Expression of Endochitinase from Trichoderma harzianum in Transgenic Apple Increases Resistance to Apple Scab and Reduces Vigor.

ABSTRACT The goal of this research was to improve scab resistance of apple by transformation with genes encoding chitinolytic enzymes from the bio-control organism Trichoderma harzianum. The endochitinase gene, as cDNA and genomic clones, was transferred into apple cv. Marshall McIntosh by Agrobacterium-transformation. A total of 15 lines were identified as transgenic by NPTII enzyme-linked immunosorbent assay and polymerase chain reaction and confirmed by Southern analysis. Substantial differences in endochitinase activity were detected among different lines by enzymatic assay and western analysis. Eight lines propagated as grafted and own-rooted plants were inoculated with Venturia inaequalis. Six of these transgenic lines expressing endochitinase were more resistant than nontransformed cv. Marshall McIntosh. Disease severity compared with cv. Marshall McIntosh was reduced by 0 to 99.7% (number of lesions), 0 to 90% (percentage of leaf area infected), and 1 to 56% (conidia recovered) in the transgenic lines tested. Endochitinase also had negative effects on the growth of both inoculated and uninoculated plants. There was a significant negative correlation between the level of endochitinase production and both the amount of disease and plant growth.

Ecological Studies of Transformed Trichoderma harzianum Strain 1295-22 in the Rhizosphere and on the Phylloplane of Creeping Bentgrass.

ABSTRACT A beta-glucuronidase (GUS) reporter gene and a hygromycin B (hygB) phosphotransferase gene were integrated separately into the Trichoderma harzianum strain 1295-22 genome, using biolistic transformation. The mycelial growth and biocontrol ability of the transformed strains did not differ from that of the original strain. The transformed Gus(+)-kanamycin-resistant (Gus(+)Kan(R)) strains were used to monitor growth and interactions with Rhizoctonia solani on creeping bentgrass plants. The hygB-resistant (hygB(R)) strains were used to selectively recover strain 1295-22 from the rhizosphere soil and phylloplane of creeping bentgrass after spray applications. The population levels of two hygB(R) strains and the original strain were very similar for all treatments. All three strains persisted for the duration of the experiment (28 days) in both the rhizosphere soil and on leaves, although population levels declined somewhat over the course of the experiment in unautoclaved soils. In this study, the results demonstrated that hygB(R) strains remained dominant over time when assayed on Trichoderma-selective medium containing hygB. The hygB(R) strains were not displaced by strains that colonized untreated plants. Microscopic observation showed that the Gus(+)Kan(R) strains colonized the rhizoplane, seed coat, and phylloplane of creeping bentgrass. These results supported our earlier observation that strain 1295-22 was rhizosphere and phyllo-plane competent. Interactions between T. harzianum and R. solani were readily observed in situ and changed over time. Two types of reactions were found in these experiments. In the first type, sections of hyphae of R. solani near the hyphae of T. harzianum appeared damaged, and the pathogen appeared necrotic when viewed with a microscope. The second type, observed less frequently than the first type, was typical of myco-parasitism. The findings in this study provide new insight into the interactions between R. solani and T. harzianum, providing a basis for future research.

Molecular cloning and expression of the nag1 gene (N-acetyl-beta-D-glucosaminidase-encoding gene) from Trichoderma harzianum P1.

A 72-kDa N-acetyl-beta-D-glucosaminidase was purified from the mycoparasitic fungus Trichoderma harzianum P1; antibodies were raised against it, and aa-sequences were obtained. The antibody reacted with a single 72-kDa protein band in culture filtrates of T. harzianum grown on chitin, and was subsequently used to clone the corresponding nag1 gene from a lambdagt11 cDNA expression library. It was interrupted by two short introns and encoded a protein of 580 amino acids. The deduced protein sequence contained aa-sequence areas of high similarity to N-acetyl-glucosaminidases from other eukaryotes such as Candida albicans, and invertebrate and vertebrate animal tissues. The highest similarity was observed with the corresponding gene from the silkworm. The aa-sequence of a tryptic fragment of purified N-acetyl-beta-D-glucosaminidase from T. harzianum corresponded to a deduced aa sequence from a portion of the cloned gene, thus verifying that the protein is encoded by nag 1. Southern analysis showed that nag 1 is present as a single-copy gene in T. harzianum. Expression of nag1-mRNA was strongly induced upon growth on chitin, N-acetyl-glucosamine and the cell walls of Botrytis cinerea used as a carbon source. The appearance of the corresponding N-acetyl-beta-D-glucosaminidase protein, as determined by Western analysis, paralleled the pattern of nag 1 expression, thereby suggesting that its formation is regulated at the level of transcription.

Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei.

The chromosomal endochitinase gene (ThEn-42) of the mycoparasite fungus Trichoderma harzianum P1 was isolated and overexpressed in the filamentous fungus Trichoderma reesei under the promoter of the major cellulase gene cbhl1. The host strain RutC-30 did not produce any endogenous endochitinase activity. The prepro region of the T harzianum endochitinase was correctly processed in T. reesei. No differences in expression were observed when the prepro region was replaced with the CBHI signal sequence. Shake flask cultivation yielded 130 mg of active enzyme per liter, which in terms of activity represents about a 20-fold increase over the endochitinase activity produced by T. harzianum. The presence of multiple copies of the expression cassette in the transformant resulted in limitation in transcription and/or regulation factors needed for full activity of the cbh1 promoter, although this was not the major limiting factor for higher expression of endochitinase. The endochitinase was very sensitive to an acidic protease at the late stages of T. reesei cultivation. T. reesei RutC-30 appeared to be tolerant of the endochitinase and can be used as a production host for this enzyme, which has antifungal activity toward plant pathogens.

Isolation and characterization of a cDNA from Trichoderma harzianum P1 encoding a 14-3-3 protein homolog.

A full-length cDNA close, Th1433, (GenBank accession No. U24158), was isolated and characterized from the filamentous fungus, Trichoderma harzianum. The deduced amino acid (aa) sequence showed an acidic 30-kDa protein homologous to the 14-3-3 proteins, a family of putative kinase regulators originally characterized in mammalian brain tissue. The greatest homology, 71% identical aa, was found to BMH1, the corresponding protein from Saccharomyces cerevisiae and to the epsilon isoform from sheep brain. Southern analysis of genomic DNA indicated that Th1433 is a member of a small genomic family. At least two genes encoding 14-3-3-like proteins exist in T. harzianum. Northern analysis showed the highest level of expression during the first day after inoculation of the culture with conidial spores.

Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi.

Chitinase, beta-1,3-glucanase, and protease activities were formed when Trichoderma harzianum mycelia, grown on glucose as the sole carbon source, were transferred to fresh medium containing cell walls of Botrytis cinerea. Chitobiohydrolase, endochitinase, and beta-1,3-glucanase activities were immunologically detected in culture supernatants by Western blotting (immunoblotting), and the first two were quantified by enzyme-linked immunosorbent assay. Under the same conditions, exogenously added [U-14C]valine was incorporated in acetone-soluble compounds with an apparent M(r) of < 2,000. These compounds comigrated with the peptaibols trichorzianines A1 and B1 in thin-layer chromatography and released [U-14C]valine after incubation in 6N HCl. Incorporation of radioactive valine into this material was stimulated by the exogenous supply of alpha-aminoisobutyric acid, a rare amino acid which is a major constituent of peptaibols. The obtained culture supernatants inhibited spore germination as well as hyphal elongation of B. cinerea. Culture supernatants from mycelia placed in fresh medium without cell walls of B. cinerea did not show hydrolase activities, incorporation of [U-14C]valine into peptaibol-like compounds, and inhibition of fungal growth. Purified trichorzianines A1 and B1 as well as purified chitobiohydrolase, endochitinase, or beta-1,3-glucanase inhibited spore germination and hyphal elongation, but at concentrations higher than those observed in the culture supernatants. However, when the enzymes and the peptaibols were tested together, an antifungal synergistic interaction was observed and the 50% effective dose values obtained were in the range of those determined in the culture supernatants. Therefore, the parallel formation and synergism of hydrolytic enzymes and antibiotics may have an important role in the antagonistic action of T. harzianum against fungal phytopathogens.

Potential of genes and gene products from Trichoderma sp. and Gliocladium sp. for the development of biological pesticides.

Fungal cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes from T. harzianum were able to improve substantially the antifungal ability of a biocontrol strain of Enterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library of T. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.

Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination.

Different classes of cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gliocladium virens inhibited spore germination of Botrytis cinerea in a bioassay in vitro. The addition of any chitinolytic or glucanolytic enzyme to the reaction mixture synergistically enhanced the antifungal properties of five different fungitoxic compounds against B. cinerea. The chemicals tested were gliotoxin, flusilazole, miconazole, captan and benomyl. Dose response curves were determined for each combination of toxin and enzyme, and in all cases the ED50 values of the mixtures were substantially lower than ED50 values of the two compounds used alone. For instance, the addition of endochitinase from T. harzianum at a concentration of 10 micrograms ml-1 reduced the ED50 values of toxins up to 86-fold. The level of synergism appeared to be higher when enzymes were combined with toxins having primary sites of action associated with membrane structure, compared with pesticides having multiple or cytoplasmic sites of action. Among enzymes tested, the highest levels of synergism with synthetic fungicides were detected for the endochitinase from T. harzianum strain P1, which, when used alone, was the most effective chitinolytic enzyme against phytopathogenic fungi of those tested. The use of hydrolytic enzymes to synergistically enhance the antifungal ability of fungitoxic compounds may reduce the impact of some chemical pesticides on plants and animals.

Isolation and sequence of an endochitinase-encoding gene from a cDNA library of Trichoderma harzianum.

There are no reports of gene sequences coding for extracellular chitinolytic enzymes from filamentous fungi, even though these enzymes are considered critical to the biological control of plant pathogenic fungi. The purpose of this paper was to report the isolation of a gene (ThEn-42) encoding endochitinase (Ech) from Trichoderma harzianum strain P1, describe its sequence, and to determine whether it was related to genes coding for enzymes with similar functions from prokaryotic or other eukaryotic sources. A clone containing a 1096-bp foreign cDNA fragment was isolated from thalli grown under induced conditions. This cDNA molecule was sequenced and found to lack a portion of the 5' terminus. Polymerase chain reaction (PCR) was used to isolate a fragment from the lambda gt11 library which contained the 5' terminus plus an overlap region with the 1096-bp cDNA clone. The full-length cDNA sequence, consisting of 1554 bp, contained an open reading frame (ORF) expressing a protein of 424 amino acids (aa). Southern analysis of genomic DNA indicated that there is only a single gene in strain P1 with sequence identity to the sequence described in this report. One region within the protein, thought to be required for catalytic activity of the enzyme, was highly conserved between genes coding for Ech from Th, Serratia marcescens, Bacillus circulans, Streptomyces plicatus, Vibrio parahemolyticus and Kluyveromyces lactis.

Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA.

Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl2 protoplast fusion protocol. Hygromycin B-resistant (HygBR) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygBR progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus.

Methods for electrophoretic karyotyping of filamentous fungi in the genus Trichoderma.

Methods for electrophoretic karyotyping of filamentous fungi in the genus Trichoderma were developed. These techniques permitted the separation and visualization of intact chromosomes from viable protoplasts. Three strains were analyzed: Trichoderma harzianum strains T12 his-2 and T95 lys-1, and a prototrophic strain (1295-22) produced by protoplast fusion of T12 his-2 with T95 lys-1. Four chromosome bands ranging in size from 2.2 Mb (megabase pairs) to 5.4 Mb were visualized with strain T95 lys-1, whereas two chromosome bands were visualized for strains T12 his-2 and 1295-22. The largest chromosome of all three strains seems to be similar in size and has been estimated to be approximately 5.4 Mb. All remaining chromosomes observed were dissimilar in size. Methods for protoplast isolation, protoplast embedding, and electrophoretic conditions useful for separation of intact chromosomes ranging in size from 50 kb (kilobase pairs) up to approximately 6.0 Mb utilizing transverse alternating field electrophoresis (TAFE) will be discussed. The techniques provided should be applicable to a variety of lower eukaryotic organisms when using the TAFE system.