Bacterial wilt (BW) is a devastating plant disease caused by the soil-borne bacterium Ralstonia solanacearum species complex (Rssc). Numerous efforts have been exerted to control BW, but effective, economical, and environmentally friendly approaches are still not available. Bacteriophages are a promising resource for the control of bacterial diseases, including BW. So, in this study, a crop BW pathogen of lytic bacteriophage was isolated and named PQ43W. Biological characterization revealed PQ43W had a short latent period of 15 min, 74 PFU/cell of brust sizes, and good stability at a wide range temperatures and pH but a weak resistance against UV radiation. Sequencing revealed phage PQ43W contained a circular double-stranded DNA genome of 47,156 bp with 65 predicted open reading frames (ORFs) and genome annotation showed good environmental security for the PQ43W that no tRNA, antibiotic resistance, or virulence genes contained. Taxonomic classification showed PQ43W belongs to a novel genus of subfamily Kantovirinae under Caudoviricetes. Subsequently, a dose of PQ43W for phage therapy in controlling crop BW was determined: 108 PFU*20 mL per plant with non-invasive irrigation root application twice by pot experiment. Finally, a field experiment of PQ43W showed a significantly better control effect in crop BW than the conventional bactericide Zhongshengmycin. Therefore, bacteriophage PQ43W is an effective bio-control resource for controlling BW diseases, especially for crop cultivation.

Ethylene response factors (ERFs) have been associated with biotic stress in Arabidopsis, while their function in non-model plants is still poorly understood. Here we investigated the role of potato ERF StPti5 in plant immunity. We show that StPti5 acts as a susceptibility factor. It negatively regulates potato immunity against potato virus Y and Ralstonia solanacearum, pathogens with completely different modes of action, and thereby has a different role than its orthologue in tomato. Remarkably, StPti5 is destabilised in healthy plants via the autophagy pathway and accumulates exclusively in the nucleus upon infection. We demonstrate that StEIN3 and StEIL1 directly bind the StPti5 promoter and activate its expression, while synergistic activity of the ethylene and salicylic acid pathways is required for regulated StPti expression. To gain further insight into the mode of StPti5 action in attenuating potato defence responses, we investigated transcriptional changes in salicylic acid deficient potato lines with silenced StPti5 expression. We show that StPti5 regulates the expression of other ERFs and downregulates the ubiquitin-proteasome pathway as well as several proteases involved in directed proteolysis. This study adds a novel element to the complex puzzle of immune regulation, by deciphering a two-level regulation of ERF transcription factor activity in response to pathogens.

As important transcription factors, WRKYs play a vital role in the defense response of plants against the invasion of multiple pathogens. Though some WRKY members have been reported to participate in pepper immunity in response to Ralstonia solanacearum infection, the functions of the majority of WRKY members are still unknown. Herein, CaWRKY22b was cloned from the pepper genome and its function against R. solanacearum was analyzed. The transcript abundance of CaWRKY22b was significantly increased in response to the infection of R. solanacearum and the application of exogenous methyl jasmonate (MeJA). Subcellular localization assay in the leaves of Nicotiana benthamiana showed that CaWRKY22b protein was targeted to the nuclei. Agrobacterium-mediated transient expression in pepper leaves indicated that CaWRKY22b overexpression triggered intensive hypersensitive response-like cell death, H2O2 accumulation, and the up-regulation of defense- and JA-responsive genes, including CaHIR1, CaPO2, CaBPR1, and CaDEF1. Virus-induced gene silencing assay revealed that knock-down of CaWRKY22b attenuated pepper\'s resistance against R. solanacearum and the up-regulation of the tested defense- and jasmonic acid (JA)-responsive genes. We further assessed the role of CaWRKY22b in modulating the expression of JA-responsive CaDEF1, and the result demonstrated that CaWRKY22b trans-activated CaDEF1 expression by directly binding to its upstream promoter. Collectively, our results suggest that CaWRKY22b positively regulated pepper immunity against R. solanacearum in a manner associated with JA signaling, probably by modulating the expression of JA-responsive CaDEF1.

Ralstonia solanacearum species complex (RSSC) includes soilborne bacterial plant pathogens with worldwide distribution and wide host ranges. Virulence factors are regulated via four hierarchically organized cell-cell contact independent quorum-sensing (QS) signalling systems: the Phc, which uses as signals (R)-methyl 3-hydroxypalmitate [(R)-3-OH PAME] or (R)-methyl 3-hydroxymyristate [(R)-3-OH MAME], the N-acyl homoserine lactone (AHL)-dependent RasI/R and SolI/R systems, and the recently identified anthranilic acid-dependent system. The unique Phc QS system has been extensively studied; however, the role of the two AHL QS systems has only recently been addressed. In this microreview, we present and discuss current data of the SolI/R and RasI/R QS systems in the RSSC. We also present the distribution and frequency of these AHL QS systems in the RSSC, discuss possible ecological roles and evolutive implications. The complex QS hierarchical networks emphasizes the crucial role of cell-cell signalling in the virulence of the RSSC.

The presence of soil-borne disease obstacles and antibiotic resistance genes (ARGs) in soil leads to serious economic losses and health risks to humans. One area in need of attention is the evolution of ARGs as pathogenic soil gradually develops, which introduces uncertainty to the dynamic ability of conventional farming models to predict ARGs. Here, we investigated variations in tomato bacterial wilt disease accompanied by the resistome by metagenomic analysis in soils over 13 seasons of monoculture. The results showed that the abundance and diversity of ARGs and mobile genetic elements (MGEs) exhibited a significant and positive correlation with R. solanacearum. Furthermore, the binning approach indicated that fluoroquinolone (qepA), tetracycline (tetA), multidrug resistance genes (MDR, mdtA, acrB, mexB, mexE), and β-lactamases (ampC, blaGOB) carried by the pathogen itself were responsible for the increase in overall soil ARGs. The relationships between pathogens and related ARGs that might underlie the breakdown of soil ARGs were further studied in R. solanacearum invasion pot experiments. This study revealed the dynamics of soil ARGs as soil-borne diseases develop, indicating that these ecological trends can be anticipated. Overall, this study enhances our understanding of the factors driving ARGs in disease-causing soils.

Plant bacterial wilt caused by Ralstonia solanacearum results in huge losses. Accordingly, developing an effective control method for this disease is urgently required. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential biocontrol solution. A filamentous phage RSCq that infects R. solanacearum was isolated in this study through genome mining. We constructed engineered filamentous phages based on RSCq by employing our proposed approach with wide applicability to non-model phages, enabling the exogenous genes delivery into bacterial cells. CRISPR-AsCas12f1 is a miniature class 2 type V-F CRISPR-Cas system. A CRISPR-AsCas12f1-based gene editing system that targets the key virulence regulator gene hrpB was developed, generating the engineered phage RSCqCRISPR-Cas. Similar to the Greek soldiers in the Trojan Horse, our findings demonstrated that the engineered phage-delivered CRISPR-Cas system could disarm the key \"weapon,\" hrpB, of R. solanacearum, in medium and plants. Remarkably, pretreatment with RSCqCRISPR-Cas significantly controlled tobacco bacterial wilt, highlighting the potential of engineered filamentous phages as promising biocontrol agents against plant bacterial diseases.IMPORTANCEBacterial disease, one of the major plant diseases, causes huge food and economic losses. Phage therapy, an environmentally friendly control strategy, has been frequently reported in plant bacterial disease control. However, host specificity, sensitivity to ultraviolet light and certain conditions, and bacterial resistance to phage impede the widespread application of phage therapy in crop production. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential solution to overcome the limitations of lytic phage biocontrol. This study developed a genetic engineering approach with wide applicability to non-model filamentous phages and proved the application possibility of engineered phage-based gene delivery in plant bacterial disease biocontrol for the first.

BACKGROUND: The dirigent (DIR) genes encode proteins that act as crucial regulators of plant lignin biosynthesis. In Solanaceae species, members of the DIR gene family are intricately related to plant growth and development, playing a key role in responding to various biotic and abiotic stresses. It will be of great application significance to analyze the DIR gene family and expression profile under various pathogen stresses in Solanaceae species.
RESULTS: A total of 57 tobacco NtDIRs and 33 potato StDIRs were identified based on their respective genome sequences. Phylogenetic analysis of DIR genes in tobacco, potato, eggplant and Arabidopsis thaliana revealed three distinct subgroups (DIR-a, DIR-b/d and DIR-e). Gene structure and conserved motif analysis showed that a high degree of conservation in both exon/intron organization and protein motifs among tobacco and potato DIR genes, especially within members of the same subfamily. Total 8 pairs of tandem duplication genes (3 pairs in tobacco, 5 pairs in potato) and 13 pairs of segmental duplication genes (6 pairs in tobacco, 7 pairs in potato) were identified based on the analysis of gene duplication events. Cis-regulatory elements of the DIR promoters participated in hormone response, stress responses, circadian control, endosperm expression, and meristem expression. Transcriptomic data analysis under biotic stress revealed diverse response patterns among DIR gene family members to pathogens, indicating their functional divergence. After 96 h post-inoculation with Ralstonia solanacearum L. (Ras), tobacco seedlings exhibited typical symptoms of tobacco bacterial wilt. The qRT-PCR analysis of 11 selected NtDIR genes displayed differential expression pattern in response to the bacterial pathogen Ras infection. Using line 392278 of potato as material, typical symptoms of potato late blight manifested on the seedling leaves under Phytophthora infestans infection. The qRT-PCR analysis of 5 selected StDIR genes showed up-regulation in response to pathogen infection. Notably, three clustered genes (NtDIR2, NtDIR4, StDIR3) exhibited a robust response to pathogen infection, highlighting their essential roles in disease resistance.
CONCLUSIONS: The genome-wide identification, evolutionary analysis, and expression profiling of DIR genes in response to various pathogen infection in tobacco and potato have provided valuable insights into the roles of these genes under various stress conditions. Our results could provide a basis for further functional analysis of the DIR gene family under pathogen infection conditions.

Concern over nano- and microplastic contamination of terrestrial ecosystems has been increasing. However, little is known about the effect of nano- and microplastics on the response of terrestrial ecosystems already under biotic stress. Here, nano- and microplastics at 150-500 mg·kg-1 were exposed to tomatoes (Solanum lycopersicum L.), and the results demonstrate that the presence of nano- and microplastics increased the occurrence of bacterial wilt caused by Ralstonia solanacearum in tomatoes as a function of contaminant concentration, surface modification, and size. Our work shows that nanoplastics (30 nm, 250 mg·kg-1) increased the disease incidence by 2.19-fold. The disease severities in amino- and carboxyl-modified nanoplastic treatments were 30.4 and 21.7% higher than that in unmodified nanoplastic treatment, respectively. The severity of disease under the influence of different-sized nano- and microplastic treatments followed the order 30 > 100 nm > 1 > 50 μm. Mechanistically, nanoplastics disrupted the structure of the tomato rhizosphere soil bacterial community and suppressed the induced systemic resistance in tomato; nanoplastics in planta decreased the salicylic acid and jasmonic acid content in tomatoes, thus inhibiting systemic acquired resistance; and microplastics increased the soil water retention, leading to increased pathogen abundance in the rhizosphere. Additionally, the leachates from nano- and microplastics had no effect on disease occurrence or the growth of tomatoes. Our findings highlight a potential risk of nano- and microplastic contamination to agriculture sustainability and food security.

The NAC (NAM, ATAF, and CUC) is one of the largest transcription factor gene families in plants. In this study, 180, 141, and 131 NAC family members were identified from Saccharum complex, including S. officinarum, S. spontaneum, and Erianthus rufipilus. The Ka/Ks ratio of ATAF subfamily was all less than 1. Besides, 52 ATAF members from 12 representative plants were divided into three clades and there was only a significant expansion in maize. Surprisingly, ABA and JA cis-elements were abundant in hormonal response factor, followed by transcriptional regulator and abiotic stressor. The ATAF subfamily was differentially expressed in various tissues, under low temperature and smut pathogen treatments. Further, the ScATAF1 gene, with high expression in leaves, stem epidermis, and buds, was isolated. The encoded protein, lack of self-activation activity, was situated in the cell nucleus. Moreover, SA and JA stresses down-regulated the expression of this gene, while ABA, NaCl, and 4°C treatments led to its up-regulation. Interestingly, its expression in the smut susceptible sugarcane cultivars was much higher than the smut resistant ones. Notably, the colors presented slight brown in tobacco transiently overexpressing ScATAF1 at 1 d after DAB staining, while the symptoms were more obvious at 3 d after inoculation with Ralstonia solanacearum, with ROS, JA, and SA signaling pathway genes significantly up-regulated. We thus speculated ScATAF1 gene could negatively mediate hypersensitive reactions and produce ROS by JA and SA signaling pathways. These findings lay the groundwork for in-depth investigation on the biological roles of ATAF subfamily in sugarcane.

MYC2 is a class of bHLH family transcription factors and a major regulatory factor in the JA signaling pathway, and its molecular function in tobacco has not been reported. In this study, CRISPR/Cas9-mediated MYC2 gene NtMYC2a knockout mutants at tobacco was obtained and its agronomic traits, disease resistance, and chemical composition were identified. Comparing with the WT, the leaf width of the KO-NtMYC2a was narrowed, the nornicotine content and mecamylamine content increased significantly and the resistance to Ralstonia solanacearum significantly decreased. The transcriptome sequencing results showed that DEGs related to immunity, signal transduction and growth and development were enriched between KO-NtMYC2a and WT. NtJAR1 and NtCOI1 in KO-NtMYC2a were down-regulated to regulating the JA signaling pathway, result in a significant decrease in tobacco\'s resistance to R. solanacearum. Our research provides theoretical support for the functional research of MYC2 and the study of the mechanism of tobacco bacterial wilt resistance.