Bacteriophages are the most abundant biological entities on earth and known to play a crucial role in microbial ecology, physiology, virulence, and nutrient cycling (1). Most studies on bacteriophage abundance, diversity, genetic content, host-specificity, and their effect on microbial communities are currently focused on the limited viral taxa, specifically within the double-strand DNA (dsDNA) phages. However, recent studies on metatranscriptome revealed the actual ubiquity and diversity of non-dsDNA viral particles are largely understudied, and in particular single strand RNA (ssRNA) phages are especially prominent in soil ecosystems (2-6). Despite their importance, shotgun RNA sequencing efforts only provide limited and partial information about these novel ssRNA phages, and especially fail to associate these phages with their host bacteria, infectivity pathways, mechanism of host cell take-over and host lysis. For example, though ssRNA phages have been known to bind conjugative pili elements for infectivity, the knowledge and genetic basis of their infection and resistance has been limited to a couple of canonical phages (7). These early studies primarily isolated phage-resistant host mutants and characterized them using classical genetic approaches. With the recent discovery of tens of thousands of novel ssRNA phages there is a need for high-throughput technologies to characterize ssRNA phage-host interactions. Here, we adopt recently developed two high-throughput genetic technologies (8), Random barcode transposon site sequencing (RB-TnSeq) and Dual-barcoded shotgun expression library sequencing (Dub-seq) to a model E. coli strain for discovering host factors, and gene dosage barriers crucial in ssRNA phage infection and bacterial resistance. We resourced a collection of ssRNA phages including three model ssRNA phages, MS2, fr, and Qβ to map genetic landscape important in phage infection. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm the importance of conjugative pili elements as well as uncover other host factors playing an important role in ssRNA phage infective pathways. To the best of our knowledge, this is the first such report to systematically characterize non-dsDNA phage-host interactions and opens up an avenue to extend it to the other E. coli strains and non-E. coli phages.