Data_Sheet_1_Amino Acid Polymorphisms in the VHIID Conserved Motif of Nodulation Signaling Pathways 2 Distinctly Modulate Symbiotic Signaling and Nodule Morphogenesis in Medicago truncatula.DOCX

Legumes establish an endosymbiotic association with nitrogen-fixing soil bacteria. Following the mutual recognition of the symbiotic partner, the infection process is controlled by the induction of the signaling pathway and subsequent activation of symbiosis-related host genes. One of the protein complexes regulating nitrogen-fixing root nodule symbiosis is formed by GRAS domain regulatory proteins Nodulation Signaling Pathways 1 and 2 (NSP1 and NSP2) that control the expression of several early nodulation genes. Here, we report on a novel point mutant allele (nsp2-6) affecting the function of the NSP2 gene and compared the mutant with the formerly identified nsp2-3 mutant. Both mutants carry a single amino acid substitution in the VHIID motif of the NSP2 protein. We found that the two mutant alleles show dissimilar root hair response to bacterial infection. Although the nsp2-3 mutant developed aberrant infection threads, rhizobia were able to colonize nodule cells in this mutant. The encoded NSP2 proteins of the nsp2-3 and the novel nsp2 mutants interact with NSP1 diversely and, as a consequence, the activation of early nodulin genes and nodule organogenesis are arrested in the new nsp2 allele. The novel mutant with amino acid substitution D244H in NSP2 shows similar defects in symbiotic responses as a formerly identified nsp2-2 mutant carrying a deletion in the NSP2 gene. Additionally, we found that rhizobial strains induce delayed nodule formation on the roots of the ns2-3 weak allele. Our study highlights the importance of a conserved Asp residue in the VHIID motif of NSP2 that is required for the formation of a functional NSP1-NSP2 signaling module. Furthermore, our results imply the involvement of NSP2 during differentiation of symbiotic nodule cells.

豆科植物可与固氮土壤细菌建立内共生联合体。在共生双方相互识别后，侵染过程受信号通路诱导及后续共生相关宿主基因激活的调控。调控固氮根瘤共生的一类蛋白复合物由GRAS结构域（GRAS domain）调控蛋白结瘤信号通路1和2（Nodulation Signaling Pathways 1 and 2，NSP1和NSP2）构成，二者可调控多个早期结瘤基因的表达。本研究报道了一个影响NSP2基因功能的新型点突变等位基因（nsp2-6），并将该突变体与此前鉴定的nsp2-3突变体进行比较。两种突变体的NSP2蛋白VHIID基序均存在单氨基酸替换。研究发现，两个突变等位基因在根毛对细菌侵染的响应上存在显著差异。尽管nsp2-3突变体形成了异常侵染线，但根瘤菌仍可在该突变体的根瘤细胞中定殖。nsp2-3与新型nsp2突变体编码的NSP2蛋白与NSP1的互作模式存在差异，进而导致新型nsp2等位基因中早期结瘤素基因的激活与根瘤器官发生被阻滞。该携带NSP2蛋白D244H氨基酸替换的新型突变体，其共生响应缺陷与此前鉴定的携带NSP2基因缺失的nsp2-2突变体相似。此外，研究发现根瘤菌菌株可在弱效等位基因nsp2-3的根系诱导延迟结瘤。本研究凸显了NSP2蛋白VHIID基序中保守天冬氨酸残基的重要性，该残基是形成功能型NSP1-NSP2信号模块所必需的。此外，本研究结果暗示NSP2在共生根瘤细胞的分化过程中亦发挥作用。