A vigilant gliding bird protects plants.

The plant hormone salicylic acid (SA) receptor NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) plays a critical role for plant defense against biotrophic and hemi-biotrophic pathogens. In a milestone paper, Kumar, Zavaliev, Wu et al. unraveled the structural basis for the assembly of an enhanceosome by NPR1 in activating the expression of plant defense genes.

Depositing centromere repeats induces heritable intragenic heterochromatin establishment and spreading in Arabidopsis.

Stable transmission of non-DNA-sequence-based epigenetic information contributes to heritable phenotypic variants and thus to biological diversity. While studies on spontaneous natural epigenome variants have revealed an association of epialleles with a wide range of biological traits in both plants and animals, the function, transmission mechanism, and stability of an epiallele over generations in a locus-specific manner remain poorly investigated. Here, we invented a DNA sequence deposition strategy to generate a locus-specific epiallele by depositing CEN180 satellite repeats into a euchromatic target locus in Arabidopsis. Using CRISPR/Cas9-mediated knock-in system, we demonstrated that depositing CEN180 repeats can induce heterochromatin nucleation accompanied by DNA methylation, H3K9me2, and changes in the nucleosome occupancy at the insertion sites. Interestingly, both DNA methylation and H3K9me2 are restricted within the depositing sites and depletion of an H3K9me2 demethylase IBM1 enables the outward heterochromatin propagation into the neighboring regions, leading to inheritable target gene silencing to persist for at least five generations. Together, these results demonstrate the promise of employing a cis-engineering system for the creation of stable and site-specific epialleles and provide important insights into functional epigenome studies and locus-specific transgenerational epigenetic inheritance.

Regulatory Effects of Diverse DSF Family Quorum-Sensing Signals in Plant-Associated Bacteria.

Numerous bacterial species employ diffusible signal factor (DSF)-based quorum sensing (QS) as a widely conserved cell-cell signaling communication system to collectively regulate various behaviors crucial for responding to environmental changes. cis-11-Methyl-dodecenoic acid, known as DSF, was first identified as a signaling molecule in Xanthomonas campestris pv. campestris. Subsequently, many structurally related molecules have been identified in different bacterial species. This review aims to provide an overview of current understanding regarding the biosynthesis and regulatory role of DSF signals in both pathogenic bacteria and a biocontrol bacterium. Recent studies have revealed that the DSF-based QS system regulates antimicrobial factor production in a cyclic dimeric GMP-independent manner in the biocontrol bacterium Lysobacter enzymogenes. Additionally, the DSF family signals have been found to be involved in suppressing plant innate immunity. The discovery of these diverse signaling mechanisms holds significant promise for developing novel strategies to combat stubborn plant pathogens. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

In Situ Forming Gel Polymer Electrolyte for High Energy-Density Lithium Metal Batteries.

In situ forming gel polymer electrolyte (GPE) is one of the most feasible ways to improve the safety and cycle performances of lithium metal batteries with high energy density. However, most of the in situ formed GPEs are not compatible with high-voltage cathode materials. Here, this work provides a novel strategy to in situ form GPE based on the mechanism of Ritter reaction. The Ritter reaction in liquid electrolyte has the advantage of appropriate reaction temperature and no additional additives. The polymer chains are cross-linked by amide groups with the formation of GPE with superior electrochemical properties. The GPE has high ionic conductivity (1.84 mS cm-1 ), wide electrochemical stability window (>5.25 V) and high lithium ion transference number (≈0.78), compatible with high-voltage cathode materials. The Li|LiNi0.6 Co0.2 Mn0.2 O2 batteries with in situ formed GPE show excellent long-term cycle stability (93.4%, 300 cycles). The density functional theory calculation and X-ray photoelectron spectroscopy results verify that the amide and nitrile groups are beneficial for stabilizing cathode structure and promoting uniform Li deposition on Li anode. Furthermore, the in situ formed GPE exhibits excellent electrochemical performance in Graphite|LiMn2 O4 and Graphite|LiNi0.5 Co0.2 Mn0.3 O2 pouch batteries. This approach is adaptable to current battery technologies, which will be sure to promote the development of high energy-density lithium-ion batteries.

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

Unveiling the Role of Diffusible Signal Factor-Family Quorum Sensing Signals in Regulating Behavior of Xanthomonas and Lysobacter.

Diffusible signal factor (DSF) family signals represent a unique group of quorum sensing (QS) chemicals that modulate a wide range of behaviors for bacteria to adapt to different environments. However, whether DSF-mediated QS signaling acts as a public language to regulate the behavior of biocontrol and pathogenic bacteria remains unknown. In this study, we present groundbreaking evidence demonstrating that RpfFXc1 or RpfFOH11 could be a conserved DSF-family signal synthase in Xanthomonas campestris or Lysobacter enzymogenes. Interestingly, we found that both RpfFOH11 and RpfFXc1 have the ability to synthesize DSF and BDSF signaling molecules. DSF and BDSF positively regulate the biosynthesis of an antifungal factor (heat-stable antifungal factor, HSAF) in L. enzymogenes. Finally, we show that RpfFXc1 and RpfFOH11 have similar functions in regulating HSAF production in L. enzymogenes, as well as the virulence, synthesis of virulence factors, biofilm formation, and extracellular polysaccharide production in X. campestris. These findings reveal a previously uncharacterized mechanism of DSF-mediated regulation in both biocontrol and pathogenic bacteria.

Biocontrol Methods for the Management of Sclerotinia sclerotiorum in Legumes: A Review.

Sclerotinia sclerotiorum is an economically damaging fungal pathogen that causes Sclerotinia stem rot in legumes, producing enormous yield losses. This pathogen is difficult to control due to its wide host spectrum and ability to produce sclerotia, which are resistant bodies that can remain active for long periods under harsh environmental conditions. Here, the biocontrol methods for the management of S. sclerotiorum in legumes are reviewed. Bacillus strains, which synthesized lipopeptides and volatile organic compounds, showed high efficacies in soybean plants, whereas the highest efficacies for the control of the pathogen in alfalfa and common bean were observed when using Coniothyrium minitans and Streptomyces spp., respectively. The biocontrol efficacies in fields were under 65%, highlighting the lack of strategies to achieve a complete control. Overall, although most studies involved extensive screenings using different biocontrol agent concentrations and application conditions, there is a lack of knowledge regarding the specific antifungal mechanisms, which limits the optimization of the reported methods.

DNA methyltransferase CHROMOMETHYLASE3 prevents ONSEN transposon silencing under heat stress.

DNA methylation plays crucial roles in transposon silencing and genome integrity. CHROMOMETHYLASE3 (CMT3) is a plant-specific DNA methyltransferase responsible for catalyzing DNA methylation at the CHG (H = A, T, C) context. Here, we identified a positive role of CMT3 in heat-induced activation of retrotransposon ONSEN. We found that the full transcription of ONSEN under heat stress requires CMT3. Interestingly, loss-of-function CMT3 mutation led to increased CHH methylation at ONSEN. The CHH methylation is mediated by CMT2, as evidenced by greatly reduced CHH methylation in cmt2 and cmt2 cmt3 mutants coupled with increased ONSEN transcription. Furthermore, we found more CMT2 binding at ONSEN chromatin in cmt3 compared to wild-type accompanied with an ectopic accumulation of H3K9me2 under heat stress, suggesting a collaborative role of H3K9me2 and CHH methylation in preventing heat-induced ONSEN activation. In summary, this study identifies a non-canonical role of CMT3 in preventing transposon silencing and provides new insights into how DNA methyltransferases regulate transcription under stress conditions.

Evaluating the Efficacy of Hymexazol in Controlling Globisporangium spinosum, the Newly Identified Pathogen Causing Root Rot in Houttuynia cordata.

Houttuynia cordata is a prevalent vegetable in several Asian countries and is commonly used as a traditional Chinese medicinal herb for treating various diseases in China. Unfortunately, its yield and quality are adversely affected by root rot. However, the pathogen responsible for the losses remains unidentified, and effective fungicides for its management have not been thoroughly explored. In this work, we demonstrate the first report of Globisporangium spinosum as the causative agent causing root rot of H. cordata. Moreover, we evaluated the efficacy of hymexazol to manage the disease, which displayed remarkable inhibitory effects against mycelial growth of G. spinosum in vitro, with EC50 values as low as 1.336 µg/ml. Furthermore, hymexazol completely inhibited sporangia in G. spinosum at a concentration of 0.3125 µg/ml. Specifically, we observed that hymexazol was highly efficacious in reducing the incidence of H. cordata root rot caused by G. spinosum in a greenhouse setting. These findings offer a potential management tool for utilization of hymexazol in controlling H. cordata root rot in field production.

PBS3: a versatile player in and beyond salicylic acid biosynthesis in Arabidopsis.

AVRPPHB SUSCEPTIBLE 3 (PBS3) belongs to the GH3 family of acyl acid amido synthetases, which conjugates amino acids to diverse acyl acid substrates. Recent studies demonstrate that PBS3 in Arabidopsis plays a key role in the biosynthesis of plant defense hormone salicylic acid (SA) by catalyzing the conjugation of glutamate to isochorismate to form isochorismate-9-glutamate, which is then used to produce SA through spontaneous decay or ENHANCED PSEUDOMONAS SUSCEPTIBILITY (EPS1) catalysis. Consistent with its function as an essential enzyme for SA biosynthesis, PBS3 is well known to be a positive regulator of plant immunity in Arabidopsis. Additionally, PBS3 is also involved in the trade-off between abiotic and biotic stress responses in Arabidopsis by suppressing the inhibitory effect of abscisic acid on SA-mediated plant immunity. Besides stress responses, PBS3 also plays a role in plant development. Under long-day conditions, PBS3 influences Arabidopsis flowering time by regulating the expression of flowering regulators FLOWERING LOCUS C and FLOWERING LOCUS T. Taken together, PBS3 functions in the signaling network of plant development and responses to biotic and/or abiotic stresses, but the molecular mechanisms underlying its diverse roles remain obscure.

TrpR-Like Protein PXO_00831, Regulated by the Sigma Factor RpoD, Is Involved in Motility, Oxidative Stress Tolerance, and Virulence in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a Gram-negative bacterium that causes bacterial leaf blight in rice. In this study, we identified a putative TrpR-like protein, PXO_TrpR (PXO_00831), in Xoo. This protein contains a tryptophan (Trp) repressor domain and is highly conserved in Xanthomonas. Auxotrophic assays and RT-qPCR confirmed that PXO_TrpR acts as a Trp repressor, negatively regulating the expression of Trp biosynthesis genes. Pathogenicity tests showed that PXO_trpR knockout in Xoo significantly reduced lesion development and disease symptoms in the leaves of susceptible rice. RNA-seq analysis and phenotypic tests revealed that the PXO_trpR mutant exhibited impaired cell motility and was more sensitive to H2O2 oxidative stress than the wild-type strain. Furthermore, we found that the sigma 70 factor RpoD controlled the transcription of PXO_trpR by directly binding to its promoter region. This study demonstrates the biological function and transcriptional mechanism of PXO_TrpR as a Trp repressor in Xoo and evaluates its novel pathogenic roles by regulating flagellar motility and the oxidative stress response.

Current Situation of Fire Blight in China.

Fire blight, caused by the plant-pathogenic bacterium Erwinia amylovora, is a devastating disease that occurs on rosaceous plants, including pears and apples. E. amylovora is indigenous to North America and was spread to the Eurasian continent in the second half of the 20th century through contaminated plant materials. In 2016, fire blight was first observed in Yili, Xinjiang Province, in Northwestern China. Since then, it has spread to most pear-producing regions in Xinjiang Province and parts of Gansu Province. The disease has caused severe damage to China's pear and apple industries, including the 2017 disease epidemic in Korla, Xinjiang, which caused an overall yield reduction of 30 to about 50% in Korla and the destruction of over 1 million pear trees. Over the past few years, a combined effort of research, extension, and education by the Chinese government, scientists, and fruit growers has greatly alleviated outbreaks and epidemics in affected regions while successfully limiting the further spread of fire blight to new geographical regions. Here, we review the occurrence, spread, and damage of this disease to the Chinese fruit industry, as well as the management options used in China and their outcomes. We also discuss future perspectives for restraining the spread and alleviating the damage of fire blight in China.

First Report of Bacterial Fruit Blotch caused by Kosakonia cowanii on Trichosanthis fructus in Jiangsu Province, China.

Trichosanthis fructus is one of the most common medicinal plants in China. In September 2022, T. fructus fruit showed black necrotic spots and surface irregularities, with water-soaked lesions (Fig 1). The affected T. fructus fruit (five weeks after blossom) were located in a field in Huai'an Municipality, Jiangsu Province (33.85°N, 119.00°E). The incidence was approximately 50%, causing great losses in fruit production. To isolate the causal agent, two symptomatic fruit from different plants were surface-disinfested with 75% (v/v) ethanol for 1 min, washed three times with sterile distilled water, and cultured on Nutrient agar (NA) plates at 28°C for 24 h. The obtained colonies were light yellow and transferred to fresh NA plates using the conventional repetitive streaking technique to obtain pure cultures. The purified bacterial cells were rod shaped, 1.64 to 2.47 µm long (n = 45), and 0.58 to 0.74 µm wide (n = 45) (Figure S2). Three isolates were used for further characterization. Biochemical tests indicated that the three isolates were Gram negative. DNA was extracted from the three bacterial isolates and used to amplify the16S rRNA (27F/1492R primers) and partial gyrB (UP1/Up2r primers) genes (Marchesi et al. 1998; Yamamoto and Harayama 1995). PCR products were purified using the DNA Clean-up Kit (CW2301, CWBIO), ligated into the PMD-19 vector (6013, Takara), and sequenced by Beijing Tsingke Biotech. The obtained 16S rRNA (GenBank accessions: OQ923996-OQ923998) and gyrB sequences (OR140942-OR140944) showed the best match, over 99%and 98% identity with 100% coverage to the K. cowanii type strain JCM 10956 (CP019445.1). To fulfill Koch's postulates, pathogenicity tests were conducted on healthy T. fructus fruit. T. fructus fruit showed no wounds or lesions, and were surface disinfected with 75% alcohol. The three isolates were grown in nutrient broth at 200 rpm in 28 oC for 24 h and re-suspended in sterilized ddH2O to OD600 = 0.6~1.0 (108~109cfu/mL). Five µL of bacterial suspension was inoculated into the healthy fruit surface with a sterile knife. For the control experiment, the same volume of sterilized ddH2O was used. In each treatment, four healthy T. fructus fruit were treated. All samples were incubated at 25°C and 75% humidity in a plant incubator (Bluepard, MGC-350BP-2). After 12 days, bacterial fruit blotch symptoms were observed in all the inoculated fruit. The pathogen was recovered from the infected fruit, and its identity was confirmed by 16S rRNA/gyrB sequencing and morphological analysis. To further investigate the pathogenicity, four-week-old T. fructus plant leaves were inoculated with the above three isolated suspension (OD600=0.6~1.0) using the leaf cutting method (Kauffman et al. 1973). Sterilized ddH2O was used as mock control. After 10 days, bacterial blight symptoms were observed in all inoculated leaves. To our knowledge, this is the first report of K. cowanii causing fruit blotch on T. fructus worldwide. This species has been previously associated with acute cholecystitis in humans (Berinson et al. 2020; Petrzik et al. 2021), but it was recently identified as the causal agent of bacterial wilt on patchouli, bacterial blight on soybean, and stalk rot in foxtail millet (Han et al. 2023; Krawczyk and Borodynko-Filas 2020; Zhang et al. 2022). China is the largest producer of T. fructus. This report reveals that K. cowanii has a greater host range than was known. This report will help to better understand the pathogens that affects T. fructus production in China.

First Report of Bacterial Leaf Blight caused by Enterobacter asburiae on Sorghum in Jiangsu Province, China.

Sorghum (Sorghum bicolor [L.] Moench) is a major cereal crop in China, with a planting area of more than 674666 ha in 2021. In August 2022, bacterial leaf blight symptoms were observed on sorghum plants grown in a field in Huai'an (119.30437 ºE, 33.999644 ºN), in Jiangsu Province (Fig. 1). To determine the causal agent, four symptomatic leaves from different plants were surface sterilized with 75% (v/v) ethanol for 1 min and washed three times with ddH2O. The surface-sterilized plant tissues were cut into small pieces (4 × 4 mm in size) and cultured on Nutrient Agar (NA) plates at 28ºC for 24 h. To obtain pure cultures, these colonies were transferred to fresh NA plates by using the conventional streak plate method. The purified bacterial cells were rod-shaped, from 1.14 to 1.66 µm long, and from 0.61 to 0.86 µm wide (number of observations = 31) (Fig. 2). Three isolates were used for further characterization. The Gram stain test indicated that the three isolates were Gram negative. 16S rRNA (27F/1492R primers) and gyrB (UP1/Up2r) genes were amplified and sequenced (Marchesi et al. 1998; Yamamoto and Harayama 1995). The obtained 16S rRNA (0R143361-0R143363) and gyrB sequences (0R146993-0R146995) were submitted to GenBank. The 16S rRNA sequences of the three isolated strains showed over 98% identity (1447/1462, 1438/1462 and 1443/1460 bp) to the E. asburiae reference strains ENIPBJ CG1, CAV1043 and 1808 013 (CP014993.1, CP011591.1 and AP019632.1, respectively). Similarly, the gyrB sequences of the three strains showed 98% identity (1103/1129, 1105/1129 and 1108/1129 bp) to the same E. asburiae reference strains. Four-week-old sorghum plants were used in the pathogenicity tests. A phylogenetic tree was constructed with reference strains (Hoffmann et al., 2005). The healthy leaves were inoculated with bacterial suspensions of the three bacterial isolates (OD600 = 0.6~1.0) using the leaf cutting method (Kauffman et al. 1973). For the control group, sterilized ddH2O was used. Each isolate was inoculated in three healthy plants. Inoculated plants were incubated at 28ºC and 75% humidity with alternating 12-h light and 12-h dark cycles with a photon flux density of 200 mmol/m2/s. After 10 days, bacterial leaf blight symptoms were observed in all the inoculated leaves. The inoculated leaves showed severe browning near the inoculation site (1-2 cm), and advanced yellowing from 2 to 7 cm from the inoculation site, while no symptoms were found in control group. The pathogen was recovered from the infected leaves, and its identity was confirmed by 16S rRNA/gyrB sequencing and morphological analysis, fulfilling Koch's postulates (Fig 2). To our knowledge, this is the first report of E. asburiae causing bacterial leaf blight on sorghum worldwide. This species is a well-known pathogen of humans that can cause nosocomial infections (Markovska et al. 2019; Zhu et al. 2017). Recently, E. asburiae was identified as the causal agent of bacterial blight on rice and tuber rot on radish (Wang et al. 2023; Yu et al. 2021). The emergence E. asburiae as a plant pathogen may be produced by the numerous resistant strains reported during recent years. Pantoea ananatis has been reported as a common companion pathogen of E. asburiae (Xue et al. 2021). This report will help to better understand the host promiscuity of E. asburiae and reveals a new pathogen that affects sorghum production in China. This study also serves as a basis for future studies to develop management strategies and cultivation for the disease to prevent sorghum yield loss. As far as we know, no control method for the management of this new plant pathogen was reported to date, which highlights the potential hazard of this discovery. Reference Hoffmann, H., et al. 2005. Syst. Appl. Microbiol. 28:196. Kauffman, H. E., et al. 1973. Plant Dis. Rep. 57:537. Marchesi, J. R., et al. 1998. Appl. Environ. Microbiol. 64:795. Markovska, R., et al. 2019. Infect. Dis. 51:627. Wang, R., et al. 2023. Plant Dis. in press. https://doi.org/10.1094/PDIS-11-22-2650-PDN Xue, Y., et al. 2021. Plant Dis. 105:2078. Yamamoto, S., et al. 1995. Appl. Environ. Microbiol. 61:1104. Yu, L., et al. 2021. Plant Dis. 106:310. Zhu, B., et al. 2017. J. Glob. Antimicrob. Resist. 8:104.

Application of chitosan nanoparticles in quality and preservation of postharvest fruits and vegetables: A review.

Chitosan is an interesting alternative material for packaging development due to its biodegradability. However, its poor mechanical properties and low permeability limit its actual applications. Chitosan nanoparticles (CHNPs) have emerged as a suitable solution to overcome these intrinsic limitations. In this review, all studies regarding the use of CHNPs to extend the shelf life and improve the quality of postharvest products are covered. The characteristics of CHNPs and their combinations with essential oils and metals, along with their effects on postharvest products, are compared and discussed throughout the manuscript. CHNPs enhanced postharvest antioxidant capacity, extended shelf life, increased nutritional quality, and promoted tolerance to chilling stress. Additionally, the CHNPs reduced the incidence of postharvest phytopathogens. In most instances, smaller CHNPs (<150 nm) conferred higher benefits than larger ones (>150 nm). This was likely a result of the greater plant tissue penetrability and surface area of the smaller CHNPs. The CHNPs were either applied after preparing an emulsion or incorporated into a film, with the latter often exhibiting greater antioxidant and antimicrobial activities. CHNPs were used to encapsulate essential oils, which could be released over time and may enhance the antioxidant and antimicrobial properties of the CHNPs. Even though most applications were performed after harvest, preharvest application had longer lasting effects.

Diguanylate Cyclase and Phosphodiesterase Interact To Maintain the Specificity of Cyclic di-GMP Signaling in the Regulation of Antibiotic Synthesis in Lysobacter enzymogenes.

Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. A large number of c-di-GMP-related diguanylate cyclases (DGCs), phosphodiesterases (PDEs), and effectors are responsible for the complexity and dynamics of c-di-GMP signaling. Some of these components employ various methods to avoid undesired cross talk to maintain signaling specificity. The synthesis of the antibiotic HSAF (heat-stable antifungal factor) in Lysobacter enzymogenes is regulated by a specific c-di-GMP signaling pathway that includes a PDE, LchP, and a c-di-GMP effector, Clp (also a transcriptional regulator). In the present study, from among 19 DGCs, we identified a diguanylate cyclase, LchD, that participates in this pathway. Subsequent investigation indicates that LchD and LchP physically interact and that the catalytic center of LchD is required for both the formation of the LchD-LchP complex and HSAF production. All the detected phenotypes support that LchD and LchP display local c-di-GMP signaling to regulate HSAF biosynthesis. Although direct evidence is lacking, our investigation, which shows that the interaction between a DGC and a PDE maintains the specificity of c-di-GMP signaling, suggests the possibility of the existence of local c-di-GMP pools in bacteria. IMPORTANCE Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. The signaling of c-di-GMP is complex and dynamic, and it is mediated by a large number of components, including c-di-GMP synthases (diguanylate cyclases [DGCs]), c-di-GMP-degrading enzymes (phosphodiesterases [PDEs]), and c-di-GMP effectors. These components deploy various methods to avoid undesired cross talk to maintain signaling specificity. In the present study, we identified a DGC that interacted with a PDE to specifically regulate antibiotic biosynthesis in L. enzymogenes. We provide direct evidence to show that the DGC and PDE form a complex and also indirect evidence to argue that they may balance a local c-di-GMP pool to control antibiotic production. These results represent an important finding regarding the mechanism of a DGC and PDE pair to control the expression of specific c-di-GMP signaling pathways.

The Xanthomonas type III effector XopAP prevents stomatal closure by interfering with vacuolar acidification.

Plant stomata close rapidly in response to a rise in the plant hormone abscisic acid (ABA) or salicylic acid (SA) and after recognition of pathogen-associated molecular patterns (PAMPs). Stomatal closure is the result of vacuolar convolution, ion efflux, and changes in turgor pressure in guard cells. Phytopathogenic bacteria secrete type III effectors (T3Es) that interfere with plant defense mechanisms, causing severe plant disease symptoms. Here, we show that the virulence and infection of Xanthomonas oryzae pv. oryzicola (Xoc), which is the causal agent of rice bacterial leaf streak disease, drastically increased in transgenic rice (Oryza sativa L.) plants overexpressing the Xoc T3E gene XopAP, which encodes a protein annotated as a lipase. We discovered that XopAP binds to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ), a membrane phospholipid that functions in pH control in lysosomes, membrane dynamics, and protein trafficking. XopAP inhibited the acidification of vacuoles by competing with vacuolar H+ -pyrophosphatase (V-PPase) for binding to PtdIns(3,5)P2 , leading to stomatal opening. Transgenic rice overexpressing XopAP also showed inhibition of stomatal closure when challenged by Xoc infection and treatment with the PAMP flg22. Moreover, XopAP suppressed flg22-induced gene expression, reactive oxygen species burst and callose deposition in host plants, demonstrating that XopAP subverts PAMP-triggered immunity during Xoc infection. Taken together, these findings demonstrate that XopAP overcomes stomatal immunity in plants by binding to lipids.

An Antifungal Polycyclic Tetramate Macrolactam, Heat-Stable Antifungal Factor (HSAF), Is a Novel Oxidative Stress Modulator in Lysobacter enzymogenes.

Polycyclic tetramate macrolactams (PoTeMs) are a fast-growing family of antibiotic natural products found in phylogenetically diverse microorganisms. Surprisingly, none of the PoTeMs have been investigated for potential physiological functions in their producers. Here, we used heat-stable antifungal factor (HSAF), an antifungal PoTeM from Lysobacter enzymogenes, as a model to show that PoTeMs form complexes with iron ions, with an association constant (Ka ) of 2.71 × 106 M-1 The in vivo and in vitro data showed formation of 2:1 and 3:1 complexes between HSAF and iron ions, which were confirmed by molecular mechanical and quantum mechanical calculations. HSAF protected DNA from degradation in high concentrations of iron and H2O2 or under UV radiation. HSAF mutants of L. enzymogenes barely survived under oxidative stress and exhibited markedly increased production of reactive oxygen species (ROS). Exogenous addition of HSAF into the mutants significantly prevented ROS production and restored normal growth in the mutants under the oxidative stress. The results reveal that the function of HSAF is to protect the producer microorganism from oxidative damage rather than as an iron-acquisition siderophore. The characteristic structure of PoTeMs, a 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. The study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damage to the cells.IMPORTANCE PoTeMs are a family of structurally distinct metabolites that have been found in a large number of bacteria. Although PoTeMs exhibit diverse therapeutic properties, the physiological function of PoTeMs in the producer microorganisms had not been investigated. HSAF from Lysobacter enzymogenes is an antifungal PoTeM that has been subjected to extensive studies for mechanisms of biosynthesis, regulation, and antifungal activity. Using HSAF as a model system, we here showed that the characteristic structure of PoTeMs, a 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. In L. enzymogenes, HSAF functions as a small-molecule modulator for oxidative damage caused by iron, H2O2, and UV light. Together, the study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damage to the cells. HSAF represents the first member of the fast-growing PoTeM family of microbial metabolites whose potential biological function has been studied.

F-box protein CFK1 interacts with and degrades de novo DNA methyltransferase in Arabidopsis.

DNA methylation plays crucial roles in cellular development and stress responses through gene regulation and genome stability control. Precise regulation of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), the de novo Arabidopsis DNA methyltransferase, is crucial to maintain DNA methylation homeostasis to ensure genome integrity. Compared with the extensive studies on DRM2 targeting mechanisms, little information is known regarding the quality control of DRM2 itself. Here, we conducted yeast two-hybrid screen assay and identified an E3 ligase, COP9 INTERACTING F-BOX KELCH 1 (CFK1), as a novel DRM2-interacting partner and targets DRM2 for degradation via the ubiquitin-26S proteasome pathway in Arabidopsis thaliana. We also performed whole genome bisulfite sequencing (BS-seq) to determine the biological significance of CFK1-mediated DRM2 degradation. Loss-of-function CFK1 leads to increased DRM2 protein abundance and overexpression of CFK1 showed reduced DRM2 protein levels. Consistently, CFK1 overexpression induces genome-wide CHH hypomethylation and transcriptional de-repression at specific DRM2 target loci. This study uncovered a distinct mechanism regulating de novo DNA methyltransferase by CFK1 to control DNA methylation level.

The Arabidopsis zinc finger proteins SRG2 and SRG3 are positive regulators of plant immunity and are differentially regulated by nitric oxide.

Nitric oxide (NO) regulates the deployment of a phalanx of immune responses, chief among which is the activation of a constellation of defence-related genes. However, the underlying molecular mechanisms remain largely unknown. The Arabidopsis thaliana zinc finger transcription factor (ZF-TF), S-nitrosothiol (SNO) Regulated 1 (SRG1), is a central target of NO bioactivity during plant immunity. Here we characterize the remaining members of the SRG gene family. Both SRG2 and, especially, SRG3 were positive regulators of salicylic acid-dependent plant immunity. Analysis of SRG single, double and triple mutants implied that SRG family members have additive functions in plant immunity and, surprisingly, are under reciprocal regulation. SRG2 and SRG3 localized to the nucleus and functioned as ethylene-responsive element binding factor-associated amphiphilic repression (EAR) domain-dependent transcriptional repressors: NO abolished this activity for SRG3 but not for SRG2. Consistently, loss of GSNOR function, resulting in increased (S)NO concentrations, fully suppressed the disease resistance phenotype established from SRG3 but not SRG2 overexpression. Remarkably, SRG3 but not SRG2 was S-nitrosylated in vitro and in vivo. Our findings suggest that the SRG family has separable functions in plant immunity, and, surprisingly, these ZF-TFs exhibit reciprocal regulation. It is remarkable that, through neofunctionalization, the SRG family has evolved to become differentially regulated by the key immune-related redox cue, NO.