Milpas are traditional Mesoamerican agroecosystems maintained with ancestral practices. Maize landraces are grown in polyculture, creating highly productive and diverse ecosystems. Recent studies suggest that milpas maintain beneficial plant-microbe interactions that are probably absent in modern agroecosystems; however, direct comparisons of the microbiome of plants between traditional and modern agroecosystems are still needed. Here, we studied seed-endophytic bacterial communities from native maize landraces from milpas and hybrid varieties. First, we quantified the abundance of culturable endophytic microbes; next, we assessed pairwise antagonistic interaction networks between bacterial isolates; finally, we compared bacterial community structure by 16S rRNA amplicon sequencing. We found that seeds from native maize landraces harbour a higher endophytic microbial load, including more bacterial strains with antagonistic activity against soil-borne bacteria, and overall harbour more diverse bacterial communities than the hybrid varieties. Noteworthy, most of the seedendophytic strains with antagonistic activity corresponded to Burkholderia spp. that were only found in native maize seeds, through both culturedependent and independent strategies. Altogether, our results support that crop modernization alters the functions and structure of plant-associated microbes; we propose native maize from milpas could serve as a model for understanding plant-microbe interactions and the effect of modernization.

Quorum sensing (QS) is a mechanism of synthesis and detection of signaling molecules to regulate gene expression and coordinate behaviors in bacterial populations. In Bacillus subtilis, multiple paralog Rap-Phr QS systems (receptor-signaling peptides) are highly redundant and multifunctional, interconnecting the regulation of differentiation processes such as sporulation and competence. However, their functions in the Bacillus cereus group are largely unknown. We evaluated the functions of Rap proteins in Bacillus thuringiensis Bt8741, which codes for eight Rap-Phr systems; these were individually overexpressed to study their participation in sporulation, biofilm formation, spreading, and extracellular proteolytic activity. Our results show that four Rap-Phr systems (RapC, RapK, RapF, and RapLike) inhibit sporulation, two of which (RapK and RapF) probably dephosphorylate Spo0F from the Spo0A phosphorelay; these two Rap proteins also inhibit biofilm formation. Four systems (RapC, RacF1, RacF2, and RapLike) participate in spreading inhibition; finally, six systems (RapC, -F, -F2, -I, and -I1 and RapLike) decrease extracellular proteolytic activity. We foresee that functions performed by Rap proteins of Bt8741 could also be carried out by Rap homologs in other strains within the B. cereus group. These results indicate that Rap-Phr systems constitute a highly multifunctional and redundant regulatory repertoire that enables B. thuringiensis and other species from the B. cereus group to efficiently regulate collective functions during their life cycle in the face of changing environments. IMPORTANCE The Bacillus cereus group of bacteria includes species of high economic, clinical, biological warfare, and biotechnological interest, e.g., B. anthracis in bioterrorism, B. cereus in food intoxications, and B. thuringiensis in biocontrol. Knowledge about the ecology of these bacteria is hindered by our limited understanding of the regulatory circuits that control differentiation and specialization processes. Here, we uncover the participation of eight Rap quorum-sensing receptors in collective functions of B. thuringiensis. These proteins are highly multifunctional and redundant in their functions, linking ecologically relevant processes such as sporulation, biofilm formation, spreading, extracellular proteolytic activity, and probably other functions in species from the B. cereus group.

In bacterial populations, quorum sensing (QS) systems participate in the regulation of specialization processes and regulate collective behaviors that mediate interactions and allow survival of the species. In Gram-positive bacteria, QS systems of the RRNPP family (Rgg, Rap, NprR, PlcR, and PrgX) consist of intracellular receptors and their cognate signaling peptides. Two of these receptors, Rap and NprR, have regained attention in Bacillus subtilis and the Bacillus cereus group. Some Rap proteins, such as RapH and Rap60, are multifunctional and/or redundant in function, linking the specialization processes of sporulation and competence, as well as global expression changes in the transition phase in B. subtilis. NprR, an evolutionary intermediate between Rap and RRNPP transcriptional activators, is a bifunctional regulator that modulates sporulation initiation and activates nutrient scavenging genes. In this review, we discuss how these receptors switch between functions and connect distinct signaling pathways. Based on structural evidence, we propose that RapH and Rap60 should be considered moonlighting proteins. Additionally, we analyze an evolutionary and ecological perspective to understand the multifunctionality and functional redundancy of these regulators in both Bacillus spp. and non-Bacillus Firmicutes. Understanding the mechanistic, structural, ecological, and evolutionary basis for the multifunctionality and redundancy of these QS systems is a key step for achieving the development of innovative technologies for health and agriculture.

20 Quorum Sensing (QS) are mechanisms of synthesis and detection of signaling molecules to 21 regulate gene expression and coordinate behaviors in bacterial populations. In Bacillus 22 subtilis (Bs), multiple paralog Rap-Phr QS systems (receptor-signaling peptide) are highly Importance 41 The Bacillus cereus group of bacteria includes species of high economic, clinical, 42 biological warfare and biotechnological interest, e.g. B. anthracis in bioterrorism, B. 43 cereus in food intoxications and B. thuringiensis in biocontrol. Knowledge on the ecology 44 of these bacteria is hindered due to our limited knowledge of the regulatory circuits that 45 control differentiation and specialization processes. Here, we uncover the participation of 46 eight Rap quorum-sensing receptors in collective functions of B. thuringiensis. These proteins are highly multifunctional and redundant in their functions, linking ecologically 48 relevant processes such as sporulation, biofilm formation, extracellular proteolytic activity 49 and spreading, and probably other additional functions in species from the B. cereus group. 50 51