Gene silencing mechanisms in Phytophthora infestans

Abstract: The hemibiotrophic oomycete, Phytophthora infestans, causes late blight on potato and tomato. This destructive pathogen is well known for the rapidity with which it overcomes host resistance. This thesis focuses on the molecular basis of gene (RNA) silencing in P. infestans and the role it may have in its genome biology and pathogenesis. A comparative genomic approach identified 51 genes from nine gene families, encoding Dicer-like (PiDcl1), Argonaute (PiAgo), RNA-dependent RNA polymerase (PiRdr1), Histone eacetylases, histone methyltransferases, DEAD helicases, chromodomain proteins,and a class 1 RNaseIII with a possible role in gene silencing. It was shown by knockdown of PiDcl1, PiAgo1, and PiHda1 (Histone deacetylase) expression, that these components were involved in maintaining transcriptional (heterochromatic) silencing in P. infestans. The large genome of P. infestans (~ 240 Mb) is composed of approximately 75% transposons and repeats. The genome has a bimodal architecture, consisting of a highly conserved and tightly packed core genome that is interrupted by transposons and repeats. The genes encoding the RxLR and Crinkler (CRN) effector classes that are critical to plant infection are predominantly located in the repeat and transposon rich regions. Small RNAs (sRNA) of 40 nt, homologous to a non-autonomous short interspersed retrotransposable element infSINEm were found. A sense orientation transcriptional fusion of infSINEm to the PiAvr3a (RxLR) effector gene led to silencing of both the introduced fusion and endogenous homologous sequences. Hence, it was concluded that P. infestans likely uses RNA silencing to minimize the activity of transposons and might influence expression of effector encoding genes in close proximity. Deep sequencing of sRNA from different life cycle stages of two P. infestans isolates, that differ in their disease causing abilities, revealed 21, 25/26, and 32 nt size classes of sRNAs that were predominantly derived from transposons. Effector gene-derived sRNAs were also present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Knockdown of Dicer and Argonaute gene expression provided evidence that biogenesis of 21 nt sRNAs is Dicerdependent,while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, six miRNA candidates, and sRNAs that mapped to mitochondrial, tRNA and genomic hotspots were identified. sRNAs and RNA silencing in P. infestans have features characteristic of both plants and animals.