Root nodule symbiosis (RNS) is one of the most productive and economical systems for nitrogen fixation, and previous studies have shown that several nodule-specific C2H2-zinc finger proteins (ZFPs) play important roles in symbiosis establishment and nodule function. However, C2H2-ZFPs are the most widespread ZFPs in eukaryotes, and a great variation of structure and function exist among the family members. It remains largely unclear whether or not special types of C2H2-ZF genes participate in symbiosis, especially in soybean. In the present study, we performed a genome-wide survey of soybean C2H2-ZF genes, and 321 soybean C2H2-ZF genes were identified and classified into 11 clearly distinguishable subsets (Gm-t1-SF, Gm-t2-SF, Gm-1i-Q-SF, Gm-1i-M-SF, Gm-1i-Z-SF, Gm-1i-D-SF, Gm-2i-Q-SF, Gm-2i-M-SF, Gm-2i-Mix-SF, Gm-3i-SF, and Gm-4i-SF) based on the arrangements, numbers, and types of C2H2-ZF domains. Phylogenetic and gene ontology analyses were carried out to assess the conserved sequence and GO function among these subsets, and the results showed that the classification of soybean C2H2-ZFPs was reasonable. The expression profile of soybean C2H2-ZFPs in multiple tissues showed that nearly half of soybean C2H2-ZFPs within different subsets had expressions in nodules, including a clustering branch consisting of 11 Gm-1i-Q-SF genes specifically expressed in symbiotic-relative tissues. RNA-Seq was used to identify symbiosis-related soybean C2H2-ZFPs, and the expression pattern of the soybean C2H2-ZFPs in roots and nodules at different development stages showed that soybean C2H2-ZFPs mainly played roles in nodule development or nodule function rather than nodulation signal transduction, and nearly half of these genes had high expressions and/or different expression patterns during soybean nodule development, especially for the six clustering branches of genes consisting of different subsets of C2H2-ZFPs. Furthermore, the selected symbiosis-related soybean C2H2-ZFPs might function in legume-rhizobium symbiosis through regulating or interacting with other key proteins. Taken together, our findings provided useful information for the study on classification and conservative function of C2H2-ZFPs, and offered solid evidence for investigation of rhizobium symbiosis-related C2H2-ZFPs in soybean or other legumes.

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.

Plants regulate various biological activities in cells by producing reactive oxygen species (ROS) as signal molecules. Plant NADPH oxidases, respiratory burst oxidase homologues (Rbohs), are the main source of ROS and participate in a variety of plant physiological processes. However, there are few studies regarding the function of Rbohs in rhizobium–legume symbiotic nodulation. A Rbohs subfamily gene GmRbohK was cloned and the gene expression and its subcellular localization were studied. The GmRbohK gene was highly expressed in soybean roots and nodules and greatly induced by rhizobia. GmRbohK was located at the cell membrane of Arabidopsis protoplasts. Moreover, RNA interference (RNAi) of GmRbohK resulted in a decrease in the number of nodules and nitrogenase activity. Meanwhile, the expression levels of nodulation marker genes decreased in association with the decrease in GmRbohK expression level. RNAi of GmRbohK inhibited the production of ROS and blocked infection events at the initial stage of nodule formation. Further analysis of nodule tissue sections revealed that RNAi of GmRbohK significantly reduced the number of bacteroids in the nodule infection area. In conclusion, GmRbohK is heavily involved in the regulation of soybean nodule formation.