Complementation of Ustilago maydis MAPK mutants by a wheat leaf rust, Puccinia triticina homolog: potential for functional analyses of rust genes.

From a large expressed sequence tag (EST) database representing several developmental stages of Puccinia triticina, we discovered a mitogen-activated protein kinase (MAPK) with homology to kinases with known pathogenic functions in other fungi. This PtMAPK1 is similar to the Ustilago maydis MAPK, Ubc3/Kpp2, but has a longer N-terminal extension of 43 amino acids (aa) with identities to U. maydis Kpp6, a homolog of Ubc3/Kpp2 with a 170-aa N-terminal extension. Ubc3/Kpp2 is involved in mating and subsequent pathogenic development, whereas Kpp6 functions during invasive growth in corn tissue. PtMAPK1, expressed from a Ustilago sp.-specific promoter, was able to complement a ubc3/kpp2 deletion mutant and restore mating. It also substantially increased virulence on corn, measured as tumor formation, of a kpp6 deletion mutant. Moreover, this construct restored to near-full pathogenicity a ubc3/kpp2 kpp6 nonpathogenic double deletion mutant. Complementation of the ubc3/kpp2 mutant with the complete PtMAPK gene and verification of expression by reverse-transcription polymerase chain reaction indicated that the rust promoter is recognized in U. maydis. Phylogenetically, these basidiomycete plant pathogens are related, which was reflected in comparison of P. triticina ESTs to U. maydis gene sequences. The U. maydis heterologous expression system allows functional analysis of rust genes, currently frustrated by the lack of efficient transformation and selection procedures.

Protein analysis in a pleomorphically deteriorated strain of the insect-pathogenic fungus Metarhizium anisopliae.

Pleomorphic deterioration is a process where a fungal isolate loses the ability to produce conidia during repeated subculturing. We have previously isolated strains of the entomopathogenic fungus Metarhizium anisopliae that have irreversibly lost the ability to produce conidia and only produce mycelia when grown on agar. Gel electrophoresis was used to examine differences in intracellular protein patterns (urea-soluble proteins and urea-insoluble proteins (i.e., hydrophobins)) in conidiating and mycelial cultures of M. anisopliae. Two major proteins present in a conidiating culture and one from a mycelial culture were N-terminally sequenced but showed no homologies to known proteins. The presence of hydrophobins in conidiating and mycelial cultures was also examined, and it was shown that these proteins were abundant in conidiating cultures but not in mycelial cultures. We also used primers designed from regulatory genes involved in conidiation in Aspergillus nidulans. The amplified fragments were not homologous to A. nidulans genes.

Genetic groups of the insect-pathogenic fungus Beauveria bassiana are associated with habitat and thermal growth preferences.

A persistent paradigm in insect pathology is one that relates the insect host to certain genetic groups of insect-pathogenic fungi. This paradigm assumes that the genotype of an insect-pathogenic fungus coevolves with a certain taxon of insect host that it infects. The insect-pathogenic fungus Beauveria bassiana shows a wide host range and is considered to be a facultative insect pathogen. In this study, a population genetics analysis of B. bassiana from forested and agricultural habitats as well as from the Canadian Arctic showed distinct genetic groups associated with the three different habitats. Within each group, recombining population structures and clonally reproducing lineages were observed. The B. bassiana isolates were also assessed for their abilities to grow at 8, 15, 25 and 37 degrees C and for their tolerances to UV exposure. The genetic groups from the Arctic and from the forested habitats grew at lower temperatures, while the genetic group from the agricultural habitat grew at 37 degrees C and was tolerant to UV exposure. There were no clear associations between the genetic group and the ability to infect coleopteran or lepidopteran insect larvae. There is increasing evidence that such studies represent a significant paradigm shift; habitat selection, not insect host selection, drives the population structure of deuteromycetous insect-pathogenic fungi. We suggest that adaptation to a certain habitat type is an important criterion for identifying insect-pathogenic fungal strains for use in insect biocontrol efforts.

Attenuation of fungal infection in thermoregulating Locusta migratoria is accompanied by changes in hemolymph proteins.

Hemolymph proteins in the locust, Locusta migratoria migratorioides infected with the fungus Metarhizium anisopliae var acridum were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under conditions that allowed locusts to thermoregulate, 2 proteins, ITB1 (ca. 18kDa) and ITB2 (ca. 13kDa) were induced 48h post inoculation. In contrast, under non-thermoregulating conditions, only 1 band, INTB1 (ca. 18kDa) was induced with similar molecular mass to ITB1. ITB1 and ITB2 were N-terminally sequenced but showed little homology to known proteins. The induction of hemolymphal proteins in infected, thermoregulating locusts and implication in insect immune defence are discussed.