Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.

Rice domestication has dramatically improved its agronomic traits, albeit with unavoidable significantly reduced genetic diversity. Dongxiang common wild rice, the wild rice species distributed in northernmost China, exhibits excellent resistance against stress and diseases and provides a rich genetic resource for rice breeding. Most of the studies focus on the function of the plant genes, often disregarding the role of the root microbes associated with the plants. In this work, we isolated a Burkholderia strain from the root of Dongxiang wild rice, which we identified as Burkholderia cepacia BRDJ, based on a phylogenetic analysis. This strain promoted the rice growth under greenhouse conditions. The grain yield was higher in a rice line containing a small genomic fragment derived from the Dongxiang wild rice, compared to the indica rice cultivar Zhongzao 35. This new strain also increased the plant biomass under limiting nitrogen conditions. Interestingly, this strain had a differential effect on indica and japonica rice varieties under full nitrogen supply conditions. By genome sequencing and comparison with another two B. cepacia strains, we observed enriched genes related with nitrogen fixation and phytohormone and volatiles biosynthesis that may account for the growth-promoting effects of the BRDJ. BRDJ has the potential to be used as a biofertilizer in promoting nitrogen use efficiency and overall growth in rice.

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1 -transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.

The mitogen-activated protein kinase (MAPK) LjMPK6 is a phosphorylation target of SIP2, a MAPK kinase that interacts with SymRK (symbiosis receptor-like kinase) for regulation of legume-rhizobia symbiosis. Both LjMPK6 and SIP2 are required for nodulation in Lotus japonicus. However, the dephosphorylation of LjMPK6 and its regulatory components in nodule development remains unexplored. By yeast two-hybrid screening, we identified a type 2C protein phosphatase, LjPP2C, that specifically interacts with and dephosphorylates LjMPK6 in vitro. Physiological and biochemical assays further suggested that LjPP2C phosphatase is required for dephosphorylation of LjMPK6 in vivo and for fine-tuning nodule development after rhizobial inoculation. A non-phosphorylatable mutant variant LjMPK6 (T224A Y226F) could mimic LjPP2C functioning in MAPK dephosphorylation required for nodule development in hairy root transformed plants. Collectively, our study demonstrates that interaction with LjPP2C phosphatase is required for dephosphorylation of LjMPK6 to fine tune nodule development in L. japonicus.

Bacterial microcompartments (BMCs) are intracellular structures for compartmentalizing specific metabolic pathways in bacteria. As a unique type of BMCs, carboxysomes utilize protein shells to sequester ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase for efficient carbon dioxide (CO₂) fixation. This study aims to reconstruct an α-carboxysome in Saccharomyces cerevisiae and investigate its metabolic effects. Here, genes of the cso operon from Halothiobacillus neapolitanus, Calvin cycle-related enzyme phosphoribulokinase (PRK) from Spinacia oleracea, and two S. cerevisiae chaperone genes, HSP60 and HSP10, were introduced into S. cerevisiae. The engineered yeast strain demonstrated assembled and enzymatically active Rubisco, significant increase in ethanol production and reduction in the byproduct glycerol. Formation of the α-carboxysome structures was observed after purification by sucrose density gradient centrifugation. The engineered yeast strain harboring functional α-carboxysome has the potential for enhancing bioethanol production.

Heme is biosynthesized in legume root nodules to meet the demand for leghemoglobins (Lbs) and other heme-binding proteins. However, the main source of nodule heme remains unknown. Both the plant host and rhizobia possess a complete heme biosynthetic pathway, differing slightly in the production of 5-aminolevulinic acid (ALA), a key regulatory step catalyzed by glutamyl-tRNA reductase (GluTR) in the plant and by HemA in the rhizobia. Transcriptomic analysis revealed that many plant heme biosynthetic genes, including GluTR2 but not GluTR1, are upregulated in nodules compared to roots, whereas expression of related rhizobial genes, including both HemA1 and HemA2, is generally inhibited under symbiotic conditions compared to free-living conditions. Knockout of Lotus japonicus GluTR2, but not of HemA1 and HemA2, led to a significant decrease (∼50%) in nodule heme content. The stable heterozygous mutant of GluTR1 or transient knockdown of GluTR1 exhibited a ∼20% reduction in nodule heme content. Overexpression of Fluorescent in blue light (FLU), a feedback inhibitor of GluTR activity, caused a much greater reduction in nodule heme content (∼75%) and an increased level of apo-Lb and, in combination with the hemA1 hemA2 mutant, a drastic inhibition of nitrogenase activity (>90%). This study provides genetic evidence supporting a major role of plant GluTRs in coordinating heme biosynthesis between the two symbionts by supplying heme to assemble with cytoplasmic apo-Lbs and by providing ALA for heme synthesis in the bacteroids.