A non-Dicer RNase III and four other novel factors required for RNAi-mediated transposon suppression in the human pathogenic yeastC. neoformans

The human pathogenic yeastCryptococcus neoformanssilences transposable elements using endo-siRNAs and an Argonaute, Ago1. Endo-siRNAs production requires the RNA-dependent RNA polymerase, Rdp1, and two partially redundant Dicer enzymes, Dcr1 and Dcr2, but is independent of histone H3 lysine 9 methylation. We describe here an insertional mutagenesis screen for factors required to suppress the mobilization of theC. neoformans HARBINGERfamily DNA transposonHAR1. Validation experiments uncovered five novel genes (RDE1-5) required forHAR1suppression and global production of suppressive endo-siRNAs. Loss of theRDEgenes does not impact transcript levels, suggesting the endo-siRNAs do not act by impacting target transcript synthesis or turnover.RDE3encodes a non-Dicer RNase III related toS. cerevisiaeRnt1,RDE4encodes a predicted terminal nucleotidyltransferase, whileRDE5has no strongly predicted encoded domains. Affinity purification-mass spectrometry studies reveal that Rde3 and Rde5 are physically associated.RDE1encodes a G-patch protein homologous to theS. cerevisiaeSqs1/Pfa1, a nucleolar protein that directly activates the essential helicase Prp43 during rRNA biogenesis. Rde1 copurifies Rde2, another novel protein obtained in the screen, as well as Ago1, a homolog of Prp43, and numerous predicted nucleolar proteins. We also describe the isolation of conditional alleles ofPRP43, which are defective in RNAi. This work reveals unanticipated requirements for a non-Dicer RNase III and presumptive nucleolar factors for endo-siRNA biogenesis and transposon mobilization suppression inC. neoformans.

Threonine synthase CoTHR4 is involved in infection-related morphogenesis during the pre-penetration stage in Colletotrichum orbiculare

Upon recognition of host plants, Colletotrichum orbiculare, an anthracnose disease fungus of cucurbitaceous plants, initiates morphological differentiation, including conidial germination and appressorium formation on the cuticle layer. The series of infection processes of C. orbiculare requires enormous nutrient and energy, but the surface of the cucurbitaceous hosts is hardly nutrient-rich. Hence, C. orbiculare must exert tight management of its intracellular nutrients in order to properly induce infection-related morphogenesis. Here, we carried out a large-scale insertional mutagenesis screen using Agrobacterium tumefaciens-mediated transformation to identify novel genes involved in the pathogenicity of C. orbiculare and found that CoTHR4-encoded threonine synthase, a homolog of Saccharomyces cerevisiae THR4, is required for pathogenicity and conidiation in C. orbiculare. Threonine supplementation allowed the cothr4 mutant to produce conidia to a level equivalent to that of the wild-type. The conidia produced from the threonine-treated cothr4 mutant failed to germinate in the absence of threonine, but retained the ability to germinate and to form appressoria in the presence of threonine. However, the conidia produced from the threonine-treated cothr4 mutant remained attenuated in pathogenicity on cucumber cotyledons even in the presence of threonine. Cytorrhysis assays revealed that appressoria of the cothr4 mutant induced by exogenous threonine treatment showed low turgor generation. Taken together, these results showed that threonine synthase CoThr4 plays a pivotal role in infection-related morphogenesis during the pre-penetration stage of C. orbiculare.