Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection.

The 5' noncoding region of clpg2, an endopolygalacturonase gene of the bean pathogen Colletotrichum lindemuthianum, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed that clpg2 was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant.

Endopolygalacturonase genes from Colletotrichum lindemuthianum: cloning of CLPG2 and comparison of its expression to that of CLPG1 during saprophytic and parasitic growth of the fungus.

Following the previous isolation of CLPG1, a gene encoding an endopolygalacturonase (endoPG) secreted into the culture filtrate of Colletotrichum lindemuthianum, we have isolated and sequenced an additional endoPG gene, CLPG2. This gene is present as a single copy in the genome of the fungus. At the amino acid level, CLPG2 shows 61% identity to CLPG1 and between 37 to 59% identity to other fungal endoPGs. RNA blot analyses of endoPG gene expression were followed with specific probes during in vitro culture of the fungus. When conidia were used to inoculate a synthetic medium containing pectin as sole carbon source, only CLPG1 was found to be expressed after 3 days of culture. However, transferring the mycelium grown on glucose for 4 days to a pectin-containing medium allowed the detection of CLPG1 and CLPG2 transcripts as early as 12 h after transfer on this substrate. Expression of CLPG2 was transient while that of CLPG1 was more prolonged. Immunocytological localization of endoPG in C. lindemuthianum-infected bean tissues with antibodies against CLPG1 confirmed that the protein is produced in planta and is associated with extensive degradation of the host cell wall. Detection of endoPG transcripts by reverse transcription-polymerase chain reaction revealed that CLPG1, but not CLPG2, is expressed at the beginning of the necrotrophic stage of infection. These results show that the two endoPG genes are differentially expressed and that CLPG1 encodes the major secreted endoPG both during saprophytic growth and during plant infection.

Induction by (alpha)-L-Arabinose and (alpha)-L-Rhamnose of Endopolygalacturonase Gene Expression in Colletotrichum lindemuthianum.

The production of endopolygalacturonase (endoPG) by Colletotrichum lindemuthianum, a fungal pathogen causing anthracnose on bean seedlings, was enhanced when the fungus was grown in liquid medium with L-arabinose or L-rhamnose as the sole carbon source. These two neutral sugars are present in plant cell wall pectic polysaccharides. The endolytic nature of the enzyme was demonstrated by its specific interaction with the polygalacturonase-inhibiting protein of the host plant as well as by sugar analysis of the products released from its action on oligogalacturonides. Additional characterization of the protein was achieved with an antiserum raised against the pure endoPG of the fungus. Induction by arabinose and rhamnose was more prolonged and led to a level of enzyme activity at least five times higher than that on pectin. Northern blot experiments showed that this effect was correlated to the induction of a 1.6-kb transcript. A dose-response study indicated that the endoPG transcript level was already increased at a concentration of each sugar as low as 2.75 mM in the medium and was maximum at 55 mM arabinose and 28 mM rhamnose. Glucose, the main plant cell wall sugar residue which is also present in the apoplast, prevented endoPG gene expression, partially when added to pectin at concentrations ranging from 5 to 110 mM and totally when added at 55 mM to arabinose. Inhibition by glucose of the rhamnose-induced endoPG was correlated to nonuptake of rhamnose. This is the first report that arabinose and rhamnose stimulate endoPG gene expression in a fungus. The possible involvement of these various sugars on endoPG gene expression during pathogenesis is discussed.

Isolation and sequence analysis of Clpg1, a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum lindemuthianum.

Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race beta and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of Cl. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed on ORF encoding a 363-amino-acid (aa) polypeptide beginning with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5' non-coding region. This genomic clone was thereafter designated Clpg1. Southern analysis, performed with a Clpg1-specific probe, showed that this gene is present as a single copy in the Cl genome.