Browsing by Author "Monte, Enrique"
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- Diagnosis of ink disease of chestnut by molecular identification of associated phytophthora speciesPublication . Gouveia, Maria Eugénia; Coelho, Valentim; Choupina, Altino; Abreu, Carlos Gomes; Hermosa, R.; Monte, EnriqueFor diagnostic proposes of ink disease, chestnut orchards with symptoms of decline or sudden death of trees were sampled by soil baiting techniques and selective agar media (P10VPH). Thirty-six Phytophthora isolates were obtained. One isolate per tree and three or two isolates from the soil of the same plant were considered for molecular identification. Genomic DNA was extracted from all the isolates and from the reference strain P. cinnamomi CECT 2965. The ribosomal regions ITS1, 5.8S and ITS2 were amplified with the universal primer pair ITS6 (Cooke and Duncan, 1997) and ITS4 (White et al., 1990) by PCR. The amplified fragment (900 pb) was digested with restriction enzymes MspI, AluI and TaqI. Two different patterns of fingerprinting were obtained with enzymes TaqI and AluI (type I and II) and three different patterns with MspI (type I, Ia, II). The fingerprinting of each isolate was compared with database of CABI by public web access. Type I and Ia (14 isolates) were assigned to P. cinnamomi and type II (4 isolates) was assigned to P. cambivora. Molecular methods provide a rapid means of Phytophthora species identification associated with ink disease of chestnut and will provide a useful tool for etiological and epidemiological studies of this important disease of chestnut.
- HE-TAIL PCR, a powerfull tool for identification of Trichoderma harzianum lip1 genePublication . Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, AltinoTrichoderma harzianum is a widespread soil fungus, known as a biocontrol agent against soilborne plant pathogens. Species of Trichoderma are commercially applied as biological control agents against plant fungal pathogens based on different mechanisms, such as antibiosis, competition for nutrients and mycoparasitism. One of the mechanisms involved is the production of several lytic enzymes. In T. harzianum has been identified several glucanases, celulases, chitinases and proteases, but nothing are already known about its lipolytic system. In this work we described the identification of lip1 from T. harzianum, a gene encoding the first extracellular triacylglicerol lipase known in this specie. A cDNA library of T. harzianum CECT 2413 cloned in pBluescript SK+ was screened, and the expressed sequence tags (ESTs) with more similarity against lipase gene sequences in international databases selected and sequenced. Elucidation of complete gene nucleotide sequence of lip1, including 52 bp of the open reading frame at the N-terminal region and 693 bp of the promoter region were achieved by high-efficiency thermal asymmetric interlaced (HE-TAIL) PCR (Michiels et al., 2003), which confirmed to be a powerful tool to identify flanking regions from short known sequences. Lip1 codifies a 558 amino acids protein, with 59322,6 KDa and a calculated global iso-electric point value of 4,56. The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AM18087.
- Identification of Trichoderma harzianum lip2 gene by HE-TAIL PCRPublication . Jorge, Lurdes; Monte, Enrique; Gonzalez, Francisco J.; Choupina, AltinoTrichoderma harzianum is a widespread soil fungus, known as a biocontrol agent against soil borne plant pathogens. Its biological control against plant fungi pathogens is based on different mechanisms, namely the production of several lytic enzymes. In T. harzianum several glucanases, cellulases, chitinases and proteases, has been identified but little is known about its lipolytic system. The aim of this work was to achieve the complete elucidation of T. harzianum lip2 by HE-TAIL PCR (High-Efficiency Thermal Asymetric Interlaced PCR), a method described as efficient to identify flanking regions from short known DNA sequences. From a cDNA library of T. harzianum CECT 2413, obtained by NewBiotechnic, it has been selected an EST who showed lipase homology in agreement with the program FASTA: EST-1279. DNA sequencing was performed using an ABI 373 automated sequencer. After two sequencing rounds, EST-1279 had 1168bp, and a great homology (1,8e-80 ) with a hypothetical lipase of Fusarium graminearum. Comparison between two sequences, suggested that still are lacking 300bp of the beginning of the ORF. In order to determine the lacking bases, it was used an HE-TAIL PCR, that seemed suitable to the problem resolution. Degenerated 16bp primers R1 (5'- NGTCGASWGAMAWGAA-3'), R2 (5'-GTNCGASWCANAWGTT-3'), R3 (5'- WGTGNAGWANCANAGA-3') and R4 (5'-NCAGCTWSCTMTSCTT-3'), were used. Gene-specific primers, lip2a (5'-CTGGCAGAACCGATTCCCGAGCGC TT-3'), lip2b (5'-ACGCAACTACGATGGCGCCTTGCTCG-3'), lip2c (5'-TGC GATGAACCCACAGCTATCGCCGA-3') and lip2d (5'-GAGAAAGCCTGTACT CCACGTAGAGG-3') with 26bp and melting temperatures of 70-72ºC were designed in the incomplete ORF of lip2. From genomic DNA three rounds of PCR were performed on a MyCycler Thermal Cycler (BIORAD), using the product of the previous PCR as a template for the next. In primary and secondary PCR reactions, a single-step annealing-extension at 62ºC-66ºC was used. Separation and identification of DNA products of the tertiary reaction, including the controls, were made by agarose gel electrophoresis. At tertiary PCR reaction none amplification products were obtained with degenerate primers R1, R2 or R3. Bands only appear in the combinations R4+lip2c and R4+lip2d. A 2000bp band was selected, purified by geneclean and partially sequenced, who allowed the elucidation of lip2 ORF beginning and part of the promoter region. In the end, lip2 sequence had a total of 1992bp, including 548bp of the promoter, 1215bp of ORF and 227bp of terminator. The complete gene sequence was submitted to EMBL databases (Accession number AM774154). Nucleotide and deduced amino acid sequences were analyzed using FASTA programs from EMBL databases. Lip2 codifies a 404 amino acids protein, with 44.6KDa. The UniProt accession number for the amino-acid sequence reported in this paper is B7ZET5_TRIHA. Elucidation of complete gene nucleotide sequence of lip2, including part of the open reading frame at the N-terminal region and 548bp of the promoter region was achieved by HE-TAIL PCR, which confirmed to be a powerful tool to identify flanking regions from previous known ones.
- Identification of Trichoderma harzianum lip2 gene by HE-TAIL PCRPublication . Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, AltinoTrichoderma harzianum is a widespread soil fungus, known as a biocontrol agent against soilborne plant pathogens. Its biological control against plant fungi pathogens is based on different mechanisms, namely the production of several lytic enzymes. In T. harzianum several glucanases, celulases, chitinases and proteases has been identified but little is known about its lipolytic system. The aim of this work was to achieve the complete elucidation of T. harzianum lip2 by HE-TAIL PCR (High-Efficiency Thermal Asymetric Interlaced PCR), a method described as efficient to identify flanking regions from short known DNA sequences
- Isolation and analysis of lip2 gene from Trichoderma harzianumPublication . Vaz, Madalena; Belo, Hélio; Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, AltinoTrichodorma spp. covers a group of fungi extremely common in soils of all climatic areas. These fungi arc efficient enzyme producers, with industrial applications, or in nature, involved in the degradation of the coli wall of the phytopathogens as well as in the degradation of other fungi, organic matter and nutrients secreted by roots.
- Isolation and analysis of lip2 gene from Trichoderma harzianumPublication . Vaz, Madalena; Belo, Hélio; Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, AltinoThe genus Trichoderma is cosmopolitan in soils, wood decomposition and plant material. Species of Trichoderma are often dominant components of the soil microflora in various habitats. This is due to different metabolic capacity of the Trichoderma species and its aggressive competitiveness in nature. The genus Trichoderma are frequently used in biological control because of its antagonist ability of phytopathogenic fungi. The mechanisms employed by Trichoderma spp. to antagonize other fungi are competition (for space and / or nutrients), antibiosis and microparasites, while in the latter case, involved lytic enzymes such as proteases, glucanases, chitinases and lipases. Some of these proteins have a large agricultural potential, especially as active components of new formulations of fungicides. Trichoderma harzianum Rifai (Ascomycota, Hypocreales, Hypocreaceae) is a filamentous fungus, asexual, commonly isolated of tropical soil of plant material, rhizosphere ecosystems and decomposing organic material a ratio of 101-103 spores per gram of soil (Figure 1).
- Trichoderma harzianum Lip1 genePublication . Jorge, Lurdes; Choupina, Altino; Monte, EnriqueThe nucleotide sequence of T. harzianum Lip1 gene can be accessed in EMBL database (AM180877.1), including the 5’ upstream and the 3’ downstream regions. Lip1 open reading frame (ORF) has 1667 bp. However, according to the predictive analysis of introns made in the application AUGUSTUS (Stanke et al., 2008) restricted to fungi, and based on the comparison with sequences of Fusarium graminearum genes, its entire nucleotide sequence is not converted into amino acids, having an intron of 44 bp detected at positions 1576 to 1619 of the ORF. The protein encoded by T. harzianum Lip1 (Lip1) has a carboxylesterase type-B signature, with a serine active site (PROSITE PS00122) (Sigrist et al., 2002). As in lipases and serine proteases, the catalytic triad of esterases is formed by three amino acids: a serine, a glutamic or aspartic acid, and a histidine. Sequence around the serine-containing active center serine is well preserved, and is used as a signature pattern: F-[GR]-G-x(4)-[LIVM]-x-[LIV]-x-G-x-S-[STAG]-G. As secondary pattern was selected a conserved region located at the N-terminal region, which contains a cysteine involved in a disulfide bond, the sequence is [EDA]-[DG]-C-L-[YTF]-[LIVT]-DNS]-[LIV]-[LIVFYW]-x-[PQR]. In Lip 1 are present the sequences FGGDPDKVTLWGFSAG, and EDCLTLNVQRP, in the the amino acid positions 216-231 and 115-125. The serine at the active center of Lip 1 corresponds to residue 229, with a relative position similar to that existing in other lipases and a primary structure coincident with the consensus G-x-S-x-G, described as an active center of lipases. The other components of the catalytic triad are the residues E361 and H474. The oxyanion hole, critical for catalysis, is located in residues 134-144. The three-dimensional structural prediction made on the Phyre2 server (Kelley & Sternberg, 2009), based on the homology of Lip1 with the crystallized protein 4BE4 of the fungus Ophiostoma picea in closed conformation (Gutiérrez-Fernández et al., 2014), evidences the Lip1 "lid" region, constituted by an α-helix (residues 99-107) flanked by two "loops" that end in a disulfide bridge (Cys 83-Cys117). Lip1 was cloned in Pichia pastoris, and lipolytic activities of transformants were evaluated. The presence of homologous genes was searched in the genomes of T. atroviride, T. reesei and T. virens by Southern Blot, but hybridization only occurred in T. harzianum.
- Trichoderma harzianum Lip1 genePublication . Jorge, Lurdes; Choupina, Altino; Monte, EnriqueAn extraordinary panoply of cell-wall degrading enzymes has been related to the mycoparasitism process of Trichoderma sp. However, the role of lipolytic enzymes in this process is less known. The aim of this study is to characterize the first extracellular triacylglicerol lipase described in T. harzianum . The nucleotide sequence of Lip1 gene from T. harzianum CECT 2413 (T34) can be accessed in EMBL database (AM180877.1), including the 5’ upstream and the 3’ downstream regions. Lip1 open reading frame (ORF) has 1667 bp, encoding a predicted protein of 532 amino acids (Lip1), that can be accessed in UniProtKB (B0B099_TRIHA).
- Trichoderma harzianum lipolyitic enzymes - a contributionPublication . Jorge, Lurdes; Monte, Enrique; Choupina, AltinoTrichoderma sp. has been related to the mycoparasitism process due to an extraordinary range of cell-wall degrading enzymes (CWDE): chitinases, β-1,3 and β-1,6 glucanases, celulases and proteases. However, the role of lipases and carboxylesterases in this process is less known, although lipids were up to 3% of fungal CW (Feofilova, 2010). According to Silva et al. (2009) and Lopes et al. (2012), in experiments involving T. reesei and Pythium ultimum, it seems that lipases are implicated in mycoparasitism and are secreted in a phytopathogen-dependent manner, as they, like most of the CWDE already described, are inducible by substrate. Here we present our contribution to the knowledge of T. harzianum T34 lipolytic enzymes (EC 3.1.1). Some ESTs of a T. harzianum cDNA library coming from the EU-funded TRICHOEST project and obtained under mycoparasitism, nutritional stress and plant interaction conditions were BLAST screened, and two of them were completed by HE-TAIL PCR (Michiels et al., 2003) and sequenced, showing significant homology with EC 3.1.1 enzymes. The Lip1 gene, with a 1677 bp ORF and accessed in EMBL database (AM180877.1), codifies a deduced 56,3 kDa protein of 532 amino acids (B0B099_TRIHA), a carboxylesterase_B_1 (EC 3.1.1.1; PROSITE PS00122, E-value 2,9 e-128), with affinity for short chain fatty acids (C<10) and soluble substrates. The Lip2 gene (AM774154.1) has a 1215 bp ORF and codifies a deduced 44 kDa protein (B7ZET5_TRIHA), a triacylglycerol lipase (EC 3.1.1.3; Pfam PF01764), with affinity for long chain fatty acids (C>10) and acting on insoluble substrates. These results shows that T. harzianum has different kind of lipolytic enzymes that could have synergistic effects with other hydrolytic enzymes in the process of mycoparasitism, although its role is not so well-known.
- Trichoderma harzianum lipolytic enzymes – a contributionPublication . Jorge, Lurdes; Monte, Enrique; Choupina, AltinoTrichoderma has been related to the mycoparasitism process due to an extraordinary range of cell-wall degrading enzymes (CWDE): chitinases, β-1,3 and β-1,6 glucanases, celulases and proteases. However, the role of lipases and carboxylesterases in this process is less known, although lipids were up to 3% of fungal CW (Feofilova, 2010). According to Silva et al. (2009) and Lopes et al. (2012), in experiments involving T. reesei and Pythium ultimum, it seems that lipases are implicated in mycoparasitism and are secreted in a phytopathogen-dependent manner, as they, like most of the CWDE already described, are inducible by substrate. One of the purposes of this work was to find if the lipolytic enzymes of T. harzianum were alike homologous enzymes of other Trichoderma species. Here we present our contribution to the knowledge of T. harzianum T34 lipolytic enzymes (EC 3.1.1).