Factors governing attachment of Rhizobium leguminosarum to legume roots

Primary attachment of rhizobia to host legume roots depends on pH and is the first physical interaction during nodulation. Genome-wide insertion sequencing, luminescence-based attachment assays and proteomic analysis demonstrate primary attachment of Rhizobium leguminosarum biovar viciae 3841 to Pisum sativum (pea) roots is more complex than previously thought. In total, 115 proteins are needed for initial attachment under one or more test conditions (acid, neutral or alkaline pH), with 22 required under all conditions. These include cell-surface filamentous hemagglutinin adhesin (RL4382) and its transporter (RL4381), transmembrane protein RL2400, RL3752 (PssA, glycosyl transferase) affecting capsular polysaccharide and transcriptional regulator RL4145 (PckR). RNASeq was used to determine targets of RL4145 (PckR) and regulator RL3453. The 54 proteins required for attachment at pH 7.0 were investigated for nodulation phenotypes. Glucomannan biosynthesis protein A (GmsA) is needed at pH 6.5 and pH 7.0. Membrane proteins DgkA and ImpA are required specifically at pH 6.5, and RpoZ at pH 7.5. Sonicated cell surface fractions inhibited root attachment at alkaline pH but no overlap between proteins identified by proteomic and INseq analysis, suggests there is no single rhicadhesin needed for alkaline attachment. Our results demonstrate the complexity of primary root attachment and diversity of mechanisms involved.