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Publikation

Penselin, D., Münsterkötter, M., Kirsten, S., Felder, M., Taudien, S., Platzer, M., Ashelford, K., Paskiewicz, K. H., Harrison, R. J., Hughes, D. J., Wolf, T., Shelest, E., Graap, J., Hoffmann, J., Wenzel, C.: Wöltje, N., King, K. M., Fitt, B. D. L., Güldener, U., Avrova, A. & Knogge, W. Comparative genomics to explore phylogenetic relationship, cryptic sexual potential and host specificity of Rhynchosporium species on grasses BMC Genomics 17, 953, (2016) DOI: 10.1186/s12864-016-3299-5

Background

The Rhynchosporium species complex consists of hemibiotrophic fungal pathogens specialized to different sweet grass species including the cereal crops barley and rye. A sexual stage has not been described, but several lines of evidence suggest the occurrence of sexual reproduction. Therefore, a comparative genomics approach was carried out to disclose the evolutionary relationship of the species and to identify genes demonstrating the potential for a sexual cycle. Furthermore, due to the evolutionary very young age of the five species currently known, this genus appears to be well-suited to address the question at the molecular level of how pathogenic fungi adapt to their hosts.

Results

The genomes of the different Rhynchosporium species were sequenced, assembled and annotated using ab initio gene predictors trained on several fungal genomes as well as on Rhynchosporium expressed sequence tags. Structures of the rDNA regions and genome-wide single nucleotide polymorphisms provided a hypothesis for intra-genus evolution. Homology screening detected core meiotic genes along with most genes crucial for sexual recombination in ascomycete fungi. In addition, a large number of cell wall-degrading enzymes that is characteristic for hemibiotrophic and necrotrophic fungi infecting monocotyledonous hosts were found. Furthermore, the Rhynchosporium genomes carry a repertoire of genes coding for polyketide synthases and non-ribosomal peptide synthetases. Several of these genes are missing from the genome of the closest sequenced relative, the poplar pathogen Marssonina brunnea, and are possibly involved in adaptation to the grass hosts. Most importantly, six species-specific genes coding for protein effectors were identified in R. commune. Their deletion yielded mutants that grew more vigorously in planta than the wild type.

Conclusion

Both cryptic sexuality and secondary metabolites may have contributed to host adaptation. Most importantly, however, the growth-retarding activity of the species-specific effectors suggests that host adaptation of R. commune aims at extending the biotrophic stage at the expense of the necrotrophic stage of pathogenesis. Like other apoplastic fungi Rhynchosporium colonizes the intercellular matrix of host leaves relatively slowly without causing symptoms, reminiscent of the development of endophytic fungi. Rhynchosporium may therefore become an object for studying the mutualism-parasitism transition.

Publikation

Siersleben, S., Penselin, D., Wenzel, C., Albert, S. & Knogge, W. PFP1, a gene encoding an Epc-N domain-containing protein, is essential forpathogenicity of the barley pathogen Rhynchosporium commune. Eukaryot. Cell 13, 1026-1035, (2014) DOI: 10.1128/EC.00043-14

Scald caused by Rhynchosporium commune is an important foliar disease of barley. Insertion mutagenesis of R. commune generated a non-pathogenic fungal mutant, which carries the inserted plasmid in the upstream region of a gene named PFP1. The characteristic feature of the gene product is an Epc-N domain. This motif is also found in homologous proteins shown to be components of histone acetyltransferase (HAT) complexes of fungi and animals. Therefore, PFP1 is suggested to be the subunit of a HAT complex in R. commune with an essential role in the epigenetic control of fungal pathogenicity. Targeted PFP1 disruption also yielded non-pathogenic mutants, which showed wild-type like growth ex planta except for the occurrence of hyphal swellings. Complementation of the deletion mutants with the wild-type gene reestablished pathogenicity and suppressed the hyphal swellings. However, despite wild-type level PFP1 expression the complementation mutants did not reach wild-type level virulence. This indicates that the function of the protein complex and, thus, fungal virulence is influenced by a position-affected long-range control of PFP1 expression. 

Publikation

Hawkins, N.J., Cools, H.J., Sierotzki, H., Shaw, M.W., Knogge, W., Kelly, S.L., Kelly, D.E. & Fraaije, B.A. Paralogue re-emergence: a novel, historically-contingent mechanism in the evolution of anti-microbial resistance. Mol Biol Evol 31, 1793-1802, (2014) DOI: 10.1093/molbev/msu134

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51 , and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R.commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B , but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by ho rizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.

Publikation

Torriani, S.F.F., Penselin, D., Knogge, W., Felder, M., Taudien, S., Platzer, M., McDonald, B.A. & Brunner, P.C. Comparative analysis of mitochondrial genomes from closely related Rhynchosporium species reveals extensive intron invasion Fungal Gen Biol 62, 34-42, (2014) DOI: 10.1016/j.fgb.2013.11.001

We sequenced and annotated the complete mitochondrial (mt) genomes of four closely related Rhynchosporium species that diverged ∼14,000–35,000 years ago. During this time frame, three of the mt genomes expanded significantly due to an invasion of introns into three genes (cox1, cox2, and nad5). The enlarged mt genomes contained ∼40% introns compared to 8.1% in uninvaded relatives. Many intron gains were accompanied by co-conversion of flanking exonic regions. The comparative analysis revealed a highly variable set of non-intronic, free-standing ORFs of unknown function (uORFs). This is consistent with a rapidly evolving accessory compartment in the mt genome of these closely related species. Only one free-standing uORF was shared among all mt genomes analyzed. This uORF had a mutation rate similar to the core mt protein-encoding genes, suggesting conservation of function among the species. The nucleotide composition of the core protein-encoding genes significantly differed from those of introns and uORFs. The mt mutation rate was 77 times higher than the nuclear mutation rate, indicating that the phylogeny inferred from mt genes may better resolve the phylogenetic relationships among closely related Rhynchosporium species than phylogenies inferred from nuclear genes.

Publikation

Navarro-Quezada, A. & Schumann, N. & Quint, M. Plant F-Box protein evolution is determined by lineage-specific timing of major gene family expansion waves PLoS One 8, e68672, (2013) DOI: org/10.1371/journal.pone.0068672

F-box proteins (FBPs) represent one of the largest and fastest evolving gene/protein families in the plant kingdom. The FBP superfamily can be divided in several subfamilies characterized by different C-terminal protein-protein interaction domains that recruit targets for proteasomal degradation. Hence, a clear picture of their phylogeny and molecular evolution is of special interest for the general understanding of evolutionary histories of multi-domain and/or large protein families in plants. In an effort to further understand the molecular evolution of F-box family proteins, we asked whether the largest subfamily in Arabidopsis thaliana, which carries a C-terminal F-box associated domain (FBA proteins) shares evolutionary patterns and signatures of selection with other FBPs. To address this question, we applied phylogenetic and molecular evolution analyses in combination with the evaluation of transcriptional profiles. Based on the 2219 FBA proteins we de novo identified in 34 completely sequenced plant genomes, we compared their evolutionary patterns to a previously analyzed large subfamily carrying C-terminal kelch repeats. We found that these two large FBP subfamilies generally tend to evolve by massive waves of duplication, followed by sequence conservation of the F-box domain and sequence diversification of the target recruiting domain. We conclude that the earlier in evolutionary time a major wave of expansion occurred, the more pronounced these selection signatures are. As a consequence, when performing cross species comparisons among FBP subfamilies, significant differences will be observed in the selective signatures of protein-protein interaction domains. Depending on the species, the investigated subfamilies comprise up to 45% of the complete superfamily, indicating that other subfamilies possibly follow similar modes of evolution.

Publikation

Avrova, A. & Knogge, W. Rhynchosporium commune: a persistent threat to barley cultivation Mol. Plant Pathol. 13, 986-997, (2012) DOI: 10.1111/j.1364-3703.2012.00811.x

Summary

Rhynchosporium commune is a haploid fungus causing scald or leaf blotch on barley, other Hordeum spp. and Bromus diandrus.

Taxonomy

Rhynchosporium commune is an anamorphic Ascomycete closely related to the teleomorph Helotiales genera Oculimacula and Pyrenopeziza.

Disease symptoms

Rhynchosporium commune causes scald-like lesions on leaves, leaf sheaths and ears. Early symptoms are generally pale grey oval lesions. With time, the lesions acquire a dark brown margin with the centre of the lesion remaining pale green or pale brown. Lesions often merge to form large areas around which leaf yellowing is common. Infection frequently occurs in the leaf axil, which can lead to chlorosis and eventual death of the leaf.

Life cycle

Rhynchosporium commune is seed borne, but the importance of this phase of the disease is not fully understood. Debris from previous crops and volunteers, infected from the stubble from previous crops, are considered to be the most important sources of the disease. Autumn-sown crops can become infected very soon after sowing. Secondary spread of disease occurs mainly through splash dispersal of conidia from infected leaves. Rainfall at the stem extension growth stage is the major environmental factor in epidemic development.

Detection and quantification

Rhynchosporium commune produces unique beak-shaped, one-septate spores both on leaves and in culture. The development of a specific polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) has allowed the identification of asymptomatic infection in seeds and during the growing season.

Disease control

The main measure for the control of R. commune is the use of fungicides with different modes of action, in combination with the use of resistant cultivars. However, this is constantly under review because of the ability of the pathogen to adapt to host plant resistance and to develop fungicide resistance.

Publikation

Kirsten, S., Navarro-Quezada, A., Penselin, D., Wenzel, C., Matern, A., Leitner, A., Baum, T., Seiffert, U. & Knogge, W. Necrosis-inducing proteins of Rhynchosporium commune, effectors in quantitative disease resistance Mol. Plant-Microbe Interact. 25, 1314-1325, (2012) DOI: 10.1094/MPMI-03-12-0065-R

The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an “active” NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner.

Publikation

Schumann, N., Navarro-Quezada, A.R., Ullrich, K., Kuhl, C. & Quint, M. Molecular Evolution and Selection Patterns of Plant F-box Proteins with C-terminal Kelch Repeats Plant Physiol 155, 835-850, (2011)

The F-box protein superfamily represents one of the largest families in the plant kingdom. F-box proteins phylogenetically organize into numerous subfamilies characterized by their carboxyl (C)-terminal protein-protein interaction domain. Among the largest F-box protein subfamilies in plant genomes are those with C-terminal kelch repeats. In this study, we analyzed the phylogeny and evolution of F-box kelch proteins/genes (FBKs) in seven completely sequenced land plant genomes including a bryophyte, a lycophyte, monocots, and eudicots. While absent in prokaryotes, F-box kelch proteins are widespread in eukaryotes. Nonplant eukaryotes usually contain only a single FBK gene. In land plant genomes, however, FBKs expanded dramatically. Arabidopsis thaliana, for example, contains at least 103 F-box genes with well-conserved C-terminal kelch repeats. The construction of a phylogenetic tree based on the full-length amino acid sequences of the FBKs that we identified in the seven species enabled us to classify FBK genes into unstable/stable/superstable categories. In contrast to superstable genes, which are conserved across all seven species, kelch domains of unstable genes, which are defined as lineage specific, showed strong signatures of positive selection, indicating adaptational potential. We found evidence for conserved protein features such as binding affinities toward A. thaliana SKP1-like adaptor proteins and subcellular localization among closely related FBKs. Pseudogenization seems to occur only rarely, but differential transcriptional regulation of close relatives may result in subfunctionalization.

Publikation

Kirsten, S., Siersleben, S. & Knogge, W. A GFP-based assay to quantify the impact of effectors on the ex planta development of the slowly growing barley pathogen Rhynchosporium commune. Mycologia 103, 1019-1027, (2011) DOI: 10.3852/10-306

A growth assay was established for the barley pathogen Rhynchosporium commune with EGFP-tagged fungal mutants. This assay was used to study the effect of four antibiotics (hygromycin B, nourseothricin, kanamycin, phleomycin) and a herbicide (phosphinothricin) on fungal development. Fitting the growth curves to the modified Gompertz model allowed calculating growth parameters, such as lag periods of fungal colony formation and mycelial growth rates as well as EC50 values. Except kanamycin all compounds were efficient inhibitors so that the corresponding resistance-conferring genes can be used as markers for selection of fungal transformation-based mutants. In addition the assay was used to quantify the inhibitory activity of a barley secondary metabolite, the indole alkaloid gramine.

Publikation

Horbach, R., Navarro-Quesada, A.R., Knogge, W. & Deising, H.B. When and how to kill a plant cell: Infection strategies of plant pathogenic fungi. J Plant Physiol. 168, 51-62, (2011)

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