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Background/Objectives: The balanced regulation of innate immunity plays essential roles in rhizobial infection and the establishment and maintenance of symbiosis. The evolutionarily conserved cell death suppressor Bax inhibitor-1 plays dual roles in nodule symbiosis, providing a valuable clue in balancing immunity and symbiosis, while it remains largely unexplored in the legume Lotus japonicus. Methods/Results: In the present report, the BI-1 gene family of L. japonicus was identified and characterized. We identified 6 BI-1 genes that translate into peptides containing 240–255 amino acids with different structural characteristics and isoelectric points. We performed phylogenetic analyses and detected evolutionary conservation and divergence among BI-1 proteins from L. japonicus, Glycine max, Medicago truncatula, Arabidopsis thaliana, and Oryza sativa. Expression profiles among different roots indicated that the inoculation of MAFF303099 significantly increased the expression of most of the L. japonicus BI-1 family genes. We down-regulated the transcripts of LjBI-1a by RNA interference and observed that LjBI-1a promotes nodulation and nodule formation. Conclusions: These discoveries shed light on the functions of BI-1 genes in L. japonicus, and simultaneously emphasize the potential application of LjBI-1a in enhancing the symbiotic nitrogen fixation ability of legumes. [ABSTRACT FROM AUTHOR]

Simple Summary: The brown planthopper is a major pest threatening rice crops in Asia, making it essential to identify target genes for RNAi-based pest control strategies. The Cleavage Stimulation Factor (CstF) complex, which plays a key role in mRNA 3′ end processing, is composed of several critical genes, and mutations in these genes can cause severe developmental defects, indicating their potential as targets. In this study, we identified homologs of the human CstF complex genes (NlCstF50, NlCstF64, and NlCstF77) in Nilaparvata lugens and investigated their functions using RNAi techniques. These genes are expressed throughout all developmental stages and various tissues, with particularly high levels in eggs and testes. Targeting these genes with RNAi resulted in significant mortality, decreased egg production, and lower hatch rates. These results suggest that NlCstF50, NlCstF64, and NlCstF77 could be effective targets for RNAi-based pest control strategies. The Cleavage Stimulation Factor (CstF) complex, consisting of three subunits, is essential for the 3′ end processing of precursor messenger RNA (pre-mRNA). In mammals, this complex includes CstF50, CstF64, and CstF77, named according to their molecular weights, and these proteins are conserved across many organisms. However, the functional roles of the three CstF genes (NlCstF50, NlCstF64, and NlCstF77) in Nilaparvata lugens, a major rice pest, have not been fully explored. This study identified and characterized the sequences of these genes, with proteins encoded by NlCstF50, NlCstF64, and NlCstF77 consisting of 439, 419, and 732 amino acids, respectively. These proteins are conserved among various insect species. Spatio-temporal expression analysis revealed that these genes are expressed at all developmental stages and in various tissues, with peak levels in eggs and testes. RNA interference (RNAi) targeting one or all three NlCstF genes resulted in a reduction in gene expression by 68% to 90% at 72 h post-injection, indicating that multi-gene dsRNA can achieve similar silencing outcomes as single-gene dsRNA. Knocking down one or all three NlCstF genes caused significant lethal phenotypes and molting disruptions. Mortality rates increased from 62.5% (dsNlCstF50) to 95.4% (dsNlCstF(50+64+77)). Additionally, silencing these genes reduced the number of eggs laid per female and hatch rates. These results highlight the critical role of NlCstF genes in the development and reproduction of N. lugens, suggesting their potential as targets for RNAi-based pest control strategies. [ABSTRACT FROM AUTHOR]

Transcription factor complex formation is a central step in regulating gene expression. In this report, a novel MYB coiled-coil transcription factor referred to as IPN2, for Interacting Protein of NSP2, is described., The interaction between IPN2 and NSP2 was examined by protein pull-down assays and bimolecular fluorescence complementation ( Bi FC). Subcellular localization of proteins, gene expression and gene function were assessed in transgenic hairy roots expressing tagged recombinant proteins, promoter-reporter and RNA interference ( RNAi) constructs, respectively., The GRAS domain of NSP2 and the coiled-coil domain of IPN2 were found to be responsible for the interaction between the two proteins. IPN2 had strong transcription activation activity, bound directly to the NIN gene promoter, and was localized to the nuclei of Lotus japonicus root cells. The expression of IPN2 was elevated during nodule development, coinciding with increased NSP2 gene expression during nodule organogenesis. RNAi-mediated knockdown expression of IPN2 did not affect arbuscular mycorrhizal development, but had deleterious effects on rhizobial infection and nodule formation in L. japonicus., These results demonstrate an important role of IPN2 in nodule organogenesis and place a new MYB transcription factor in the Nod signaling pathway. [ABSTRACT FROM AUTHOR]

The symbiosis receptor kinase, SymRK, is required for root nodule development. A SymRK-interacting protein (SIP2) was found to form protein complex with SymRK in vitro and in planta. The interaction between SymRK and SIP2 is conserved in legumes. The SIP2 gene was expressed in all Lotus japonicus tissues examined. SIP2 represents a typical plant mitogen-activated protein kinase kinase (MAPKK) and exhibited autophosphorylation and transphosphorylation activities. Recombinant SIP2 protein could phosphorylate casein and the Arabidopsis thaliana MAP kinase MPK6. SymRK and SIP2 could not use one another as a substrate for phosphorylation. Instead, SymRK acted as an inhibitor of SIP2 kinase when MPK6 was used as a substrate, suggesting that SymRK may serve as a negative regulator of the SIP2 signaling pathway. Knockdown expression of SIP2 via RNA interference (RNAi) resulted in drastic reduction of nodules formed in transgenic hairy roots. A significant portion of SIP2 RNAi hairy roots failed to form a nodule. In these roots, the expression levels of SIP2 and three marker genes for infection thread and nodule primordium formation were downregulated drastically, while the expression of two other MAPKK genes were not altered. These observations demonstrate an essential role of SIP2 in the early symbiosis signaling and nodule organogenesis. [ABSTRACT FROM AUTHOR]