Comparative genomics of Knoxdaviesia species in the Core Cape Subregion

Aylward, Janneke (2017-03)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Knoxdaviesia capensis and K. proteae are saprotrophic fungi that inhabit the seed cones (infructescences) of Protea plants in the Core Cape Subregion (CCR) of South Africa. Arthropods, implicated in the pollination of Protea species, disperse these native fungi from infructescences to young flower heads (inflorescences). Knoxdaviesia proteae is a specialist restricted to one Protea species, while the generalist K. capensis occupies a range of Protea species. Within young flower heads, Knoxdaviesia species grow vegetatively, but switch to sexual reproduction once flower heads mature into enclosed infructescences. Nectar becomes depleted and infructescences are colonised by numerous other organisms, including the arthropod vectors of the fungi. The aim of this dissertation was to study the ecology of K. capensis and K. proteae by making use of their genome sequences. Knoxdaviesia belongs to the family Gondwanamycetaceae, for which no genomes were available at the start of this study. Using Illumina technology, we determined the genome sequences of both CCR Knoxdaviesia species and applied them to investigate reproduction, substrate use and tolerance to competition. Population genetic studies on K. capensis and K. proteae have revealed massive diversity, suggesting an outcrossing reproductive strategy. To determine the genetic basis for this diversity, we used the genomes to identify and characterise their mating type (MAT) loci. Each species contained only a single MAT idiomorph per isolate, indicating that they require an individual of the opposite mating type for sexual reproduction. The MAT loci of the two Knoxdaviesia species were almost identical, reflecting their phylogenetic relatedness. The features of the Knoxdaviesia MAT1-2-7 gene also suggested a historic recombination event between the MAT1-1 and MAT1-2 idiomorphs. The carbon resources that Knoxdaviesia species utilise were investigated with phenome assays and compared to the carbon usage profile of a Protea pathogen, Ceratocystis albifundus. Knoxdaviesia capensis, the generalist, utilised the widest range of substrates, whereas the pathogen utilised the least. The Knoxdaviesia species were able to grow on all monosaccharides that occur in Protea nectar. The predicted proteins in the Knoxdaviesia and C. albifundus genomes suggested that cell wall degradation is important to the nutrition of Knoxdaviesia species in infructescences, whereas the pathogen prefers plant storage polysaccharides. Overall, carbon metabolism in three ecologically different, but related fungi reflected their ecological adaptations. Knoxdaviesia species appear to be effective competitors in infructescences. Few secondary metabolite biosynthesis clusters were, however, detected from the K. capensis and K. proteae genomes. This may suggest that it is the antimicrobial products of Streptomyces bacteria that rid infructescences of fungal competitors. The few secondary metabolite clusters of the Knoxdaviesia species likely produce compounds that enable them to tolerate these bacteria and arthropod and nematode predation. Proteins involved in cell defence were also detected among the predicted secreted proteins of K. capensis and K. proteae. Knoxdaviesia proteae appears to have some non-functional secondary metabolite clusters and secretes less cell defence proteins than K. capensis, suggesting that its specialisation on a single host has resulted in the loss of some functions.

AFRIKAANS OPSOMMING: Knoxdaviesia capensis en K. proteae is saprofitiese fungi wat die saadkeëls van Protea plante in die Kern Kaapse Substreek (KKS) van Suid-Afrika bewoon. Artropodes, geïmpliseer in die bestuiwing van Protea spesies, verprei hierdie inheemse fungi van saadkeëls na jong blomkoppe. Knoxdaviesia proteae is ʼn spesialis wat beperk is tot een Protea spesie, terwyl die meer algemene K. capensis in ʼn reeks Protea spesies voorkom. Binne jong blomkoppe groei Knoxdaviesia spesies vegetatief, maar skakel om na seksuele voortplanting sodra die blomkoppe tot geslote saadknoppe ontwikkel. Nektar raak uitgeput, en saadknoppe word gekoloniseer deur verskeie ander organismes, insluitend die artropode vektore van die fungi. Die mikpunt van hierdie dissertasie was om die ekologie van K. capensis en K. proteae te bestudeer deur van genoom basisvolgordes gebruik te maak. Knoxdaviesia behoort aan die familie Gondwanamycetaceae, waarvoor geen genome beskikbaar was aan die begin van hierdie studie nie. Deur van Illumina tegnologie gebruik te maak het ons die genoom basisvolgordes van beide KKS Knoxdaviesia spesies bepaal, en hulle toegepas om die reproduksie, substraat-gebruik en toleransie teen kompetisie te ondersoek. Populasie genetiese studies op K. capensis en K. proteae het massiewe diversiteit geopenbaar, wat ʼn kruistelende reproduktiewe strategie voorstel. Om die genetiese basis vir hierdie diversiteit te bepaal het ons die genome gebruik om hul paringsmaat-tipe (MAT) lokusse te bepaal en te karakteriseer. Elke spesie het slegs een MAT idiomorf per isolaat bevat, wat aandui dat hulle ʼn individu van die teenoorgestelde paringstipe benodig vir seksuele voortplanting. Die MAT lokusse van die twee Knoxdaviesia spesies was amper identies, wat hulle filogenetiese verwantskap reflekteer. Die kenmerke van die Knoxdaviesia MAT1-2-7 geen suggereer ook ʼn historiese rekombinasie gebeurtenis tussen die MAT1-1 en MAT1-2 idiomorfe. Die koolstofbronne wat Knoxdaviesia spesies gebruik is ondersoek met fenoom toetse en vergelyk met die koolstofgebruik-profiel van ʼn Protea patogeen, Ceratocystis albifundus. Die algemene Knoxdaviesia capensis het die wydste reeks substrate gebruik, terwyl die patogeen minste gebruik het. Die Knoxdaviesia spesies kon groei op al die monosakkariede wat in Protea nektar voorkom. Die voorspelde proteïene in die Knoxdaviesia en C. albifundus genome stel voor dat selwand degradasie belangrik is vir voeding van Knoxdaviesia spesies in saadkoppe, terwyl die patogeen plant storingspolisakkariede verkies. Oor die algemeen, reflekteer die koolstofmetabolisme in drie ekologies verskillend, maar verwante fungi hulle ekologiese aanpassings. Dit blyk of Knoxdaviesia effektiewe kompeteerders is in saadknoppe. Min sekondêre metaboliet biosintese groeperings is egter in die K. capensis en K. proteae genome opgespoor. Dit mag suggereer dat dit die antimikrobiese produkte van Streptomyces bakterieë is wat saadknoppe van fungus-kompeteerders ontneem. Die paar sekondêre metaboliet biosintese groeperings van die Knoxdaviesia spesies produseer waarskynlik verbindings wat hulle in staat stel om hierdie bakterieë sowel as artropode- en nematode-predasie te verdra. Proteïene betrokke by selbeskerming is ook waargeneem tussen die voorspelde gesekreteerde proteïene van K. capensis en K. proteae. Knoxdaviesia proteae blyk ook om nie-funksionele sekondêre metaboliet groeperings te hê, en sekreteer minder selbeskermingsproteïene as K. capensis, wat voorstel dat sy spesialisasie op ʼn enkele gasheer die verlies van sekere funksies tot gevolg gehad het.

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