Mungbean (Vigna radiata) is an important cash crop, yet the production is significantly compromised by continuous cropping. Beneficial microbial inoculation offers a promising strategy to alleviate the stresses through rhizosphere modulation and host physiological reprogramming. This study evaluated the efficacy of two biological control agents, Bacillus subtilis (B. subtilis) and Trichoderma harzianum (T. harzianum), in promoting mungbean growth under continuous-cropping conditions. Both individual applications of B. subtilis and T. harzianum significantly improved plant biomass, root system architecture, and yield. Combined metagenomic and transcriptomic analyses were conducted to unravel the underlying mechanisms. According to metagenomic analysis, both B. subtilis and T. harzianum were responsible for significant changes in beta diversity without significantly affecting the alpha diversity of the rhizosphere microbial community. T. harzianum recruited Chitinophagaceae unclassified, Abditibacterium, Hydrogenophilaceae unclassified, Methylophilaceae unclassified, and Chimaeribacter, while Bs recruited Candidatus Saccharibacteria unclassified. Transcriptomic analysis indicated that T. harzianum induced more extensive transcriptional reprogramming than B. subtilis. The enrichment analysis revealed both shared and distinct responses triggered by the two treatments. These findings suggest that B. subtilis and T. harzianum alleviate continuous-cropping stress through distinct yet complementary mechanisms involving rhizosphere microbiome modulation and mungbean transcriptional reprogramming. This study provides a sustainable strategy for legume cultivation.

Mungbean is an important legume and protein source, but its productivity is severely limited by the mungbean yellow mosaic India virus (MYMIV), and no MYMIV resistance gene has been cloned and functionally validated in mungbean. This study conducted extensive phenotypic variation research on the resistance of a newly developed mungbean multiparent advanced-generation intercross (MAGIC) population. Through an integrated genome-wide association study (GWAS), transcriptome analysis, and induced expression analysis, the candidate gene for MYMIV resistance was identified as VrADH, encoding alcohol dehydrogenase. Haplotype analysis revealed natural variation in VrADH, with VrADH Hap1 being the elite haplotype that has undergone selection in regions severely affected by MYMIV. Functional validation demonstrated that VrADH significantly enhanced resistance by limiting excessive reactive oxygen species accumulation and reducing viral proliferation. Collectively, our results indicate that VrADH can contribute to MYMIV resistance, providing a valuable genetic resource for future molecular breeding and resistance improvement in mungbean.

[Objective]Protein and threonine are the core nutrients of mung bean seeds,which have a critical impact on the nutritional quality of mung beans.Among them,mung bean protein is a high-quality protein source for vegetarians due to its easy digestion,absorption,and rich biological activity,which is of great value to human health.This study aims to explore genetic loci and candidate genes significantly associated with the protein content and relative content of threonine in mung bean seeds,providing a theoretical basis for genetic improvement of protein and threonine content in mung bean seeds.[Method]This study used Kjeldahl nitrogen determination method and liquid chromatography-mass spectrometry to determine the protein content and relative content of threonine in 279 mung bean varieties planted in 2022.The general linear model of Tassel5 software was used for genome-wide association study to explore genetic loci related to protein content and relative content of threonine in mung bean seeds.By combining linkage disequilibrium analysis,transcriptome comparative analysis,and comparative genomics,key candidate genes related to nitrogen metabolism were identified.[Result]Six key loci located on chromosome 3 were identified,which are related to the protein content and relative content of threonine in mung bean seeds,and can explain 6.06%to 12.20%of phenotypic variation.These 6 loci are closely linked and can be mainly divided into two haplotypes,with Hap2 being the dominant haplotype.Then,KASP molecular marker was designed for the key candidate locus SLG03_966208(A/G),which can successfully classify 80 mung bean varieties into three genotypes:AA,GG,and AG,with a detection accuracy rate of 87.5%.The key candidate gene EVM0000757 was identified within the locus linkage region,and the homologous gene AT5G65750 in Arabidopsis was significantly associated with nitrogen metabolism.Its gene expression was significantly different between high and low protein varieties(P=1.84E-03,|log2FC|=1.39),making it a reliable candidate gene.[Conclusion]279 mung bean varieties were identified for their grain protein content and relative content of threonine.Six SNP loci were detected within the 939 296-1 039 749 bp region of chromosome 3.A KASP molecular marker for the SLG03_966208(A/G)locus was developed,which can distinguish the high and low levels of protein and threonine content in mung bean grains.A key candidate gene related to nitrogen metabolism,EVM0000757,was identified.

Drought stress is a major global challenge in crop production. The occurrence of drought during seed germination directly affects crop establishment and final yield. In this work, we performed whole-genome resequencing of 414 mungbean (Vigna radiata (L.) R. Wilczek) germplasms and identified 4,875,142 high-quality single-nucleotide polymorphisms (SNPs). Two seed germination traits of the mungbean panel under drought stress across two environments were evaluated via a genome-wide association study (GWAS), and the results revealed a significant quantitative trait locus (QTL). Additionally, kompetitive allele-specific PCR (KASP) markers that can identify the germination characteristics of mungbean seeds under drought stress were developed and verified. Moreover, among the nine candidate genes near this site, the expression levels of different haplotypes of VrERF088 differed. Interestingly, the only ABA-responsive element in the VrERF088 promoter was mutated. The results demonstrated that VrABI5 activates the transcriptional activity of the Hap1 promoter by binding to the ABRE motif. The overexpression of VrERF088 in Arabidopsis thaliana significantly reduced the germination rate of plants under drought stress by increasing the expression of the ABA response genes AtPM6 and AtLEA4 and reducing the expression of the germination-related genes AtEXPAs, resulting in hypersensitivity to ABA. Our results indicate that mutation of the ABRE motif in the VrERF088 promoter results in a relatively high germination rate under drought stress.

Mungbean (Vigna radiata L.) is a nutritionally diverse food, associated with human health, such as heat dissipation. Although the genetic basis of yield-related traits in major crops has been thoroughly investigated, there is limited related gene information on mungbean. To address this issue, in this study, we first reported the high-quality genome assembly of cultivar Sulv1 Its size was 473.67 Mb, and the N50 value (11.3 Mb) was larger than those in previous studies. We then performed domestication analyses and genome-wide association studies on days to flowering and 100 seed weight. In domestication analyses, 20 differentially expressed genes were identified from 1022 selected regions and their Arabidopsis homologous genes were confirmed in Arabidopsis, such as LFR, GA20OX, and ACC2. In particular, EVM0030058 significantly increased the synthesis of isovitexin in mungbean hairy roots, and 40 μM isovitexin showed good effects in reducing inflammation in vitro. The domestication loci Chr3-47 757 894 was significantly associated with days to flowering and/or 100-seed weight. Tts candidate genes, VrLFR, a homolog of LFR in Arabidopsis, and verified by real-time PCR and haplotype analysis and found to be negatively correlated and differentially expressed in large- and small-seeded accessions. Finally, VrLFRH1 overexpression lines increased seed-size-related traits and promoted early flowering in transgenic Arabidopsis compared to Col-0. The p35S::VrLFR lines recovered the phenotype of lfr mutants. VrLFR was shown to be available for early maturing and high yielding breeding. Moreover, 100-seed weight showed a subtle positive correlative trend with isovitexin content (r = 0.385, P = 0.092). These findings establish a solid experimental foundation for anti-inflammatory studies on mungbean and provide a theoretical basis for its high-yield and high-quality molecular breeding.

Background/Objectives: Salinity stress limits agricultural production and threatens global food security. Faba bean (Vicia faba L.) is an important legume crop, and identifying salt-stress-responsive genes may support an improvement in salt response. This study aimed to identify intronless genes in faba bean, screen candidate genes associated with salt-stress responses, and develop a KASP marker for salt-response evaluation. Methods: Intronless genes were identified from the faba bean reference genome. Transcriptome analysis was conducted in roots and leaves of two cultivars, Sucan 4 and Yundou 1183, under 150 mM NaCl treatment and control conditions. Candidate genes were examined by expression analysis, functional annotation, PPI prediction, and a luciferase complementation assay. A KASP marker was developed from an SNP within the VfERF1A locus and tested in 97 accessions. Results: A total of 7581 intronless genes were identified, accounting for 20.69% of annotated genes. Fifteen intronless genes were significantly differentially expressed in both roots and leaves of the two cultivars under salt treatment. Functional annotation suggested that VfERF1A and VfHSP17.8 may be involved in salt-stress responses. PPI prediction and the LUC assay provided preliminary support for a possible association of VfERF1A with VfEIN2. The VfERF1A-based KASP marker showed clear genotype clustering, and the two homozygous classes differed significantly in QYmax, relative shoot fresh weight, and relative plant height under salt treatment (p < 0.05). The preliminary predictive accuracy for QYmax was 86.36%. Conclusions: These results provide a genome-wide resource of intronless genes in faba bean, identify candidate genes associated with salt-stress responses, and describe a preliminary KASP marker associated with salt-response traits. Further validation in independent populations, under diverse environmental conditions, and with additional functional evidence is still required.

The vernalization-responsive intronless gene in faba bean, VfERF017, functions as a flowering repressor, and its KASP marker enables early flowering selection with 83.15

Soil salinization severely impairs mungbean (Vigna radiata (L.) Wilczek) seedling uniformity and productivity. In this study, genome-wide association study (GWAS) was conducted using a natural population of 374 mungbean accessions and 4,875,143 SNPs. By evaluating the population under two independent environments and applying two statistical models, we identified a significant SNP (Chr01_26769549) associated with relative germination traits under salt stress. Based on this locus, a Kompetitive Allele-Specific PCR (KASP) marker was successfully developed for marker assisted selection. Integrated haplotype and expression analyses confirmed polygalacturonase gene VrPG1 as a key candidate gene regulating salt tolerance during seed germination. Two haplotypes of VrPG1 (Hap1/Hap2) were identified, with a mutation in the Hap1 promoter region enhancing its transcriptional activity. Overexpression of VrPG1 in Arabidopsis thaliana significantly increased germination rates under salt stress by promoting endosperm cell wall softening. Salt-tolerant mungbean varieties exhibit higher polygalacturonase activity and earlier loosening of thin-walled cell walls during the germination period, which promotes seed imbibition and radicle emergence. Collectively, these findings demonstrate that VrPG1 enhances salt tolerance during germination through cell wall remodeling. This study provides novel genetic targets and efficient marker-assisted selection tools for breeding salt-tolerant mungbean. This study provides novel genetic targets and efficient marker-assisted selection tools for breeding salt-tolerant mungbean varieties.

Pythium myriotylum Drechsler is considered to be one of the pathogens of mung bean (Vigna radiata (L.) R. Wilczek) root rot, which threatens the yield of V. radiata. Pathogenesis-related protein 1 (PR1) is an important plant defense protein against various stressors. However, the role of PR1 in V. radiata resistance to P. myriotylum remains largely unknown. In this study, we identified six PR1 family members containing the conserved CAPE1 (CAP-derived peptide 1) domain (PxGNxxxxxPY) through bioinformatic analysis of the V. radiata genome. VrPR1 expression was significantly induced after P. myriotylum infection in V. radiata plants, and three candidate genes (VrPR1-3, VrPR1-4, and VrPR1-6) were selected to investigate their molecular characteristics and roles in resistance to P. myriotylum. Subcellular localization analysis showed that these three PR1 proteins (VrPR1-3, VrPR1-4, and VrPR1-6) were localized to the extracellular space when ectopically expressed in Nicotiana benthamiana Domin leaves. VrPR1-3, VrPR1-4, and VrPR1-6 enhanced resistance to P. myriotylum, as demonstrated using Nicotiana benthamiana transient overexpression and V. radiata hairy root transformation systems. Actually, Transient overexpression of VrPR1-6 in Nicotiana benthamiana conferred enhanced resistance against P. myriotylum compared to the overexpression of VrPR1-3 or VrPR1-4. Further functional analysis using the Nicotiana benthamiana transient overexpression system indicated that the resistance conferred by PR1 depends on the C-terminal CAPE1 domain of VrPR1-6 protein. Furthermore, a comparative transcriptome analysis of hairy roots overexpressing VrPR1-6 and GFP showed that "plant-pathogen interaction" pathway genes, including 10 genes related to the Ca2+ signaling pathway, were upregulated. This study revealed that overexpression of VrPR1-3, VrPR1-4 and VrPR1-6 enhanced the resistance of V. radiata to P. myriotylum. Among them, the resistance of VrPR1-6 depends on the C-terminal CAPE1 domain and may involve Ca2 + -mediated signaling. These findings establish a foundation for further research into the immune defense pathways of V. radiata against P. myriotylum.

The number of pods per plant (PP) is strongly correlated with seed yield, and identifying genes that regulate PP could enhance the yield of mung bean (Vigna radiata (L.) Wilczek), providing valuable insights for molecular breeding. In this study, VrKNAT6 was identified through genome-wide association and multiomics analyses. Chr3-14344673 (P=3.02E-10~8.80E-07) was found to be significantly associated with PP using EMMAX, CMLM, GEMMA, GLM, and 3VmrMLM. Among the 12 genes located within a 100 kb region near Chr3-14344673 on chromosome 3, EVM0027029 (VrKNAT6) is homologous to known PP development-related genes in Oryza sativa and Arabidopsis thaliana. Overexpression of VrKNAT6H1 significantly increased rosette numbers, branch numbers, PP, and the 1,000-seed weight in transgenic Arabidopsis lines. Furthermore, when overexpressed in mung bean hairy roots and Arabidopsis, VrKNAT6H1 was found to participate in jasmonic acid (JA) synthesis through physical interaction with VrATH1. This interaction partly explains the differences in branch numbers between VrKNAT6H1-overexpressing Arabidopsis lines and the control. Additionally, the expression of JA synthetase-related genes was significantly elevated in the positive VrKNAT6H1 lines. Based on the multiomics analysis results, we propose a molecular regulatory mechanism for VrKNAT6H1, suggesting that it is a JA synthesis-related gene that could be utilized in mung bean high-yield molecular breeding.

Mungbean (Vigna radiata L. (Wilczek)) is an important food legume crop. The utilization of heterosis based on male sterile lines can help increase mungbean yields, yet genetic studies on mungbean male sterility are rare. Therefore, it is of great significance to explore the male sterility genes in mungbean. In this study, a no-pollen male sterile mutant vrnpms (Vigna radiata no pollen male sterility) was identified in mungbean. Gene mapping was conducted using F2 populations derived from the cross between vrnpms and V2709. The gene controlling the male sterility was mapped to a 426.65 kb region on chromosome 6. A candidate gene VrMYB80 (EVM0016947), encoding a protein homologous to MYB80 transcription factors, exhibits a 52-kb deletion in vrnpms, resulting in a truncated protein lacking the C’-terminus. A molecular marker linked to the male sterility phenotype was developed based on the deletion in vrnpms. Functional complementation in Arabidopsis demonstrated that VrMYB80 could restore fertility in the myb80 mutant. Subcellular localization showed that VrMYB80 was located in the nucleus. Transcriptional activation assays revealed that the C’-terminus of VrMYB80 was the transcriptional activation domain. The result of in-situ hybridization indicated that VrMYB80 is expressed in the anther tapetum. The expression level of downstream VrMS1 was down regulated in vrnpms, indicating that Vrmyb80 with the truncated C’-terminal transcriptional activation domain failed to activate downstream genes, which was the reason of sterility of vrnpms. The findings of this study contribute to unraveling the molecular genetic mechanism underlying pollen development in legume crops and pave the way for utilizing heterosis in mungbean.

Soybean (Glycine max) is one of the most important industrial and oilseed crops; however, the yield is threatened by the invasion of various pathogens. Soybean stem and root rot, caused by Phytophthora sojae, is a destructive disease that significantly damages soybean production worldwide. C2H2 zinc finger protein (C2H2-ZFP) is a large transcription factor family in plants that plays crucial roles in stress response and hormone signal transduction. Given its importance, we analyzed the expression patterns of C2H2-ZFP family genes in response to P. sojae infection and selected four candidate genes to explore their molecular characteristics and functions related to P. sojae resistance. Subcellular localization analysis indicated that three ZFPs (GmZFP2, GmZFP3, and GmZFP4) were localized in the nucleus, while GmZFP1 was found in both the nucleus and plasma membrane. Dual-luciferase transient expression analysis revealed that all four ZFPs possessed transcriptional repression activation. Further transient expression in N. benthamiana leaves demonstrated that GmZFP2 induced significant cell death and reactive oxygen species (ROS) accumulation. GmZFP2 significantly enhanced the resistance to Phytophthora pathogens in N. benthamiana leaves and soybean hairy roots. This study provides insights in to the functional characterization of soybean ZFPs in Phytophthora resistance and demonstrates that GmZFP2 plays a positive role in P. sojae resistance in soybeans.

The TATA-box binding protein (TBP) and TBP-associated factors (TAFs) constitute the transcription factor IID (TFIID), a crucial component of RNA polymerase II, essential for transcription initiation and regulation. Several TFIID subunits are shared with the Spt–Ada–Gcn5–acetyltransferase (SAGA) coactivator complex. Recent research has revealed the roles of TBP and TAFs in organogenesis and stress adaptation. In this study, we identified 1 TBP and 21 putative TAFs in the mungbean genome, among which VrTAF5, VrTAF6, VrTAF8, VrTAF9, VrTAF14, and VrTAF15 have paralogous genes. Their potential involvement in abiotic stress responses was also investigated here, including high salinity, water deficit, heat, and cold. The findings indicated that distinct genes exerted predominant influences in the response to different abiotic stresses through potentially unique mechanisms. Specifically, under salt stress, VrTBP, VrTAF2, and VrTAF15–1 were strongly induced, while VrTAF10, VrTAF11, and VrTAF13 acted as negative regulators. In the case of water-deficit stress, it was likely that VrTAF1, VrTAF2, VrTAF5–2, VrTAF9, and VrTAF15–1 were primarily involved. Additionally, in response to changes in ambient temperature, it was possible that genes such as VrTAF5–1, VrTAF6–1, VrTAF9–2, VrTAF10, VrTAF13, VrTAF14b–2, and VrTAF15–1 might play a dominant role. This comprehensive exploration of VrTBP and VrTAFs can offer a new perspective on understanding plant stress responses and provide valuable insights into breeding improvement.

Faba bean is an important legume crop consumed as a vegetable or snack food, and its green cotyledons could present an attractive color for consumers. A mutation in SGR causes stay-green in plants. In this study, vfsgr was identified from a green-cotyledon-mutant faba bean, SNB7, by homologous blast between the SGR of pea and the transcriptome of faba bean. Sequence analysis revealed that a SNP at position 513 of the CDS of VfSGR caused a pre-stop codon, resulting in a shorter protein in the green-cotyledon faba bean SNB7. A dCaps marker was developed according to the SNP that caused the pre-stop, and this marker was completely associated with the color of the cotyledon of faba bean. SNB7 stayed green during dark treatment, while the expression level of VfSGR increased during dark-induced senescence in the yellow-cotyledon faba bean HST. Transient expression of VfSGR in Nicotiana. benthamiana leaves resulted in chlorophyll degradation. These results indicate that vfsgr is the gene responsible for the stay-green of faba bean, and the dCaps marker developed in this study provides a molecular tool for the breeding of green-cotyledon faba beans.

WRKY-, PHD-, and MYB-like proteins are three important types of transcription factors in mungbeans, and play an important role in development and stress resistance. The genes’ structures and characteristics were clearly reported and were shown to contain the conservative WRKYGQK heptapeptide sequence, Cys4-His-cys3 zinc binding motif, and HTH (helix) tryptophan cluster W structure, respectively. Knowledge on the response of these genes to salt stress is largely unknown. To address this issue, 83 VrWRKYs, 47 VrPHDs, and 149 VrMYBs were identified by using comparative genomics, transcriptomics, and molecular biology methods in mungbeans. An intraspecific synteny analysis revealed that the three gene families had strong co-linearity and an interspecies synteny analysis showed that mungbean and Arabidopsis were relatively close in genetic relationship. Moreover, 20, 10, and 20 genes showed significantly different expression levels after 15 days of salt treatment (p < 0.05; Log2 FC > 0.5), respectively. Additionally, in the qRT-PCR analysis, VrPHD14 had varying degrees of response to NaCl and PEG treatments after 12 h. VrWRKY49 was upregulated by ABA treatment, especially in the beginning (within 24 h). VrMYB96 was significantly upregulated in the early stages of ABA, NaCl, and PEG stress treatments (during the first 4 h). VrWRKY38 was significantly upregulated by ABA and NaCl treatments, but downregulated by PEG treatment. We also constructed a gene network centered on the seven DEGs under NaCl treatment; the results showed that VrWRKY38 was in the center of the PPI network and most of the homologous Arabidopsis genes of the interacted genes were reported to have response to biological stress. Candidate genes identified in this study provide abundant gene resources for the study of salt tolerance in mungbeans.

YUCCA, belonging to the class B flavin-dependent monooxygenases, catalyzes the rate-limiting step for endogenous auxin synthesis and is implicated in plant-growth regulation and stress response. Systematic analysis of the YUCCA gene family and its stress response benefits the dissection of regulation mechanisms and breeding applications. In this study, 12 YUCCA genes were identified from the mungbean (Vigna radiata L.) genome and were named based on their similarity to AtYUCCAs. Phylogenetic analysis revealed that the 12 VrYUCCAs could be divided into 4 subfamilies. The evidence from enzymatic assays in vitro and transgenetic Arabidopsis in vivo indicated that all the isolated VrYUCCAs had biological activity in response to IAA synthesis. Expression pattern analysis showed that functional redundancy and divergence existed in the VrYUCCA gene family. Four VrYUCCAs were expressed in most tissues, and five VrYUCCAs were specifically highly expressed in the floral organs. The response toward five stresses, namely, auxin (indole-3-acetic acid, IAA), salinity, drought, high temperatures, and cold, was also investigated here. Five VrYUCCAs responded to IAA in the root, while only VrYUCCA8a was induced in the leaf. VrYUCCA2a, VrYUCCA6a, VrYUCCA8a, VrYUCCA8b, and VrYUCCA10 seemed to dominate under abiotic stresses, due to their sensitivity to the other four treatments. However, the response modes of the VrYUCCAs varied, indicating that they may regulate different stresses in distinct ways to finely adjust IAA content. The comprehensive analysis of the VrYUCCAs in this study lays a solid foundation for further investigation of VrYUCCA genes' mechanisms and applications in breeding.

Mung bean (Vigna radiata) production has been greatly threatened by numerous diseases. Infection with these pathogens causes extensive changes in gene expression and the activation of hormone signal transduction. Quantitative real-time PCR (qRT-PCR) is the most common technique used for gene expression validation. Screening proper reference genes for mung bean under pathogen infection and hormone treatment is a prerequisite for ensuring the accuracy of qRT-PCR data in mung bean disease-resistance research. In this study, six candidate reference genes (Cons4, ACT, TUA, TUB, GAPDH, and EF1α) were selected to evaluate the expression stability under four soil-borne disease pathogens (Pythium myriotylum, Pythium aphanidermatum, Fusarium oxysporum, and Rhizoctonia solani) and five hormone treatments (SA, MeJA, ETH, ABA, and GA3). In the samples from different treatments, the Ct value distribution of the six candidate reference genes was different. Under the condition of hormone treatment, the Ct value ranged from a minimum of 17.87 for EF1α to a maximum of 29.63 for GAPDH. Under the condition of pathogen infection, the Ct value ranged from a minimum of 19.43 for EF1α to a maximum of 31.82 for GAPDH. After primer specificity analysis, it was found that GAPDH was not specific, so the five reference genes Cons4, ACT, TUA, TUB, and EF1α were used in subsequent experiments. The software products GeNorm, NormFinder, BestKeeper and RefFinder were used for qRT-PCR data analysis. In general, the best candidates reference genes were: TUA for SA, ABA, GA3, and Pythium myriotylum treatment; TUB for ETH treatment; ACT for MeJA and Fusarium oxysporum treatment; and EF1α for Pythium aphanidermatum and Rhizoctonia solani treatment. The most stably expressed genes in all samples were TUA, while Cons4 was the least stable reference gene. Finally, the reliability of the reference gene was further validated by analysis of the expression profiles of four mung bean genes (Vradi0146s00260, Vradi0158s00480, Vradi07g23860, and Vradi11g03350) selected from transcriptome data. Our results provide more accurate information for the normalization of qRT-PCR data in mung bean response to pathogen interaction.

Crop yield can be effectively improved by changing root architecture and improving root resistance to stress. Identification of root-specific promoters is prerequisite for research into the regulation of root development and genetic manipulation of root traits. As an important crop species, researches on the genes and promoters specifically expressed in soybean roots is still devoid. In our study, genomic-scale mining of root-specific genes were performed based on the transcriptome date of soybean root, stem and leaf tissues in the seedling stage, among which 105 putative genes were further verified by RT-PCR and 33root-specific genes were identified. Then, 11 promoters（pro1 to pro 11） were cloned from soybean. GUS staining of transgenic soybean hairy roots and Nicotiana benthamiana seedlings showed that the 11 promoters had high root-specific or preferential expression activities. Besides, the GUS enzyme activities driven by pro 1, pro 2, pro 8 and pro 9 were all two folds higher than that of 35S. Our research provided a reference for the identification of tissue specific promoters in soybean and other species. In addition, the promoters identified in this study can be used to drive the specific expression of resistance genes in soybean root tissues, so as to improve the resistance of soybean to root diseases and achieve the purpose of variety improvement.

Phytophthora pathogens lead to numerous economically damaging plant diseases worldwide, including potato late blight caused by P. infestans and soybean root rot caused by P. sojae. Our previous work showed that Phytophthora pathogens may generate abundant phosphatidylinositol 3-phosphate (PI3P) to promote infection via direct association with RxLR effectors. Here, we designed a disease control strat-egy for metabolizing pathogen-derived PI3P by expressing secreted Arabidopsis thaliana phosphatidylino-sitol-4-phosphate 5-kinase 1 (AtPIP5K1), which can phosphorylate PI3P to PI(3,4)P2. We fused AtPIP5K1 with the soybean PR1a signal peptide (SP-PIP5K1) to enable its secretion into the plant apoplast. Trans -genic soybean and potato plants expressing SP-PIP5K1 showed substantially enhanced resistance to various P. sojae and P. infestans isolates, respectively. SP-PIP5K1 significantly reduced PI3P accumulation during P. sojae and soybean interaction. Knockout or inhibition of PI3 kinases (PI3Ks) in P. sojae compro-mised the resistance mediated by SP-PIP5K1, indicating that SP-PIP5K1 action requires a supply of pathogen-derived PI3P. Furthermore, we revealed that SP-PIP5K1 can interfere with the action of P. sojae mediated by the RxLR effector Avr1k. This novel disease control strategy has the potential to confer durable broad-spectrum Phytophthora resistance in plants through a clear mechanism in which catabolism of PI3P interferes with RxLR effector actions.

由于缺乏有效稳定的遗传转化体系,导致基因编辑技术在绿豆中的应用受到极大限制,也使绿豆基因功能研究受到极大阻碍.因此,建立一套基于发根农杆菌的操作简便、快速且高效的绿豆嵌合植株转化技术,可以为绿豆基因功能研究提供技术支撑.首先在 CRISPR/Cas9 载体骨架中插入 1 个绿色荧光蛋白(green fluorescent protein,GFP)表达框用于阳性发状根的快速筛选,然后将含有靶标基因gRNA的CRISPR/Cas9 质粒导入发根农杆菌K599.菌液注射侵染绿豆幼苗植株下胚轴,评价K599 在绿豆中诱导发状根发生及CRISPR/Cas9 系统在绿豆转基因发状根组织中的效率.结果显示,K599 菌液侵染种植7d的绿豆幼苗植株并在保湿条件下培养 3 周可在侵染点诱导发状根产生,此嵌合植株在切除原生根后可在霍格兰培养液中正常生长且培养 1 周后即可用于后续检测.用GFP筛选的结果显示,阳性发状根占比约为(57.8±10.0)％.随机选择 15 个荧光检测阳性的转基因发状根组织,利用测序检测靶位点的编辑效果,结果显示有 10 个靶位点的序列发生了突变,突变比例达到 67％,其中碱基缺失突变 9 个,碱基转换突变 1 个.由本研究结果可知,利用该方法可以在无菌组织培养的条件下于4 周内获得目的基因编辑的转基因组织,极大地便利了绿豆分子水平上的功能研究,也为其他尚缺乏稳定遗传转化体系的作物进行基因功能研究提供了参考.