Yongjun Feng

Pantoea ananatis YJ76 is a predominant endophytic diazotrophic bacterium isolated from rice, which can produce indole as a signal to improve stress resistance, colonization, and growth-promoting effect on the host. Methyl-accepting chemotaxis proteins (MCPs) are the main chemoreceptor in bacteria and participate in regulating various cellular activities. By constructing an mTn5 transposon mutant library of YJ76, we screened out a mutant with decreased indole production. And its ability to resist stresses and starvation, as well as colonizing and growth-promoting effect on host rice plants, was inhibited. Using the hiTAIL-PCR technique combined the genome re-sequencing, the mutation site was identified as the mcp-5685 gene with a length of 1545 bp. Bioinformatics analysis and chemotaxis experiments showed that the MCP encoded by mcp-5685 had L-serine chemotaxis functions, revealing the mechanism of the gene encoding protein to drive L-serine uptake, a key component for tryptophan synthesis, and thus promote indole synthesis in the regulatory pathways for indole synthesis. Starting from the upstream regulation direction of indole synthesis, this study breaks through the previous researching limitation of focusing only on the downstream physiological function regulation of indole and provides new ideas for studying the indole signal.

Verbasoside and hydroxysalidroside are derivatives of phenylethanol glycosides (PhGs) that exhibit a range of pharmacological activities and also serve as valuable precursors for synthesizing other more complex PhGs. However, chemical synthesis and plant extraction for large-scale production are impractical to date. Here, 79 fungi from four types of PhG-containing plants and their rhizospheric soils were screened out, and endophyte Alternaria botrytis RYF1 from Rehmannia glutinosa was identified with the ability to generate verbasoside from decaffeoyl acteoside. To mine the key enzymes concerning the biotransformation, RNA-seq was carried out on RYF1 and RSF6, from which novel feruloyl esterase DN417 was captured. DN417 possesses hydrolyzing capabilities toward several PhGs. DN417's ability to produce verbasoside was tested in a 5 L fermenter, yielding 50.37 g/L of verbasoside with a conversion rate of 94.51% from crude acteoside. Finally, a dual-enzyme synergy involving feruloyl esterase and rhamnosidase was designed to produce hydroxysalidroside. After optimization of the enzyme ratios and pH, the approach successfully yielded hydroxysalidroside at a concentration of 13.60 g/L with a conversion rate of 81.30%. The study is the first report on the feruloyl esterase for the PhGs and also laid a solid foundation for the production of value-added compounds of PhGs.

Cistanche deserticola is a precious Chinese medicinal material with extremely high health care and medicinal value. In recent years, the frequent occurrence of stem rot has led to reduced or even no harvests of C. deserticola. The unstandardized use of farm chemicals in the prevention and control processes has resulted in excessive chemical residues, threatening the fragile desert ecological environment. Therefore, it is urgent to explore safe and efficient prevention and control technologies. Biocontrol agents, with the advantages of safety and environment-friendliness, would be an important idea. The isolation, screening and identification of pathogens and antagonistic endophytic bacteria are always the primary basis. In this study, three novel pathogens causing C. deserticola stem rot were isolated, identified and pathogenicity tested, namely Fusarium solani CPF1, F. proliferatum CPF2, and F. oxysporum CPF3. For the first time, the endophytic bacteria in C. deserticola were isolated and identified, of which 37 strains were obtained. Through dual culture assay, evaluation experiment and tissue culture verification, a biocontrol candidate strain Bacillus atrophaeus CE6 with outstanding control effect on the stem rot was screened out. In the tissue culture system, CE6 showed excellent control effect against F. solani and F. oxysporum, with the control efficacies reaching 97.2% and 95.8%, respectively, indicating its great potential for application in the production. This study is of great significance for the biocontrol of plant stem rot and improvement of the yield and quality of C. deserticola.

Indole is traditionally known as a metabolite of l‐tryptophan and now as an important signaling molecule in bacteria, however, the understanding of its upstream synthesis regulation is very limited. Pantoea ananatis YJ76, a predominant diazotrophic endophyte isolated from rice (Oryza sativa), can produce indole to regulate various physiological and biochemical behaviors. We constructed a mutant library of YJ76 using the mTn5 transposon insertion mutation method, from which an indole‐deficient mutant was screened out. Via high‐efficiency thermal asymmetric interlaced PCR (hiTAIL‐PCR), the transposon was determined to be inserted in a gene (RefSeq: WP014605468.1) of unknown function that is highly conserved at the intraspecific level. Bioinformatics analysis implied that the protein (Protein ID: WP089517194.1) encoded by the mutant gene is most likely to be a new orphan substrate‐binding protein (SBP) for amino acid ABC transporters. Amino acid supplement cultivation experiments and surface plasmon resonance revealed that the protein could bind to l‐serine (K_D = 6.149 × 10⁻⁵ M). Therefore, the SBP was named as SerBP. This is the first case that a SBP responds to l‐serine ABC transports. As a precursor of indole synthesis, the transmembrane transported l‐serine was directly correlated with indole signal production and the mutation of serBP gene weakened the resistance of YJ76 to antibiotics, alkali, heavy metals, and starvation. This study provided a new paradigm for exploring the upstream regulatory pathway for indole synthesis of bacteria.

Pantoea agglomerans YS19 is a dominant endophytic bacterium isolated from rice, which is capable of promoting host plant growth by nitrogen-fixing and phytohormone secreting. We previously found that the cytidine repressor (CytR) protein conducts the regulation of indole signal in YS19. Here, we compared the whole-cell protein of the wild type YS19 and the ΔcytR mutant and subsequently identified one differential protein as alkyl hydroperoxide reductase subunit C related to oxidative stress and sulfur starvation tolerance. It was tested that cytR had a positive effect on the survival of YS19 under the oxidative stress and sulfur starvation conditions and this effect was inhibited by indole. To further understand the functional mode of indole in this regulation, we cloned the cytR promoter region (PcytR) of YS19 and tested the effect of indole on PcytR using gfp as a reporter gene. It was found that PcytR can sense indole and significantly inhibit the expression of the downstream gene. This study provided a deeper understanding of the multiple function of cytR and expanded a new research direction of how indole participates in gene regulation.

The soybean (Glycine max (L.) Merr.) is an important oil and food crop. Its growth and development is regulated by complex genetic networks, and there are still many genes with unknown functions in regulation pathways. In this study, GmNMHC5, a member of the MADS-box protein family, was found to promote flowering and maturity in the soybean. Gene expression profiling in transgenic plants confirmed that the 35S:GmNMHC5 T3 generation had early flowering and precocity. We used CRISPR-Cas9 to edit GmNMHC5 and found that late flowering and maturity occurred in Gmnmhc5 lines with stable inheritance. Remarkably, in the 35S:GmNMHC5 plants, the expression of flowering inhibitors GmFT1a and GmFT4 was inhibited. In addition, overexpression of GmNMHC5 in ft-10 (a late flowering Arabidopsis thaliana mutant lacking Flowering Locus T (FT) function) rescued the extremely late-flowering phenotype of the mutant A. thaliana. These results suggest that GmNMHC5 is a positive transcription factor of flowering and maturity in the soybean, which has a close relationship with FT homologs in the flowering regulation pathway. This discovery provides new ideas for the improvement of the flowering regulation network, and can also provide guidance for future breeding work.

As a homo‐oligomeric protein, the disassembly of Escherichia coli RbsD decamer produces a urea‐unfolded oligomeric intermediate structure, as the dissociation speed of the protein is lower than that of the unfolding process. There are five Lys2–Cl⁻–Lys2 salt linkages to connect these subunits. To explore the role of the salt linkages in these oligomeric intermediates, the Lys2Ala mutated in the N‐terminal of E. coli RbsD protein subunit was designed. It was found that the RbsD mutation protein (RbsD:K2A) loses its minor larger oligomers, which exist in RbsD, and displays other several oligomeric states (less than decamers), meanwhile the state of the oligomers depends on the protein concentration. It was also found that compared with RbsD, the crosslinking capability of the subunits of RbsD:K2A is weaker, while the crosslinking rate of dimers is higher, RbsD:K2A needs to substantially adjust its conformation to meet the space requirements when combined with d‐ribose. On the basis of these results, we suggest that Lys2–Cl⁻–Lys2 salt linkages in E. coli RbsD protein play an important role in stabilizing the intermediate products of oligomers and maintaining interaction between the intermediate products of oligomers, which may shed light on the study of these oligomeric proteins.