The AlphaproteobacteriumRhizobium radiobacter(syn.Agrobacterium tumefaciens, "A. fa brum") can live in close association with the sebacinoid fungusSerendipita(syn.Piriformospora)indicathat forms a mutualistic Sebacinalean symbiosis with a wide range of host plants. The endobacterial strainR. radiobacterF4 (RrF4), which was originally isolated from the fungus, has plant growth promotion and resistance-inducing activities resembling the beneficial activities known from the endobacteria-containingS. indica. The mechanism by which free endobacterial cells influence growth and disease resistance of colonized host plants is not fully understood. Here, we show thatRrF4 produces a spectrum of quorum sensing-mediatingN-acyl-homoserine lactones (AHLs) with acyl chains of C8, C10, and C12 as well as hydroxyl- or oxo-substitutions at the C3 position. In addition, and in line with previous findings showing that AHLs increase plant biomass and induce systemic resistance, the AHL-depleted lactonase-overexpressing transconjugantRrF4NM13 was partially compromised in promoting growth and inducing resistance against bacterial pathogens in bothArabidopsis thalianaand wheat (Triticum aestivum). Scanning and transmission electron microscopy proved thatRrF4NM13, in contrast toRrF4, does not form cellulose-like fiber scaffolds for efficient root surface attachment. Moreover,RrF4NM13 does not penetrate into the intercellular space of the cortical tissue, which in contrast is strongly colonized byRrF4. We discuss the possibility that AHLs contribute to the outcome of the Sebacinalean symbiosis.