Functional analysis of mating type genes and transcriptome analysis during fruiting body development of botrytis cinerea
Rodenburg, Sander Y.A. ; Terhem, Razak B. ; Veloso, Javier ; Stassen, Joost H.M. ; Kan, Jan A.L. van - \ 2018
mBio 9 (2018)1. - ISSN 2161-2129
Ascospore - Epigenetic regulation - Plant disease - Sexual reproduction - Transcriptome
Botrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (com-prising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction. IMPORTANCE Fungal fruiting bodies are formed by sexual reproduction. We studied the development of fruiting bodies (“apothecia”) of the ubiquitous plant-pathogenic ascomycete Botrytis cinerea. The role of mating type genes in apothecium development was investigated by targeted mutation. Two genes are essential for the initiation of sexual development; mutants in these genes are sterile. Two other genes were not essential for development of stipes; however, they were essential for stipes to develop a disk and produce sexual ascospores. We examined gene expression profiles during apothecium development, as well as in ascospores sampled from apothecia. We provide the first study ever of the transcriptome of pure ascospores in a filamentous fungus. The expression of numerous genes involved in plant infection was induced in the ascospores, implying that ascospores are developmentally primed for infection before their release from apothecia.
A novel environmental azole resistance mutation in Aspergillus fumigatus and a possible role of sexual reproduction in its emergence
Zhang, Jianhua ; Snelders, Eveline ; Zwaan, Bas J. ; Schoustra, Sijmen E. ; Meis, Jacques F. ; Dijk, Karin van; Hagen, Ferry ; Beek, Martha T. van der; Kampinga, Greetje A. ; Zoll, Jan ; Melchers, Willem J.G. ; Verweij, Paul E. ; Debets, Fons - \ 2017
mBio 8 (2017)3. - ISSN 2161-2129
Ascospores - Aspergillus fumigatus - Azole resistance - Compost heap - Conidiospores - Hot spot for resistance development - Novel mutation - Sexual reproduction
This study investigated the dynamics of Aspergillus fumigatus azoleresistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR46/Y121F/T289A genotype, 2% (1/45) harbored the TR46/Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazoleresistant mutation (TR46 3/Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinical A. fumigatus isolates showed that the novel TR46 3 mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR46 repeats. Importantly, in the laboratory, we recovered the TR46 3 mutation from a sexual cross between two TR46 isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment of Aspergillus diseases. IMPORTANCE Composting of organic matter containing azole residues might be important for resistance development and subsequent spread of resistance mutations in Aspergillus fumigatus. In this article, we show the dominance of azoleresistant A. fumigatus in azole-exposed compost and the discovery of a new resistance mutation with clinical relevance. Furthermore, our study indicates that current fungicide application is not sustainable as new resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine. We provide evidence that the sexual part of the fungal life cycle may play a role in the emergence of resistance mutations because under laboratory conditions, we reconstructed the resistance mutation through sexual crossing of two azole-resistant A. fumigatus isolates derived from the same compost heap. Understanding the mechanisms of resistance selection in the environment is needed to design strategies against the accumulation of resistance mutations in order to retain the azole class for crop protection and treatment of Aspergillus diseases.
Reproductive strategy, clonal structure and genetic diversity in populations of the aquatic macrophyte Sparganium emersum in river systems
Pollux, B.J.A. ; Jong, M.D.E. ; Steegh, A. ; Verbruggen, E. ; Groenendael, J.M. Van; Ouborg, N.J. - \ 2007
Molecular Ecology 16 (2007)2. - ISSN 0962-1083 - p. 313 - 325.
Dispersal - Hydrochory - Sexual reproduction - Vegetative reproduction - Waterfowl - Zoochory
Many aquatic and riparian plant species are characterized by the ability to reproduce both sexually and asexually. Yet, little is known about how spatial variation in sexual and asexual reproduction affects the genotypic diversity within populations of aquatic and riparian plants. We used six polymorphic microsatellites to examine the genetic diversity within and differentiation among 17 populations (606 individuals) of Sparganium emersum, in two Dutch-German rivers. Our study revealed a striking difference between rivers in the mode of reproduction (sexual vs. asexual) within S. emersum populations. The mode of reproduction was strongly related to locally reigning hydrodynamic conditions. Sexually reproducing populations exhibited a greater number of multilocus genotypes compared to asexual populations. The regional population structure suggested higher levels of gene flow among sexually reproducing populations compared to clonal populations. Gene flow was mainly mediated via hydrochoric dispersal of generative propagules (seeds), impeding genetic differentiation among populations even over river distances up to 50 km. Although evidence for hydrochoric dispersal of vegetative propagules (clonal plant fragments) was found, this mechanism appeared to be relatively less important. Bayesian-based assignment procedures revealed a number of immigrants, originating from outside our study area, suggesting intercatchment plant dispersal, possibly the result of waterfowl-mediated seed dispersal. This study demonstrates how variation in local environmental conditions in river systems, resulting in shifting balances of sexual vs. asexual reproduction within populations, will affect the genotypic diversity within populations. This study furthermore cautions against generalizations about dispersal of riparian plant species in river systems.