Identification of host-specific virulence factors of the smut fungus Sporisorium reilianum by genotype to phenotype mapping

Borah, Nilam Nayan; Schirawski, Jan (Thesis advisor); Blank, Lars Mathias (Thesis advisor)

Aachen (2018, 2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2018


The two host-adapted varieties of the smut fungus Sporisorium reilianum (namely S. reilianum f. sp. zeae, SRZ, and S. reilianum f. sp. reilianum, SRS) produce spores either on maize or on sorghum. For plant infection, mating compatible haploid sporidia need to fuse and form infectious dikaryotic filaments that infect the plant at seedling stage and cause disease phenotype in the form of spore development in male and female inflorescences of the plant. Despite both formae speciales being very closely related on the genomic level why they differ in terms of host selection remains elusive. To know the host determining factors we generated sexual spores by crossing the compatible SRZ strain SRZ1_5-2 (mating type a1b1) with the SRS strain SRS2_H2-7 (mating type a2b6). Meiotic progeny (SRSZ) with the mating type a1b1 were selected and tested for virulence on sorghum after mating with SRS_H2-7. Virulence assays showed that the progeny were either non-virulent or showed various degrees of disease phenotype. We selected 110 non-virulent offspring, 50 offspring with full virulence and 29 offspring with intermediate virulence on sorghum for genotype analysis. Genomic DNA of 191 strains (189 offspring and 2 parental strains) was isolated and sequenced using Illumina technology with a read length of 125 nt in paired end. Mapping of the reads against the two assembled sister parental genomes showed that parental origin of chromosomal regions could be un-ambiguously assigned for all SRSZ strains. Interestingly, few strains contained partially duplicated genomic regions, i.e. carrying the same chromosomal fragments from both parents. Correlation of phenotype with parental origin of genomic loci revealed a region of 35 genes in the left arm of chromosome 7 potentially associated with the virulence phenotype on sorghum. Analysis of this region revealed a cluster of 9 genes that were expressed in planta, showed low sequence conservation and coded for proteins carrying predicted secretion signal peptides. Deletion of this gene cluster in forma specialis SRS and subsequent virulence analysis showed that this cluster is essential for the spore forming capacity of SRS in sorghum. These results indicate the power of classical genetics combined with genotype to phenotype association analysis using Next generation sequencing approaches for the discovery of genes involved in determining host specificity in S. reilianum.