ABSTRACTRhizosphere colonization by plant growth-promoting rhizobacteria (PGPR) along plant roots facilitates the ability of PGPR to promote plant growth and health. Thus, an understanding of the molecular mechanisms of the root colonization process by plant-beneficialBacillusstrains is essential for the use of these strains in agriculture. Here, we observed that ansfpgene mutant of the plant growth-promoting rhizobacteriumBacillus velezensisSQR9 was unable to form normal biofilm architecture, and differential protein expression was observed by proteomic analysis. A minor wall teichoic acid (WTA) biosynthetic protein, GgaA, was decreased over 4-fold in the Δsfpmutant, and impairment of theggaAgene postponed biofilm formation and decreased cucumber root colonization capabilities. In addition, we provide evidence that the major WTA biosynthetic enzyme GtaB is involved in both biofilm formation and root colonization. The deficiency in biofilm formation of the ΔgtaBmutant may be due to an absence of UDP-glucose, which is necessary for the synthesis of biofilm matrix exopolysaccharides (EPS). These observations provide insights into the root colonization process by a plant-beneficialBacillusstrain, which will help improve its application as a biofertilizer.IMPORTANCEBacillus velezensisis a Gram-positive plant-beneficial bacterium which is widely used in agriculture. Additionally,Bacillusspp. are some of the model organisms used in the study of biofilms, and as such, the molecular networks and regulation systems of biofilm formation are well characterized. However, the molecular processes involved in root colonization by plant-beneficialBacillusstrains remain largely unknown. Here, we showed that WTAs play important roles in the plant root colonization process. The loss of thegtaBgene affects the ability ofB. velezensisSQR9 to sense plant polysaccharides, which are important environmental cues that trigger biofilm formation and colonization in the rhizosphere. This knowledge provides new insights into theBacillusroot colonization process and can help improve our understanding of plant-rhizobacterium interactions.