than on maize to 60.9 fold for the most tomato-specific gene. The other 25 host-specific transcripts were expressed more strongly in co-culture with maize than with tomato. Average transcript levels for these genes were 2.5 to 196 fold higher on maize than on tomato. CONCLUSIONS: Based on the relevant role of Trichoderma virens as a biological control agent this study provides a better knowledge of its crosstalk with plants in a host-specific manner. The differentially expressed genes encode proteins belonging to several functional classes including enzymes, transporters and small secreted proteins. Among them, glycoside hydrolases and transporters are highlighted by their abundance and suggest an important factor in the metabolism of host cell walls during colonization of the outer root layers. Host-specific gene expression may contribute to the ability of T. virens to colonize the roots of a wide range of plant species.

The Arabidopsis-related halophyte Thellungiella halophila: boron tolerance via boron complexation with metabolites?

Tolerance to boron (B) is still not completely understood. We tested here the hypothesis that Thellungiella halophila, an Arabidopsis thaliana-related 'extremophile' plant, with abundance of B in its natural environment, is tolerant to B, and examined the potential mechanisms of this tolerance. With 1-10 mm B applied ([B](ext)) to Thellungiella and Arabidopsis grown in hydroponics, the steady-state accumulated B concentration ([B](int)) in the root was below [B](ext), and was similar in both, suggesting both extrude B actively. Whether grown in soil or hydroponically, the shoot [B](int) was higher in Arabidopsis than in Thellungiella, suggesting more effective net B exclusion by Thellungiella root. Arabidopsis exhibited toxicity symptoms including reduced shoot fresh weight (FW), but Thellungiella was not affected, even at similar levels of shoot-accumulated [B](int) (about 10 to 40 mm B in 'shoot water'), suggesting additional B tolerance mechanism in Thellungiella shoot. At [B](ext) = 5 mm, the summed shoot concentration of the potentially B-binding polyhydroxyl metabolites (malic acid, fructose, glucose, sucrose and citric acid) in Arabidopsis was below [B](int) , but in Thellungiella it was over twofold higher than [B](int) , and therefore likely to allow appreciable 1:2 boron-metabolite complexation in the shoot. This, we suggest, is an important component of Thellungiella B tolerance mechanism.

Genetic interaction of the stress response factors ChAP1 and Skn7 in the maize pathogen Cochliobolus heterostrophus.

The transcription factors ChAP1 and Skn7 of the maize pathogen Cochliobolus heterostrophus are orthologs of Yap1 and Skn7 in yeast, where they are predicted to work together in a complex. Previous work showed that in C. heterostrophus, as in yeast, ChAP1 accumulates in the nucleus in response to reactive oxygen species (ROS). The expression of genes whose products counteract oxidative stress depends on ChAP1, as shown by impaired ability of a Δchap1 mutant to induce these 'antioxidant' genes. In this study, we found that under oxidative stress, antioxidant gene expression is also partially impaired in the Δskn7 mutant but to a milder extent than in the Δchap1 mutant, whereas in the double mutant - Δchap1-Δskn7 - none of the tested genes was induced, with the exception of one catalase gene, CAT2. Both single mutants are capable of infecting the plant, showing similar virulence to the WT. The double mutant, however, showed clearly decreased virulence, pointing to additive contributions of ChAP1 and Skn7. Possible mechanisms are discussed, including additive regulation of gene expression by oxidative stress.