Project description:Here we describe the identification and regulation of a novel dsRNA virus in Colletotrichum higginsianum. High throughput sequencing of small RNAs and strand-specific RNA-seq was performed on single gene knock-out mutants created for each RNAi component gene: rdr1, rdr2, rdr3, dcl1, dcl2, ago1, and ago2, and the double mutant: ∆dcl1∆dcl2. De novo assembly of the ∆dcl1 RNA-seq data identified two contigs that represented the forward and reverse strands of an uncharacterized dsRNA virus, designated here as Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1). We found increased presence of the viral RNA in the RNA-seq datasets of the ∆dcl1, ∆dcl1dcl2, and ∆ago1 strains, suggesting that these genes are required for control of the virus. We show that viral small RNAs co-immunoprecipitate with a 6xFLAG-3xHIS-tagged AGO1 protein by sequencing the small RNAs from immunoprecipitated fractions. Additionally, analyses of the small RNA datasets from the RNAi mutants revealed control of the virus through small RNA-mediated silencing required both AGO1 and DCL1.
Project description:Species from the genus Colletotrichum are the causal agents of anthracnose which contribute to significant losses to the production of commercially grown crops. The genomes of Colletotrichum orbiculare, which infects cucurbits and Nicotiana benthamiana, as well as Colletotrichum gloeosporioides, which infects a wide range of fruits and vegetables, were sequenced. A custom microarray was designed for Colletotrichum orbiculare and used to assess gene expression during infection of Nicotiana benthamiana. Gene expression of Colletotrichum orbiculare growing on its host Nicotiana benthamiana was assessed at 24 hours post inoculation, 3 days post inoculation and 7 days post inoculation. Mycelia growing in vitro and ungerminated conidia were used as controls. Three replicates were performed for each time point.