Trichoderma harzianum transcriptome in response to cadmium exposure.

Cadmium (Cd) is a heavy metal present in the environment mainly as a result of industrial contamination that can cause toxic effects to life. Some microorganisms, as Trichoderma harzianum, a fungus used in biocontrol, are able to survive in polluted environments and act as bioremediators. Aspects about the tolerance to the metal have been widely studied in other fungi although there are a few reports about the response of T. harzianum. In this study, we determined the effects of cadmium over growth of T. harzianum and used RNA-Seq to identify significant genes and processes regulated in the metal presence. Cadmium inhibited the fungus growth proportionally to its concentration although the fungus exhibited tolerance as it continued to grow, even in the highest concentrations used. A total of 3767 (1993 up and 1774 down) and 2986 (1606 up and 1380 down) differentially expressed genes were detected in the mycelium of T. harzianum cultivated in the presence of 1.0 mg mL-1 or 2.0 mg mL-1 of CdCl2, respectively, compared to the absence of the metal. Of these, 2562 were common to both treatments. Biological processes related to cellular homeostasis, transcription initiation, sulfur compound biosynthetic and metabolic processes, RNA processing, protein modification and vesicle-mediated transport were up-regulated. Carbohydrate metabolic processes were down-regulated. Pathway enrichment analysis indicated induction of glutathione and its precursor's metabolism. Interestingly, it also indicated an intense transcriptional induction, especially by up-regulation of spliceosome components. Carbohydrate metabolism was repressed, especially the mycoparasitism-related genes, suggesting that the mycoparasitic ability of T. harzianum could be affected during cadmium exposure. These results contribute to the advance of the current knowledge about the response of T. harzianum to cadmium exposure and provide significant targets for biotechnological improvement of this fungus as a bioremediator and a biocontrol agent.

Overexpression of an aquaglyceroporin gene from Trichoderma harzianum improves water-use efficiency and drought tolerance in Nicotiana tabacum.

Aquaporins (AQPs) and aquaglyceroporins (AQGPs) are integral membrane proteins that mediate the transport of water and solutes, such as glycerol and urea, across membranes. AQP and AQGP genes represent a valuable tool for biotechnological improvement of plant tolerance to environmental stresses. We previously isolated a gene encoding for an aquaglyceroporin (ThAQGP), which was up-regulated in Trichoderma harzianum during interaction with the plant pathogen Fusarium solani. This gene was introduced into Nicotiana tabacum and plants were physiologically characterized. Under favorable growth conditions, transgenic progenies did not had differences in both germination and growth rates when compared to wild type. However, physiological responses under drought stress revealed that transgenic plants presented significantly higher transpiration rate, stomatal conductance, photosynthetic efficiency and faster turgor recovery than wild type. Quantitative RT-PCR analysis demonstrated the presence of ThAQGP transcripts in transgenic lines, showing the cause-effect relationship between the observed phenotype and the expression of the transgene. Our results underscore the high potential of T. harzianum as a source of genes with promising applications in transgenic plants tolerant to drought stress.

Cloning and characterization of a protein elicitor Sm1 gene from Trichoderma harzianum.

A small protein, cysteine-rich, designated SM1, produced by Trichoderma virens and Trichoderma atroviride, acts as elicitor for triggering plant defense reactions. We analyzed Sm1 gene expression of eight different strains of Trichoderma spp. grown on glucose, seeds or roots of beans. Regardless of the carbon source, T37 strain had significantly higher Sm1 expression and was chosen for further studies. When grown on different carbon sources, Sm1 expression was highest on galactose, bean seed, glucose and starch. Sm1 gene from T37 strain was cloned; it had a single exon, and encoded a protein of 138 amino acids, showing high sequence identity with some proteins belonging to the cerato-platanin family.

Optimized microplate beta-1,3-glucanase assay system for Trichoderma spp. screening.

Beta-1,3-glucanase is an important cell wall-degrading enzyme involved in mycoparasitism by Trichoderma spp. during antagonism against phytopathogenic fungi. A simple microplate-based method to assay beta-1,3-glucanase activity is described here as an alternative to an expensive tube-assay method. The reaction volume of the micro-assay was reduced to 130 microl from the 1150 microl used in the standard beta-1,3-glucanase macro-assay. Statistical analyses showed significant difference in sensitivity between the micro- and the macro-assay. The micro-method was optimized using the Response Surface Quadratic Model. The sensitivity of the optimized micro-method was shown to be four-fold greater than the macro-assay and two-fold higher than the micro-assay. The optimized micro-assay was significantly more sensitive in all of the twenty examined isolates during Trichoderma spp. beta-1,3-glucanase screening. We conclude that this modified and optimized method is more convenient, faster, cheaper and more reproducible than the traditional tube-assay.

Involvement of G-alpha protein GNA3 in production of cell wall-degrading enzymes by Trichoderma reesei (Hypocrea jecorina) during mycoparasitism against Pythium ultimum.

The involvement of the G-alpha protein GNA3 in the production of cell wall-degrading enzymes (CWDEs) by Trichoderma reesei during antagonism against Pythium ultimum was investigated. cAMP content was 2.8-fold higher in the T. reesei mutant gna3QL than in the parental TU-6. The gna3QL, like TU-6, inhibited the growth of P. ultimum in dual culture assays. Scanning electron microscopy showed that the gna3QL promoted more morphological alterations of P. ultimum cell wall than TU-6. In general, gna3QL produced higher activities of CWDEs than TU-6. We therefore suggest that CWDEs production during mycoparasitism by T. reesei against P. ultimum may be associated with the level of GNA3 activity.

Studies on the Pycnoporus sanguineus CCT-4518 laccase purified by hydrophobic interaction chromatography.

A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by 12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K (m) values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 muM, respectively. The enzyme's pH optimum for syringaldazine was 4.2 and optimal activity was 50 degrees C. The enzyme showed to be thermostable because when kept at 50 degrees C for 24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by L: -cysteine, beta-mercaptoethanol, NaN(3), NaF, and HgCl(2).

Properties of laccases produced by Pycnoporus sanguineus induced by 2,5-xylidine.

Two isoforms of laccase produced from the culture supernatant of Pycnoporus sanguineus were partially purified by phenyl-Sepharose chromatography. Molecular masses of the enzymes were 80 kDa (Lac I) and 68 kDa (Lac II). Optimum activity of Lac I was at pH 4.8 and 30 degrees C, and Lac II was at pH 4.2 and 50 degrees C over 5 min reaction. The Km values of enzymes toward syringaldazine were 10 microM: (Lac I) and 8 microM: (Lac II). Sodium azide inhibited Lac I (85%) and Lac II (75%) activities.