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  • Author or Editor: M. Láday x
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Cellulose-acetate electrophoresis (CAE) was used to investigate isozyme polymorphisms among different isolates of Fusarium cerealis, F. culmorum, F. graminearum and F. pseudograminearum. After initial testing of 18 enzymes in three buffer systems for activity and resolution of bands, 12 proved to be appropriate for analysis of the full sample set. Comparing the different electrophoretic types (ETs), adenylate kinase (AK), NADP dependent glutamate dehydrogenase (NADP GDH), peptidase B (PEP B), peptidase D (PEP D) and phosphoglucomutase (PGM) proved to be diagnostic for at least one species examined. However, only PEP D was useful alone as a marker to distinguish the four taxa studied providing a rapid and simple CAE based diagnostic protocol.

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Species hybrids were created via fusion of zoospores of two morpho­logically distinct species, P. infestans and P. nicotianae. Sixteen putative hybrid isolates were recovered that expressed differential drug resistance of each parent. Repetitive DNA of P. nicotiane was detected readily in all of these isolates by hybridization with the species-specific DNA probe, pPP33A. DNA of P. infestans was detected in only two putative hybrid isolates using PCR and primer pair ITS3 and PINF2. The two true hybrids were more similar to P. nicotianae than to P. infestans on the basis of pathogenic, morphological and molecular evidence. Additionally, hybrids expressed modified host ranges compared to parental species. Fusion of zoospores or hyphae may contribute to formation of such hybrids, particularly in the case of heterothallic species in which the joint occurrence of compatible mating types is rare. Zoospore fusion may prove useful as a tool to study hybridization, pathogenesis, and sources of natural diversity of species.

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Based on multiple sequence alignment of mitogen activated protein kinase (MAPK) genes from 28 fungal species, we could identify twelve new hallmark sequences, specific to YSAPK (yeast and fungal stress activated protein kinases) MAPK subgroup within fungal MAPKs. Six of the motifs (I-a, I-b, IV-b, V-b, X-b1 and X-b2) showed especially high degree of specificity. Two of these six motifs, I-b (SA[RK]DQLT) and IV-b (F[IL]SPLED[IV]) were specific within eukaryotic proteins, too. The other type of motifs contained not only YSAPK specific residues but residue(s) conserved on fungal MAPK and all MAPK levels. From the viewpoint of functional role, YSAPK motif VII-a (IL[VI]NENCDL) coincided with a loop spanning 7b-8b sheets in human p38a and ERK2 MAPK proteins (consensus indicated by bold face letters). This motif was shown to be involved in interaction with L-x-L type docking motifs of activators (MAPK kinases) and transcription factors. A fungal MAPK specific signal sequence in protein kinase subdomain IX-b (AE[ML][LVI]xG[KR]PxFxG[KR]D) was also described. A subgroup specific nested PCR-based cloning approach was developed to amplify YSAPK sequences in different filamentous fungal species based on motifs I, I-b, VIII and X-b3 as primers. Putative YSAPK MAPK amplicons obtained by this approach were identified by (i) the presence of newly characterized YSAPK specific motifs and (ii) alignments to known YSAPK MAPK genes. A neighbor-joining phylogenetic tree constructed by comparing 50 fungal MAPK sequences clearly demonstrated a dichotomic origin of fungal MAPKs and separated YSAPK MAPKs from the other two fungal MAPK subgroups. Comparing the number and distribution of known MAPK genes in Fusarium and other species it is tempting to speculate that this triple structural diversity of MAPKs, at least in filamentous species is a general phenomenon. Phylogenetic analysis revealed no separation of phytopathogenic species. Blumeria graminis, Alternaria brassicicola and Aspergillus species, however were separated from the majority of filamentous Ascomycetes in all of the three MAPK subgroups.

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Fvwc1 and Fvwc2, orthologues of the wc-1 and wc-2 genes encoding for proteins of the white collar complex (WCC) in Neurospora crassa were cloned from Fusarium verticillioides and lack-of-function wc mutants were obtained by targeted gene disruption. Photo-conidiation was found to be absent in F. verticillioides, on the contrary, the wild type strain produced less conidia under continuous illumination than in the dark. Inactivation of any of the wc genes led to total female sterility, without affecting male fertility or asexual conidiation. No loss in colonization capability/invasive growth of the wc mutants was observed, when assessed on tomato fruits. Both Fvwc1 and Fvwc2 showed constitutive expression in the wild type cultures incubated in the dark and exposure to light caused only negligible increases in their transcription. Both Fvwc1 and Fvwc2 were down-regulated in a ΔFvmat1-2-1 gene disruption mutant, lacking a functional mating type (mat1-2-1) gene, suggesting that the MAT1-2-1 product has a positive regulatory effect on the white collar genes.

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