Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a significant threat to the kiwifruit industry.  The two-component signaling systems (TCSs) play a crucial role in regulating the virulence of P. syringae, yet their specific function in Psa remains largely unclear.  In this study, we found that disrupting the TCS RegAB (encoded by Psa_802/Psa_803) resulted in a notable increase in the virulence of P. syringae pv. actinidiae M228 (Psa M228) in host plant and hypersensitive reaction (HR) in nonhost plant.  Through comparative transcriptome analysis of the Psa M228 wild-type strain and the regA mutant, we identified the pivotal role of RegAB in controlling various physiological pathways, including the type III secretion system (T3SS), a key determinant of Psa virulence.  Additionally, we discovered that the RegA has binding sites in the promoter region of the hrpR/S, and the transcriptional level of the hrpR and other T3SS-related genes increased in the regA deletion strain relative to the Psa M228 wild-type.  The DNA-binding affinity of RegA, and therefore the repressor function, is enhanced by its phosphorylation.  Our findings unveil the function of TCS RegAB and the regulatory mechanism of T3SS by RegAB in Psa, highlighting the diverse functions of the RegAB system.

Increasing the oil content is a key objective in peanut breeding programs.  Accurate identification of quantitative trait loci (QTLs) with linked markers for oil content can facilitate marker-assisted selection for high-oil breeding.  In this study, a high-density bin map was constructed by resequencing a recombinant inbred line (RIL) population (ZH16×J11) consisting of 295 lines.  The bin map contained 4,212 loci and had a total length of 1,162.3 cM.  Ten QTLs for oil content were identified in six linkage groups.  Notably, two of these QTLs, qOCB03.1 and qOCB06.1, were consistently detected in a minimum of three environments and explained up to 13.62% of the phenotypic variation.  They have not been reported in previous studies and thus are novel QTLs.  The combination of favorable alleles from qOCB03.1 and qOCB06 in the RIL population could increase oil content across multiple environments from 1.50 to 2.46%.  Two insertions/deletions (InDels) markers linked to qOCB03.1 and qOCB06.1 were developed, and their association with oil content was validated in another RIL population (ZH10×ICG12625) with diverse phenotypes.  In addition, the high-resolution map allowed for the precise positioning of qOCB03.1 and qOCB06.1 within a 1.77 Mb interval on chromosome B03 and a 1.51 Mb interval on chromosome B06, respectively.  The annotation of genomic variants, analysis of transcriptome sequencing, and evaluation of the allelic effects in 292 peanut varieties revealed two candidate genes associated with oil content for each of the two QTLs.  The candidate genes identified in this study can enable the map-based cloning of key genes controlling oil content in peanut.  Furthermore, these novel and stable QTLs and their tightly linked markers are valuable for marker-assisted breeding for greater oil content in peanut.

African swine fever (ASF) is a highly lethal hemorrhagic disease of swine caused by African swine fever virus (ASFV). Development of safe and effective ASFV subunit vaccine relies on the identification of protective antigens. In this study, we systematically evaluated the antigenicity of ASFV non-structural protein pA151R recognized by T cells from immune-protected pigs. Recombinant pA151R (rpA151R) was expressed in E. coli and used to generate anti-rpA151R polyclonal antibodies (pAb). This pAb bound both eukaryotically-expressed and native viral pA151R, confirming that rpA151R retains its native antigenicity. Using ASFV attenuated vaccine-immunized pigs, we further analysed the kinetics and functions of pA151R-specific T cells as well as their epitope recognition. The results showed that pA151R-specific T cell responses peaked at 14 days post-immunization in pigs, and secreted IFN-γ, TNF-α, IL-2, and perforin simultaneously, with multifunctional characteristics. T-cell epitope mapping identified seven peptides recognized by these pA151R-specific T cells. Among them, three peptides (P2, P4, and P5) were exclusively recognized by CD4⁺ T cells, four peptides (P6, P10, P12, and P13) were specific for CD8⁺ T cells whereas P1, P7, and P9 were recognized by both CD4⁺ and CD8⁺ T cells. These peptide-specific CD4⁺ or CD8⁺ T cells showed cytotoxicity, killing peptide-pulsed autologous target cells in a dose-dependent manner. These findings demonstrated that pA151R-specific swine T cells are able to contribute to protective immunity against ASFV and pA151R is a potential protective antigen for vaccine development. This study established a benchmark for screening and defining more ASFV protective antigens.

Kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae (Psa), severely threatens the kiwifruit industry. The type III secretion system (T3SS) is a key virulence factor in Psa, but the regulatory mechanisms remain poorly understood. Polymyxin B1, the main component of polymyxin B, inhibits T3SS gene expression in Psa, yet its underlying mechanism is unclear. Cyclic diguanosine monophosphate (c-di-GMP), a crucial bacterial second messenger, is synthesized by diguanylate cyclases (DGCs) containing a GGDEF domain. In this study, we identified and characterized PSA_1379 (WspR), a GGDEF domain-containing protein in Psa. Biochemical assays demonstrated that WspR exhibits DGC activity. Virulence assays showed that WspR negatively regulates Psa virulence. RT-qPCR analyses revealed that polymyxin B induces wspR expression. Additionally, polymyxin B upregulates intracellular c-di-GMP levels and inhibits the expression of T3SS genes through WspR. Bacterial two-hybrid and GST pull-down assays confirmed that WspR interacts with the transcription factor PsrA. Both WspR and c-di-GMP inhibit the binding of PsrA to the promoter of the T3SS master regulator hrpL, thereby suppressing PsrA-mediated transcriptional activation of hrpL and ultimately repressing T3SS gene expression. This study provides new insights into Psa virulence regulation and suggests potential targets for KBC control through the WspR-c-di-GMP pathway.

Early leaf spot (ELS) is one of peanut’s prominent and widespread foliar fungal diseases, causing severe yield losses and forage quality deterioration in South China. Discovery of the genomic region and the underlying candidate gene controlling ELS resistance will promote progress in resistance breeding and facilitate uncovering its genetic basis. In this study, a major genomic region, qELSB02.1, was identified using a bulked segregant RNA-Seq (BSR-seq) approach in a RIL population derived from a cross between a susceptible cultivar ZH10 and a resistant line ICG12625. It was further confirmed via simple sequence repeat genetic map-based linkage analysis, explaining 20.13-35.27% of the phenotypic variation. Using a partial genetic map and a segregation mapping population, qELSB02.1 was fine-mapped into a 465 kb genomic region by linkage analysis and substitution mapping. Furthermore, an NB-ARC-LRR gene (Arahy.V6I7WA) was identified as the most probable candidate gene for qELSB02.1 and was named Arachis hypogaea ELS resistance 1 (AhELSR1) based on functional annotation, sequence variation analysis, expression profiling, and protein structure prediction. Allelic variation analysis using 244 global peanut germplasm accessions identified four haplotypes, providing valuable clues for understanding ELS resistance evolution mediated by AhELSR1. Five SNPs, located in the first exon of AhELSR1, altering four encoding amino acids, were used to develop a diagnostic marker. The marker was further validated using diverse peanut germplasm and through introgression of AhELSR1 into a susceptible cultivar. Our results provide new insights into the genetic basis of ELS resistance regulation and benefit the breeding efforts for developing improved cultivars with enhanced ELS resistance. 

Bacterial pathogens harbor numerous two-component systems (TCSs) in their genomes, which enable rapid sensing and response to environmental fluctuations, thereby facilitating dynamic adaptation to diverse ecological niches. Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of kiwifruit bacterial canker (KBC), a devastating disease threatening global kiwifruit production. However, the biological function of the metal-responsive TCS CzcSR in Psa remains largely uncharacterized. In this study, we demonstrated that CzcSR plays a crucial role in regulating Psa pathogenicity in the host plant and the hypersensitive response (HR) in the non-host plant. Under zinc ion (Zn²⁺) stress, Psa exhibited suppressed motility and enhanced oxidative stress tolerance; notably, this phenotype depends on the Zn²⁺-binding sites of CzcS and the phosphorylation status of CzcR. However, the key virulence factor type III secretion system (T3SS) of Psa is unaffected by Zn²⁺ stress, and CzcSR-mediated regulation of the T3SS is independent of both the Zn²⁺-binding sites of CzcS and the phosphorylation status of CzcR. Instead, CzcR controls T3SS expression by binding to the promoter region of hrpR and modulates the c-di-GMP level via interacting with diguanylate cyclase (DGC) PSA_4781. Collectively, our findings expand CzcSR’s functional repertoire, highlight TCS complexity, and deepen understanding of TCS versatility—CzcSR integrates Zn²⁺ signals for canonical regulation of phenotypes (e.g., motility, antioxidant defense) while using a signal-independent mechanism for T3SS control.

African swine fever (ASF) is a highly contagious and hemorrhagic disease caused by African swine fever virus (ASFV), with a mortality rate approaching 100% in domestic pigs. ASFV is a large DNA virus, and its genome can be recognized by the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS) following infection to trigger the production of type I interferon (IFN-I) through the cGAS-STING signaling pathway. To establish productive infection, ASFV encodes multiple proteins to negatively regulate the cGAS-STING pathway and inhibit the expression of IFN-I. However, the molecular mechanisms by which ASFV proteins negatively regulate cGAS-STING signaling pathway remain incompletely elucidated. Through screening ASFV-encoded proteins, we found that pD345L significantly inhibits IFN-I production. Furthermore, we demonstrate that ASFV pD345L inhibits the promoter activities of Interferon-β (IFN-β)-, Interferon-α (IFN-α)-, interferon-stimulated gene (ISG)-54-Luciferase (Luc), as well as the mRNA levels of IFN-β, ISG-54, ISG-56 induced by cGAS-STING in a dose-dependent manner. Moreover, our findings reveal that ASFV pD345L interacts with both stimulator of interferon genes (STING) and interferon regulatory factor 3 (IRF3), thereby disrupting the formation of the STING-IRF3 complex. This interaction leads to impaired IRF3 phosphorylation and nuclear translocation, ultimately suppressing the production of IFN-I. Collectively, our findings reveal that ASFV pD345L functions as a negative regulator of the cGAS-STING signaling pathway to inhibit IFN-I production, thereby facilitating the viral evasion of the host innate immune response.