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Cloning and expression analysis of glucanase genes from Phytophthora cinnamomi

Publication . Martins, Ivone; Meirinho, Sofia G.; Dias, Teresa; Jorge, Lurdes; Martins, Fátima; Choupina, Altino

Phytophthora cinnamomi is a soil-borne pseudofungus belonging to the Class Oomycetes or „water moulds' in the Kingdom Chromista (Figure 1). Is one among the most destructive species of Phytophthora associated to the decline of forestry, ornamental and fruit species, as well as of some 900 other woody perennial plant species Associated with this oomycete is the ink disease of Castanea Sativa Mill. Glucan endo-1,3-β-D-glucosidase (EC 3.2.1.39) catalyzes de hydrolysis of 1,3-β-D-glucoside linkages in callose, laminarin and several carbohydrates found in the cell wall of plants and fungi. It is generally thought that glucanases play a role in plant defense by digesting wall components of the fungal pathogen. In yeast, 1,3-- glucanases have been studied for their role in germination, sporulation, mating and cell growth since they are regulated in cell cycle dependent manner, and are differentially expressed during vegetative growth, mating and the late stages of sporulating diploid. In plant, 1,3--glucanases have been characterized for their major role in plant defence, as well as for their involvement in germination, microsporogenesis and embryogenesis. In oomycetes, glucanases have been studied on a biochemical level for their possible role in hyphal tip growth and branching where there is thought to be a delicate balance between cell wall synthesis and hydrolyses. In the present work, we obtained a fragment with 1231bp of the endo-1,3--glucanase gene by standard PCR, using conserved primers and the whole genomic sequence with 2586 bp was obtained by amplifying the previous sequence by asymmetric PCR. The gene expression was studied during growth in different carbon sources and was also performed a time course of endo-1,3-β-D-glucosidase production.

2010Conference object

Open access

Bioinformatics tools for identification of pathogenic factors

Publication . Choupina, Altino

The culture of the chestnut tree is extremely important in the northern region of Portugal, occupying a significant! proportion of useful agricultural area. The annual average chestnut production in Portugal can reach 20 000 tons. New plantation areas have increased in the last law decades. However the ink disease caused by the oomycete Phytophthora cinnamomi has damage and killed many trees and up to now no concrete solutions have been offered to control the illness. As a consequence, the disease propagation in the orchards 01 chestnut trees has been causing severe productivity and yield breaks. ln addition to the economical lasses, the importance of sociological and landscape aspects for the region cannot be neglected

2011Conference object

Open access

Identification of Trichoderma harzianum lip2 gene by HE-TAIL PCR

Publication . Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, Altino

Trichoderma 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

2010Conference object

Open access

Isolation and phylogenetic analysis of two actin genes from Phytophthora cinnamomi

Publication . Jorge, Lurdes; Dias, Teresa; Andrade, Maria; Vaz, Madalena; Dominguez, Ángel; Choupina, Altino

Actins, as the essential component of cellular microfilament, are ubiquitous and highly conserved proteins that play key roles in several basic functions of organism such as cytoskeleton morphology, cell division, cell motility, cellular signal transduction, cellular interaction and organelle movements, as well as locomotion, phagocytosis, endocytosis and exocytosis . Actins are highly conserved structural proteins, found in all eukaryotes. So, actin gene sequences are used as tools in scientific research, for example, for phylogenetic analysis. Actin in Phytophthora infestans is encoded by at least two genes, in contrast to unicellular and filamentous fungi (Candida albicans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Filobasidiella neoformans) where there is a single gene. These genes (designated actA and actB) have been isolated from a genomic library of P. infestans. Phytophthora cinnamomi is a host-nonspecific, soilborne, pathogen of many plant species. In Portugal it is most important as a pathogen of chestnut trees. The purpose of this study was to clone and determine the phylogenetic retionships evidencided by Phytophthora cinnamomi actins. In order to isolate the actin genes, P. cinnamomi was grown in cellophane-PDA medium and genomic DNA was used as a template in PCR amplification reactions combining degenerate primers Act1, Act2, Act3 and Act4. PCR fragments were purified, cloned into pGEM-T vector and transformants were selected. Complete open reading frames (ORFs) of act1 and act2 genes were achieved by HE-TAIL PCR, and submitted to EMBL databases (Accession numbers AM412175.1 and AM412176.1). Act1 has an 1128bp ORF, encoding a deduced protein of 375aa and 41,972kDa. Act2 ORF has 1083bp and encodes a deduced protein of 360aa and 40,237kDa. Deduced amino acid sequences were analyzed using FASTA programs from EMBL databases. Act1 showed a 98.9% identity with P. melonis actB, 94.4% with P. megasperma actin and 96.0% with P. infestans actin2. Act2 showed a 98.9% identity with Pythium splendens actin and 98.6% with P. brassicae actinA.

2010Conference object

Open access

Isolation and analysis of lip2 gene from Trichoderma harzianum

Publication . Vaz, Madalena; Belo, Hélio; Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, Altino

The 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).

2011Conference object

Open access