Evaluation of genetic and strain specific factors on root colonization in endophytic insect pathogenic fungi (EIPF) Metarhizium and Beauveria, with special emphasis on hydrophobins

The Ascomycete genera, Metarhizium and Beauveria, are traditionally known as insect pathogenic fungi and are widely used as mycopesticides in agricultural settings. More recently, an additional role of these fungi in nature as root symbionts, with the ability to transfer nitrogen from dead insects to host plants, has been recognized. In this study, the genetic as well as strain-specific factors of Metarhizium and Beauveria were assessed during interaction with the plant and insect hosts. Hydrophobins are small proteins, unique to filamentous fungi, that provide hydrophobicity to aerial hyphae and conidia, and also supports fungal attachment to host surfaces. The role of two hydrophobins (hyd1 and hyd2) in insect pathogenicity were previously described in Beauveria, but little is known about their possible role in root colonization. Gene expression and plant root colonization assays revealed that the deletion of hyd1 or hyd2 subjected Beauveria to stress, which subsequently altered the expression of genes involved in signaling pathways, pigment production, specific adhesins, as well as fungal association with the root. The involvement of six Metarhizium genes on plant root colonization and insect pathogenesis were also investigated. Nitrogen transporter genes, Mep2, MepC and Urease, were selected due to sequence similarity with previously characterized plant-associating fungal ammonium transporters. Root colonization assays showed that the targeted deletion of MepC and Mep2 genes in M. robertsii enhanced the rhizoplane colonization on barley roots and insect-derived nitrogen transfer to plant hosts. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots; these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. Root colonization assessment revealed that the loss of Hyd3, Pr1A, or the hypothetical protein gene from M. robertsii had no influence on establishing association with barley roots. We also assessed ten Metarhizium generalist and specialist strains and a related endophytic fungus Pochonia, for insect pathogenicity and their ability colonize plants; however, regardless of whether the Metarhizium species was a generalist or specialist insect pathogen all strains tested showed some ability to associate with plants. Moreover, Metarhizium spp. were able to colonize monocots better than dicots. Our data indicates that even after divergence as generalist or specialist insect pathogens, Metarhizium spp. maintain their ancestral ability to colonize plants. Overall, this study provides useful insights into the genes involved in EIPF-root interactions and also highlights the impact of gene deletion in triggering compensatory pathways