Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

Abstract

Two new proteinases secreted by Cryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that the eapB and eapC genes contain three and two introns, respectively. The products of the eapB and eapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. The eapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of the eapC coding sequence in an endothiapepsin-deficient (EapA-) C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.

Large-scale expressed sequence tag analysis for the chestnut blight fungus Cryphonectria parasitica

“…Among these, pathogenecity related genes eapB and eapC, encoding two acid proteases (Jara et al, 1996), cpc-1, encoding general amino acid control transcriptional activator (Wang et al, 1998), and a gene whose product was homologous to the carboxypeptidase Y precursor of Candida albicans (Mukhtar et al, 1992), were highly expressed in the CP-M library relative to CP-S library. It was also noted that virus infection altered a dozen of these genes at sporulation stage as revealed by the comparison of EP155-S and EP713 libraries.…”

“…These include four genes encoding acid protease A (eapC), cross-pathway control protein 1 (CPC1), mitogene-activated protein kinase (Cpmk1), and G-protein CPGB1 identified previously in C. parasitica (Choi et al, 1995(Choi et al, , 2005Jara et al, 1996;Kasahara and Nuss, 1997;Park et al, 2004;Wang et al, 1998), and 13 genes with high degree of similarity with genes found in Sordariomycetes phytopathogens F. graminearum (Guldener et al, 2006) and M. grisea (Dean et al, 2005). Among the newly identified genes were homologues of ODC1, encoding ornithine decarboxylase, that is necessary for wheat infection by the wheat pathogen Stagonospora nodorum (Bailey et al, 2000) and FOW1, encoding a mitochondrial carrier protein, which is required specifically for colonization in the plant tissue by F. oxysporum (Inoue et al, 2002).…”

“…In addition to hypovirulence, hypovirus infection results in persistent changes in multiple traits of the fungal host that include modification of colony pigment production, suppressed asexual spore production and selected enzyme activities, and altered gene expression patterns (Nuss, 2005). Studies have revealed hypovirus-mediated regulation of individual C. parasitica genes such as cpg-1, encoding a G-protein a subunit (Choi et al, 1995), and cpgb-1, encoding a G-protein b subunit (Kasahara and Nuss, 1997), key components of a trimeric G-protein signaling pathway; genes encoding pheromones (mf1-1, mf1-2, and mf2-2) (Zhang et al, 1998), and about 20 other genes involved in macromolecule metabolism (Choi et al, 2005;Kim et al, 2002;Park et al, 2004), catabolism (Choi and Nuss, 1990;Jara et al, 1996), and electron transport . A hypovirus-induced promoter have been used to dissect the modulation of cAMP-dependent signaling of the host fungus during virus infection (Parsley et al, 2002).…”

Microarray analysis of Cryphonectria parasitica G - and G -signalling pathways reveals extensive modulation by hypovirus infection

“…Intriguingly, all six are changed in both G-protein-deleted mutants (all are downregulated), but only two are similarly altered in the presence of hypovirus. Previous studies have demonstrated that null mutants of EapA and EapC produced altered extracellular proteolytic profiles, with EapA contributing 97 % of the proteolytic activity detected in C. parasitica culture medium (Jara et al, 1996;Razanamparany et al, 1992). However, information concerning the virulence of these strains is presently unavailable.…”

Identification and characterization of a bacterial glutamic peptidase

“…The characterized proteins known to be part of the G1 family are aspergilloglutamic peptidase (AGP) from Aspergillus niger [4], scytalidoglutamic peptidase (SGP) from Scytalidium lignicolum [5], acid peptidases B and C (EapB and EapC) from Cryphonectria parisitica [6], Penicillium marneffei acid proteinase (PMAP-1) [7], Talaromyces emersonii glutamic peptidase 1 (TGP1) [8] and BcACP1 from Botryotinia fuckeliana [9]. …”