The Sinorhizobium (Ensifer) fredii HH103 Nodulation Outer Protein NopI Is a Determinant for Efficient Nodulation of Soybean and Cowpea Plants

The type 3 secretion system (T3SS) is a specialized secretion apparatus commonly used by many plant and animal pathogenic bacteria to deliver proteins, termed effectors, to the interior of the host cells. These effectors suppress host defenses and interfere with signal transduction pathways to promote infection. Some rhizobial strains possess a functional T3SS, which is involved in the suppression of host defense responses, host-range determination and symbiotic efficiency. The analysis of the genome of the broad host-range rhizobial strain Sinorhizobium fredii HH103 identified eight genes that code for putative T3SS effectors. Three of these effectors, NopL, NopP and NopI are Rhizobium -specific. In this work we demonstrate for the first time that NopI, whose aminoacid sequence shows certain similarity with NopP, is secreted through the S. fredii HH103 T3SS in response to flavonoids. We also determined that NopL can be considered an effector since it is directly secreted to the interior of the host cell, as demonstrated by adenylate cyclase assays. Finally, the symbiotic phenotype of single, double and triple nopI , nopL , and nopP mutants in soybean and cowpea was assayed, showing that NopI plays an important role in the determination of the number of nodules formed in both legumes and that the absence of both NopL and NopP is highly detrimental for symbiosis. Importance The paper is focused on three Rhizobium -specific T3SS effectors of Sinorhizobium fredii HH103, NopL, NopP, and NopI. We demonstrate for the first time that S. fredii HH103 is able to secrete through the T3SS in response to flavonoids the nodulation outer protein NopI. Besides, we determined that NopL can be considered an effector since it is secreted to the interior of the host cell, as demonstrated by adenylate cyclase assays. Finally, the symbiotic phenotype of single, double and triple nopI , nopL , and nopP mutants in soybean and cowpea was assayed, showing that NopI plays an important role in the determination of the number of nodules formed in both legumes and that the absence of both NopL and NopP is highly detrimental for symbiosis.