Construction of an Efficient <i>Agrobacterium tumefaciens</i>-Based Transformation System in the Entomopathogenic fungus <i>Metahizium rileyi</i>

<i>Metarhizium rileyi</i> is a filamentous entomopathogenic fungus that is highly pathogenic to lepidopteran insects. In our study, we constructed an <i>Agrobacterium tumefaciens</i>-mediated transgene system using the hygromycin resistance gene (Hyg R) as a selection marker in <i>M. rileyi</i> through homologous recombination. Binary knockout vectors for two genes (<i>NOR_03501</i>, longevity assurance gene, and <i>NOR_03153</i>, ATP-binding domain protein domain gene) in the <i>M. rileyi</i> strain SZCY201010 were successfully developed. We compared the genetic transformation efficiency using five kinds of asexual spores. The initial genetic transformation rates using a competent blastospore for <i>NOR_03501</i> and <i>NOR_03153</i> were 54.35 and 47.19%, respectively. Subsequently, both genes were successfully knocked out, and the transformed fungi were verified by PCR, RT-qPCR, and green fluorescent protein labeling. The biological phenotypes of the two genes were analyzed. The <i>NOR_03501</i> gene plays a crucial role in carbon source utilization, stress resistance, and cuticle infection of fungal mycelium growth, while the <i>NOR_03153</i> gene is significant for conidial production, stress resistance, and body wall infection. This study provides a promising tool for gene manipulation in <i>M. rileyi</i>, enhancing research in functional genomics and the exploration of fungal gene resources.