Complementation ofUstilago maydisMAPK Mutants by a Wheat Leaf Rust,Puccinia triticinaHomolog: Potential for Functional Analyses of Rust Genes

Abstract

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.

Molecular Characterization of a Fus3/Kss1 Type MAPK from Puccinia striiformis f. sp. tritici, PsMAPK1

“…No introns were found in EFP80661. These observations were consistent with what has been reported in P. triticina and U. maydis MAPKs [13], [21]. Based on the sequence similarity and intron distribution pattern, we conclude that PsMAPK1 is more closely related to Kpp6 than to Ubc3/Kpp2 of U. maydis .…”

“…However, little is known about the role of signal transduction pathways in Pst and other rust fungi due to their obligate nature and the lack of an efficient and reliable transformation system. When expressed in Ustilago maydis , the PtMAPK1 MAPK gene of Puccinia triticina was able to partially complement the kpp2 kpp6 mutant for mating, virulence, and pathogenicity [21].…”

Interspecific Sex in Grass Smuts and the Genetic Diversity of Their Pheromone-Receptor System

“…Strikingly, most basidiomycetes are stringently heterothallic and sexual identity is determined by two specific mating type gene clusters that encode a pheromone-receptor (PR) system and heterodimerising homeodomain (HD) transcription factors. Their components are functionally conserved even across phyla [19]–[21] and transspecific polymorphism of mating type alleles has been preserved since the last common ancestor of basidiomycetes and ascomycetes [22], [23].…”

An Immunity-Triggering Effector from the Barley Smut Fungus Ustilago hordei Resides in an Ustilaginaceae-Specific Cluster Bearing Signs of Transposable Element-Assisted Evolution

“…U. hordei ORFs, either with or without the sequence coding for the SP, but without their stop codon, were amplified by PCR with a CACC tetranucleotide sequence at the 5′-end to allow for directional cloning into Gateway entry vector pENTR/D-TOPO (Invitrogen; Table S4 ). Cloned inserts were sequenced and were subsequently transferred to a designed GateWay destination vector, pUBleX1Int:GateWay:HA (a derivative of Ustilago -specific integrative expression vector pUBleX1Int [94]), using LR recombineering. For the transient assays and microscopy after bombardment, the above-mentioned pENTR clones (UHOR_10022-SP-STOP) were recombined into a modified pMCG161 vector (ChromDB at http://www.chromdb.org; NCBI accession no.…”